Method for promoting formation of nerve fascia membrane by small extracellular vesicles derived from hair follicle neural crest stem cells and application

Abstract

The invention relates to the technical field of biomedicine, in particular to a method for promoting formation of nerve fasciae and repair and regeneration after sciatic nerve defect by using small extracellular vesicles (hfNCSCs-sEVs) derived from hair follicle neural crest stem cells and application. According to the application disclosed by the invention, the effects of the hfNCSCs-sEVs on proliferation, migration, tube formation and barrier formation of nerve fascia cells in vitro are defined; the effect of hfNCSCs-sEVs on tissue regeneration and function recovery after rat sciatic nerve defect is defined, the hfNCSCs-sEVs inhibits expression of Smad7 in nerve tract membrane cells by transferring miR-21-5p, activates a TGF-beta / Smad signal channel, up-regulates expression of HAS2, promotes proliferation and migration of HAS2 and promotes expression of tight junction protein thereafter, and the hfNCSCs-sEVs can be used for treating rat sciatic nerve defect. And a new view angle and theoretical support are provided for repair and regeneration of sciatic nerve injury.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method and application of using small extracellular vesicles derived from hair follicle neural crest stem cells to promote the formation of the perineal membrane and the repair of sciatic nerve injury. Background Technology

[0002] The perineurium is a thin membrane of connective tissue composed of 7-8 layers of flattened perineurial cells. It separates nerve fibers into bundles and serves as a key component of the blood-nerve barrier. Perineural cells, with the help of tight junction proteins, effectively block harmful substances, pathogens, toxins, and other pathogens from invading nerve tissue. They play an important role in ensuring nerve signal transmission and neuronal metabolic activity (Malong et al., 2023; Lago-Baldaia et al., 2020; Yin et al., 2022; Kanda, 2013; Kucenas, 2015). Furthermore, the role of perineurial cells in nerve injury repair has attracted considerable attention. Studies have reported (Schrijder et al.) that perineurial cells play a crucial role in maintaining orderly nerve branching and stable growth during mouse nerve development and growth. Without the proper guidance of perineurial cells, nerve growth in tissues becomes disordered and branches are relatively numerous. Abnormally growing perineurial cells can interfere with Schwann cell differentiation and the formation of neuromuscular junctions. The integrity of the perineurium is essential for precise nerve fiber docking during axonal regeneration and the recovery of limb function (Clark et al., 2014; Chovatiya et al., 2023). Research has found that perineurial cells belong to glial cells with endothelial cell characteristics (Yin et al., 2022) (Kovalenko et al., 2000), and multiple studies have demonstrated that enhanced endothelial cell proliferation and migration correspondingly improve their tube-forming ability (Raposo et al., 2024; Arnaoutova and Kleinman, 2010). Several research teams have also discovered that perineurial cells possess the characteristics of mesenchymal cells, exhibiting regenerative and repair potential through proliferation and migration (Piña-Oviedo and Ortiz-Hidalgo, 2008; Zhao et al., 2022; Carr et al., 2019; Zhao et al., 2024). In nerve defect repair, they are the first to reach the damaged area, working with macrophages to clear cellular debris and playing a role in guiding Schwann cell migration and nerve fiber regeneration (Küspert et al., 2012; Lewis and Kucenas, 2014). Therefore, in the early stages of peripheral nerve defects, promoting the proliferation and migration of perineurial cells and forming a blood-nerve barrier has become crucial for repairing peripheral nerve defects.

[0003] Hair follicle neural crest stem cells (hfNCSCs) are pluripotent stem cells derived from the ectoderm within the skin, and both they and peripheral nerves develop from the neural crest. hfNCSCs have the advantage of being widely available and easily obtained (Li et al., 2021; Sieber-Blum, 2010), while other neural crest stem cells are mostly located in areas such as the central nervous system, facing numerous difficulties in sourcing them and being relatively scarce. Similarly, other ectoderm-derived stem cells, such as olfactory stem cells and dental ectoderm stem cells, also face the problem of limited availability. Researching the role of hfNCSCs in peripheral nerve injury repair and their underlying mechanisms has broad application prospects for the clinical treatment of peripheral nerve injuries (Zheng et al.).

[0004] Considering the complex risks that may arise after stem cell transplantation for nerve injury treatment, such as the risk of tumorigenesis, chromosomal aberrations, and immune rejection, our research group successfully isolated and identified sEVs secreted by hfNCSCs (hfNCSCs-sEVs) (see the invention patent "Exosomes of Hair Follicle Neural Crest Stem Cells and Their Extraction Methods and Applications", patent number: ZL 202110637001.7). Currently, some researchers have studied the role of substances secreted by hfNCSCs in nerve injury repair, but the nerve repair mechanism and the effect of hfNCSCs on perineurium cells have not yet been addressed. Summary of the Invention

[0005] The purpose of this invention is to use hfNCSCs-sEVs to deliver miR-21-5p, inhibit the expression of Smad7 in perineurium cells, activate the TGF-β / Smad pathway and upregulate the expression of hyaluronic acid synthase 2 (HAS2), promote the proliferation and migration of perineurium cells, thereby promoting the formation of tight junctions between perineurium cells and promoting the repair of sciatic nerve injury.

[0006] To achieve the above objectives, the present invention employs the following technical solutions and steps:

[0007] (a) Uptake of hfNCSCs-sEVs by perineurium cells;

[0008] (b) Study on the effects of hfNCSCs-sEVs on the proliferation and migration of perineurium cells in vitro;

[0009] (c) Effects of hfNCSCs-sEVs on the tube-forming ability, barrier function, and tight junction protein expression of nerve perimysial cells in vitro;

[0010] (d) The effect of hfNCSCs-sEVs on the expression of tight junction proteins in nerve perimysial cells in vivo;

[0011] (e) The role of hfNCSCs-sEVs in in vivo on nerve fiber regeneration and myelin formation;

[0012] (f) The role of hfNCSCs-sEVs in the recovery of nerve function and the relief of skeletal muscle atrophy in vivo;

[0013] (g) The effect of hfNCSCs-sEVs on Has2 gene expression in vivo;

[0014] (h) Effects of miR-21-5p on the TGF-β / Smad / HAS2 axis of perineurial cells in vitro and on the proliferation and migration of perineurial cells;

[0015] (i) The effect of miR-21-5p in hfNCSCs-sEVs on HAS2 expression in perineurium cells and its effect on perineurium cell proliferation, migration and tight junction protein expression.

[0016] This invention reveals that hfNCSCs-sEVs can promote the proliferation and migration of neuronal perimysial cells in vitro, enhance their tube-forming ability and barrier function, and upregulate the expression of their tight junction proteins.

[0017] This invention found that in the early stage of sciatic nerve defect repair in rats, intervention with hfNCSCs-sEVs can promote the expression of tight junction proteins in nerve perineurium cells, Schwann cell migration and nerve fiber regeneration. In the later stage of repair, it can promote nerve fiber regeneration and myelin formation, as well as the recovery of nerve function.

[0018] This invention discovers that hfNCSCs-sEVs target and inhibit the expression of Smad7 in perineurium cells by delivering miR-21-5p, activate the TGF-β / Smad signaling pathway, upregulate the expression of HAS2 and promote its proliferation and migration, and also promote the expression of tight junction proteins in perineurium cells in vitro.

[0019] Based on the above technical solution, in a first aspect, the present invention provides the use of a reagent that upregulates HAS2 expression in nerve perimysium cells in the preparation of a drug or reagent that promotes nerve perimysium formation.

[0020] Furthermore, the application of reagents that upregulate HAS2 expression in perineurium cells in the preparation of drugs or reagents that promote perineurium cell proliferation, migration, and tight junction protein expression.

[0021] Furthermore, the reagent used to upregulate HAS2 expression in nerve perimysial cells is hfNCSCs-sEVs or miR-21-5p derived from hfNCSCs-sEVs.

[0022] Furthermore, the hfNCSCs-sEVs promote the proliferation and migration of nerve perithelial cells by activating the TGF-β pathway and upregulating HAS2 expression.

[0023] Furthermore, the method for collecting and extracting hfNCSCs-sEVs includes the following steps:

[0024] (1) Pass hfNCSCs to the 4th generation. When the cell confluence reaches 70%~80%, the culture medium is replaced with passaged hfNCSCs culture medium containing exosome-free FBS. The cells are cultured for another 48 h. At this time, the cell confluence reaches 85%~95%. The cell culture medium supernatant containing hfNCSCs-sEVs is collected and centrifuged at 3000g for 10 min at 4 ℃. The supernatant is collected to remove cell debris.

[0025] (2) Mix the Exosome Concentration Solution reagent and the supernatant in the cell supernatant exosome extraction kit at a volume ratio of 1:4, vortex and mix for 1 min, and then let stand at 4 ℃ for 20 h.

[0026] (3) Centrifuge the mixture at 10,000g for 60 min at 4 ℃, discard the supernatant, add 1 / 100 volume of PBS to dissolve the precipitated hfNCSCs-sEVs, centrifuge the resuspended solution at 12,000g for 2 min at 4 ℃, transfer the supernatant to the upper chamber of the Exosome Purification Filter column, centrifuge at 3,000g for 10 min at 4 ℃, collect the liquid at the bottom of the column as the hfNCSCs-sEVs suspension, aliquot and store at -80 ℃ for later use.

[0027] A second aspect of the present invention provides the use of miR-21-5p derived from hfNCSCs-sEVs in the preparation of a medicament or reagent that promotes the formation of the perineural membrane and accelerates the repair of nerve damage.

[0028] Furthermore, hfNCSCs-sEVs can increase the content of miR-21-5p in perineurium cells in vitro. The miR-21-5p derived from hfNCSCs-sEVs can activate the TGF-β / Smad signaling pathway by inhibiting Smad7 in perineurium cells, upregulating HAS2 expression, promoting its proliferation and migration, and promoting the expression of tight junction proteins in perineurium cells.

[0029] Furthermore, miR-21-5p derived from hfNCSCs-sEVs promotes the expression of cell proliferation, migration-related proteins, and tight junction proteins between nerve perineurium cells in sciatic nerve defect repair.

[0030] A third aspect of the present invention provides a method for promoting the formation of the perineurium, which involves adding a reagent that upregulates the expression of HAS2 in the perineurium cells to the culture medium of the perineurium cells.

[0031] Furthermore, the reagent used to upregulate HAS2 expression in nerve perimysial cells is hfNCSCs-sEVs or miR-21-5p derived from hfNCSCs-sEVs.

[0032] Furthermore, the concentration of the hfNCSCs-sEVs in the culture medium is 5 × 10⁻⁶. 8 ~10×10 8 particles / mL.

[0033] In a fourth aspect, the invention provides the use of an agent that upregulates HAS2 expression in perineurium cells in the preparation of a drug or agent that promotes the repair of sciatic nerve injury.

[0034] Furthermore, the reagent that upregulates HAS2 expression in nerve perineurium cells promotes tissue repair and functional recovery after sciatic nerve defects.

[0035] Furthermore, the reagent used to upregulate HAS2 expression in nerve perimysial cells is hfNCSCs-sEVs or miR-21-5p derived from hfNCSCs-sEVs.

[0036] Furthermore, the hfNCSCs-sEVs promote the expression of tight junction proteins on nerve perisynthetic cells; increase the migration speed of Schwann cells and the extension speed of nerve fibers; promote the migration of Schwann cells and the regeneration of nerve fibers in the early stage of sciatic nerve defect repair; promote the formation of myelin sheath in the later stage of sciatic nerve defect repair; improve the signal transduction function of regenerated nerves in the later stage of sciatic nerve defect repair; and slow down the atrophy of target muscles in the later stage of sciatic nerve defect repair.

[0037] The advantages of this invention are:

[0038] 1. This invention successfully isolated and extracted hfNCSCs-sEVs, and clarified the role of hfNCSCs-sEVs in in vitro proliferation, migration, tube formation and barrier formation of nerve perimysium cells;

[0039] 2. This invention clarifies the role of hfNCSCs-sEVs in tissue regeneration and functional recovery after sciatic nerve injury in rats. Focusing on perineurium cells, this invention utilizes transcriptome sequencing and bioinformatics analysis of regenerated rat sciatic nerve tissue. In vivo and in vitro experiments revealed that hfNCSCs-sEVs can target and inhibit Smad7 via miR-21-5p, thereby activating the TGF-β / Smad signaling pathway, upregulating HAS2 expression in perineurium cells and promoting their proliferation and migration, and facilitating earlier formation of tight junction proteins between cells, thus contributing to perineurium formation and accelerating the repair process of nerve injury. These findings provide a new perspective and theoretical support for the repair and regeneration of sciatic nerve injury. This invention offers a new approach to peripheral nerve injury repair and is expected to provide a new avenue for the clinical treatment of peripheral nerve injury. Attached Figure Description

[0040] Figure 1 In vitro culture and identification of hfNCSCs

[0041] (A) Light micrograph of primary cultured rat hfNCSCs. A1: Overall view of hair follicle patch culture; A2: Showing the hair follicle bulge and emerging cells. (B) Light micrograph of 4th generation hfNCSCs (confluence approximately 70%–80%). (C) Light micrograph of 4th generation hfNCSCs (confluence reaching 85%–95%). (D) Immunofluorescence staining of hfNCSCs with the neural crest cell marker P75 (red) and the stem cell marker Nestin (green); DAPI (blue) staining shows the cell nucleus.

[0042] Figure 2 Characterization and identification of hfNCSCs-sEVs

[0043] (A) Western blotting to detect the protein expression of surface markers (CD9, CD63, TSG101) and endoplasmic reticulum marker (Calnexin) in hfNCSCs-sEVs. (B) Nanoparticle tracking analysis to determine the concentration and particle size distribution of hfNCSCs-sEVs. (C) Transmission electron microscopy images showing the typical morphology of hfNCSCs-sEVs.

[0044] Figure 3 In vitro culture and identification of perimysial cells

[0045] (A) Light micrograph of fourth-generation perimysial cells cultured in vitro. (B1~B4) Immunofluorescence staining images of fourth-generation perimysial cells cultured in vitro. The specific marker proteins Claudin1 (B1), ZO1 (B2), and GLUT1 (B3) are positive, while Schwann cell marker S100 (B4) is negative. DAPI (blue) staining indicates the cell nucleus.

[0046] Figure 4 Perineural cells take up hfNCSCs-sEVs in vitro.

[0047] Immunofluorescence staining image of ZO1 (green) positive perineurial cells and PKH26 (red) labeled hfNCSCs-sEVs; DAPI (blue) staining shows cell nuclei.

[0048] Figure 5 hfNCSCs-sEVs promote the proliferation and migration of perineurium cells in vitro.

[0049] (A) CCK-8 assay for detecting 0, 2 × 10⁻⁶ cells in vitro 8 5×10 8 10×10 8 Statistical graph of cell viability values ​​of perineurium cells in the particles / mL group at 3, 5, and 7 days. (B) Transwell assay to detect cell viability values ​​of 0, 2 × 10⁻⁶ cells in vitro. 8 5×10 8 10×10 8 The number of migrating perineurium cells in the particles / mL group at 6, 12, and 18 h. (C) Western blotting analysis of the protein expression of proliferating cell nuclear antigen (PCNA) and vimentin in perineurium cells of the PBS and hfNCSCs-sEVs groups after 5 days of in vitro culture. (D) Statistical graph of relative protein expression levels. * P < 0.05, ** P < 0.01, *** P < 0.001, ns (P > 0.05)

[0050] Figure 6 hfNCSCs-sEVs enhance the tubular formation ability of perimysial cells in vitro.

[0051] (A) Light micrographs of pericermal cell tube formation experiments in the PBS group and hfNCSCs-sEVs group, and statistical graphs of (B) the number of branching points and (C) the total lumen length. * (P < 0.05)

[0052] Figure 7 hfNCSCs-sEVs enhance barrier function of nerve perimysial cells after membrane formation in vitro.

[0053] (A) Detection of transmembrane resistance values ​​of nerve perithelial cells in the PBS group and hfNCSCs-sEVs group, and (B) Detection of cell monolayer permeability. *** P < 0.001,ns (P > 0.05)

[0054] Figure 8 hfNCSCs-sEVs promote the expression of tight junction proteins in pericerebrovascular cells in vitro.

[0055] (A) Immunofluorescence staining images of the PBS and hfNCSCs-sEVs groups, showing the expression of tight junction proteins ZO1 (green) and β-tubulin (red) on the perimysial cell membrane, and (B) a statistical graph of the cumulative fluorescence density of ZO1. (C) Western blotting analysis of the protein expression of tight junction proteins ZO1 and Claudin1 in perimysial cells of the PBS and hfNCSCs-sEVs groups after 7 days of in vitro culture, and (D) a statistical graph of the relative expression levels of ZO1 and Claudin1 proteins. *** (P < 0.001)

[0056] Figure 9 hfNCSCs-sEVs promote the expression of tight junction proteins on perineural cells during sciatic nerve defect repair.

[0057] (A) A rat model of 5mm sciatic nerve defect and in situ injection treatment after silicone tube bridging of the severed ends. (B) Immunofluorescence staining of the proximal end of the regenerated tissue in the silicone tube of rats in the PBS group and hfNCSCs-sEVs group 7 days after surgery, marking the expression of Claudin1 (red) in nerve perineurium cells, and DAPI (blue) staining indicating cell nuclei.

[0058] Figure 10 hfNCSCs-sEVs promote early Schwann cell migration and nerve fiber regeneration in the repair of sciatic nerve defects.

[0059] Immunofluorescence staining images of regenerated tissue in silicone tubes of rats in the PBS group and hfNCSCs-sEVs group at 7, 10 and 14 days after surgery, showing the expression of S100 (green) in Schwann cells and NF200 (red) in nerve fibers, and DAPI (blue) staining indicating cell nuclei.

[0060] Figure 11 hfNCSCs-sEVs promote nerve fiber regeneration and myelin sheath formation in the later stages of sciatic nerve defect repair.

[0061] (A) Immunofluorescence and HE staining images of the central cross-section of regenerated tissue in rats of the PBS group, hfNCSCs-sEVs group, autograft group, and sham operation group at 10 weeks post-operation; expression of MBP (green) labeling myelin and NF200 (red) labeling nerve fibers; and (B) statistical diagrams of the number of myelinated nerve fibers and (C) statistical diagrams of the diameter of myelinated nerve fibers. ** P < 0.01, *** P < 0.001, ns (P > 0.05)

[0062] Figure 12 hfNCSCs-sEVs promote myelin formation during the later stages of sciatic nerve defect repair.

[0063] (A) Transmission electron micrographs of the central cross-section of regenerated tissue in rats of the PBS group, hfNCSCs-sEVs group, Autograft group, and Sham group at 10 weeks post-surgery; and (B) statistical diagram of the number of myelin lamellar layers; (C) statistical diagram of G-ratio values. * P < 0.05, ** P < 0.01, *** P < 0.001, ns (P > 0.05)

[0064] Figure 13 hfNCSCs-sEVs improve the motor function of target muscles in the later stages of sciatic nerve defect repair.

[0065] (A) Gait footprints of rats in the PBS group, hfNCSCs-sEVs group, Autograft group, and Sham group at 10 weeks post-surgery; (B) Statistical graph of sciatic nerve function index. * P < 0.05, ** P < 0.01, ns (P > 0.05)

[0066] Figure 14 hfNCSCs-sEVs promote the recovery of regenerative nerve electrophysiological function in the later stages of sciatic nerve defect repair.

[0067] (A) Composite muscle action potential (CMAP) graphs of the gastrocnemius muscle on the operated side in rats of the PBS group, hfNCSCs-sEVs group, Autograft group, and Sham group 10 weeks post-surgery; (B) Statistical graphs of CMAP amplitude peak value and CMAP latency; (C) Numerical statistical graphs of motor and sensory nerve conduction velocities of the regenerated sciatic nerve on the operated side 10 weeks post-surgery. * P < 0.05,** P < 0.01, *** P < 0.001, ns (P > 0.05)

[0068] Figure 15 hfNCSCs-sEVs slow down target muscle atrophy in the later stages of sciatic nerve defect repair.

[0069] (A) Morphology of the gastrocnemius muscle in rats in the PBS group, hfNCSCs-sEVs group, Autograft group, and Sham group 10 weeks post-surgery; (B) Statistical graph of the wet weight ratio of the operated / unoperated gastrocnemius muscle; (C) Masson staining image of the cross-section of the operated gastrocnemius muscle; and (D) Statistical graph of the cross-sectional area of ​​muscle fibers. ** P < 0.01, **** P < 0.0001, ns (P > 0.05)

[0070] Figure 16 Differentially expressed genes, pathways, and functional enrichment in regenerated tissue 7 days after hfNCSCs-sEVs repair sciatic nerve defect.

[0071] (A) Volcano plot of differentially expressed genes in nerve regeneration tissues of rats in the PBS group and hfNCSCs-sEVs group 7 days after surgery, (B) heatmap of differentially expressed gene clusters, and (C) chord plot of differentially expressed gene enrichment analysis and (D) bubble plot of KEGG enrichment analysis.

[0072] Figure 17 hfNCSCs-sEVs increase the content of miR-21-5p in perineurium cells in vitro.

[0073] (A) Pie chart of the top 10 most abundant microRNAs in hfNCSCs-sEVs. (B) Schematic diagram of the binding site of the miR-21-5p target gene Smad7 predicted by TargetScan. (C) qRT-PCR detection of miR-21-5p content in perimysial cells cultured in vitro for 3 days in PBS and hfNCSCs-sEVs groups. *** (P < 0.001)

[0074] Figure 18 miR-21-5p can target and inhibit the translation of Smad7.

[0075] (A) Schematic diagram of the construction of WT and Mut SMAD7 3'-UTR luciferase reporter genes. (B) Statistical graph of relative fluorescence intensity after co-transfection of the reporter gene constructs of the Control group, WTSMAD7 3' UTR group, or Mut SMAD7 3' UTR group with miR-21-5p overexpression plasmid or negative control vector. *** P < 0.001, ns (P > 0.05)

[0076] Figure 19 miR-21-5p activates the TGF-β / Smad pathway by targeting and inhibiting Smad7 and upregulating HAS2 expression.

[0077] (A) Western blotting analysis of the expression of Smad7, Smad2 / 3, and HAS2, and the phosphorylation modification of p-Smad2 / 3 in perimysial cells cultured for 5 days in the PBS group, TGF-β group, and TGF-β+miR-21-5p mimics group; and (B) statistical graphs of the relative expression levels of Smad7 and HAS2 and the protein content ratios of p-Smad2 / Smad2 and p-Smad3 / Smad3. ** P < 0.01, *** (P < 0.001)

[0078] Figure 20 hfNCSCs-sEVs promote the proliferation and migration of HAS2 in percutaneous nerve cells in vitro by upregulating its expression.

[0079] (A) Western blotting analysis of the expression of HAS2, PCNA, and Vimentin in perineurial cells cultured in vitro for 5 days in the PBS group, si-Has2 group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+si-Has2 group, and (B) statistical graph of relative protein expression levels. (C) Cell scratch assay analysis of the migration ability of perineurial cells in the PBS group, si-Has2 group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+si-Has2 group, and (D) statistical graph of cell migration rate. (E) CCK-8 assay analysis of the absorbance values ​​of perineurial cells cultured in vitro for 5 days in the PBS group, si-Has2 group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+si-Has2 group. * P < 0.05, ** P < 0.01, *** (P < 0.001)

[0080] Figure 21miR-21-5p in hfNCSCs-sEVs promotes the expression of HAS2, PCNA, and Vimentin in perineurial cells in vitro.

[0081] (A) Western blot analysis of HAS2, PCNA, and Vimentin protein expression in perineurium cells cultured for 5 days in the PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p inhibitor group, and (B) statistical graph of relative protein expression levels. (C) Immunofluorescence staining images of perineurium cells cultured for 5 days in the PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p inhibitor group, showing the expression of HAS2 (red) and EdU (green) in perineurium cells, and (D) statistical graph of cumulative fluorescence density of HAS2 and (E) statistical graph of cell proliferation rate. ** P < 0.01, *** P < 0.001

[0082] Figure 22 miR-21-5p in hfNCSCs-sEVs promotes the expression of tight junction protein in pericergic cells in vitro.

[0083] (A) Immunofluorescence staining images of perineurium cells cultured in vitro for 7 days in the PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p inhibitor group; expression of ZO1 (green) and β-tubulin (red) in perineurium cells; and (B) statistical graph of cumulative fluorescence density of ZO1. (C) Western blot analysis of ZO1 and Claudin1 protein expression in perineurium cells cultured in vitro for 7 days in the PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p inhibitor group; and (D) statistical graph of relative protein expression levels. ** P < 0.01, *** (P < 0.001)

[0084] Figure 23 hfNCSCs-sEVs promote the expression of cell proliferation and migration-related proteins in vivo.

[0085] (A) Western blot analysis of HAS2, PCNA, and Vimentin expression in regenerated tissues of rats in the PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p inhibitor group 5 days after surgery, and (B) statistical graph of relative protein expression levels.** P < 0.01, *** (P < 0.001)

[0086] Figure 24 hfNCSCs-sEVs contain miR-21-5p, which promotes the expression of tight junction protein between nerve perithelial cells in vivo.

[0087] (A) Western blot analysis of ZO1 and Claudin1 expression in regenerated tissue of rats in the PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p inhibitor group 7 days after surgery, and (B) statistical graph of relative protein expression levels. (C) Immunofluorescence staining of the proximal end of regenerated tissue in silicone tubes of rats in the PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p inhibitor group 7 days after surgery, marking the expression of Claudin1 (red) in nerve perineurium cells, and DAPI (blue) staining to show cell nuclei. *** P < 0.001). Detailed Implementation

[0088] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0089] Example 1: Detection of uptake of hfNCSCs-sEVs by perineurium cells

[0090] 1.1 Isolation and culture of hfNCSCs

[0091] (1) Take about 80 g of male SD rats, anesthetize them with isoflurane, then kill them by cervical dislocation and immerse them in 75% alcohol for 5 min for disinfection.

[0092] (2) Take out the rat, dissect and separate the whisker pads and place them in pre-cooled PBS containing 1% bispecific antibody. Wash for 5 min × 3 times, separate the hair follicles in the whisker pads one by one, wash again with pre-cooled PBS for 5 min × 3 times, separate the hair follicle bulges and wash with PBS for 5 min × 3 times.

[0093] (3) The hair follicle protuberance was evenly applied to a 12-well plate and incubated in a saturated humidity, 5% CO2, 37 ℃ incubator for 20 min. Then, 200 μL of primary hfNCSCs culture medium [DMEM / F-12 (Corning, USA), containing 5% fetal bovine serum (Gibco, USA), 1% N2 (Invitrogen, USA), 2% B27 (Invitrogen, USA), 200 mmol / L L-glutamine (Corning, USA), 0.25% insulin-transferrin-selenium solution (ITS) (Corning, USA), 20 ng / mL epidermal growth factor (Shanghai Pufei Biotechnology Co., Ltd.), 20 ng / mL basic fibroblast growth factor (Shanghai Pufei Biotechnology Co., Ltd.), 1% penicillin / streptomycin (Corning, USA)] was added to each well. After 3 days, 200 μL of the medium was added again. μL, and then change the medium in half every 3 days. When the primary hfNCSCs cells reach 80%~90% confluence, they are passaged and cultured in passaged hfNCSCs medium (except for the fetal bovine serum concentration of 2%, the other components are the same as the primary hfNCSCs medium).

[0094] 1.2 Cell Immunofluorescence Staining Experiment

[0095] (1) Cell preparation: The cultured hfNCSCs and nerve perimysium cells were seeded in cell culture plates with coverslips placed in advance, and the cells were allowed to adhere and grow to the appropriate density.

[0096] (2) Fixation and permeabilization: Discard the culture medium, gently wash the cells with PBS for 5 min × 3 times, then add 4% paraformaldehyde fixative and fix at room temperature for 15 min, followed by washing with PBS for 5 min × 3 times. For cases requiring intracellular protein detection, permeabilization is performed using a permeabilization solution and incubated at room temperature for 10 min.

[0097] (3) Blocking: After discarding the membrane breaking solution, rinse with PBS for 5 min × 3 times, and incubate with 3% BSA blocking solution at room temperature for 30 min.

[0098] (4) Primary antibody incubation: After discarding the blocking solution, rinse with PBS for 5 min × 3 times, then add the corresponding primary antibody and incubate overnight at 4 °C.

[0099] (5) Secondary antibody incubation: After discarding the primary antibody, rinse with PBS for 5 min × 3 times, add fluorescently labeled secondary antibody with the same reactivity as the primary antibody, and incubate at room temperature in the dark for 2 h.

[0100] (6) Nuclear staining and mounting: Discard the secondary antibody, rinse with PBS for 5 min × 3 times, and mount with anti-fluorescence quenching mounting medium containing DAPI.

[0101] (7) Microscopic examination and photography: Cells were observed and images were acquired using a fluorescence microscope with an appropriate excitation wavelength. The integrated optical density (IOD) was detected and statistically analyzed using ImageJ software.

[0102] result:

[0103] Primary culture of hfNCSCs was performed in vitro using the tissue patch method. On day 4, hfNCSCs were observed to emerge from the hair follicle bulge, with cells mostly exhibiting a triangular shape. By day 7, cell confluence reached 80%–90%. Figure 1 (A1, A2), at this time, hfNCSCs were passaged. To avoid differentiation of hfNCSCs, cell culture medium containing a low concentration of FBS (2%) was used for passage to the 4th generation. When the cell confluence was about 70%~80%, ( Figure 1 B), replace the original culture medium with a medium containing 2% exosome-free FBS and continue culturing for 48 hours. When the cell confluence reaches 85%~95%, collect the cell culture medium containing hfNCSCs-sEVs. Figure 1 C), centrifuge to remove the precipitate and retain the supernatant, then store at -80 ℃. Immunofluorescence staining results showed that P75 (a neural crest cell marker) and Nestin (a stem cell marker) were highly expressed in the hfNCSCs cultured at this time. Figure 1 (D) The double positive rate is approximately 91.6%.

[0104] 1.3 Collection and Extraction of hfNCSCs-sEVs

[0105] (1) Pass hfNCSCs to the 4th generation. When the cell confluence reaches 70%~80%, the culture medium is replaced with passaged hfNCSCs medium containing exosome-free FBS. The cells are cultured for another 48 h. At this time, the cell confluence reaches 85%~95%. The cell culture supernatant containing hfNCSCs-sEVs is collected and centrifuged at 3000g for 10 min at 4 ℃. The supernatant is collected to remove cell debris.

[0106] (2) Mix the Exosome Concentration Solution reagent and the supernatant in the cell supernatant exosome extraction kit at a volume ratio of 1:4, vortex and mix for 1 min, and then let stand at 4 ℃ for 20 h.

[0107] (3) Centrifuge the mixture at 10,000g for 60 min at 4 ℃, discard the supernatant, add 1 / 100 volume of PBS to dissolve the precipitated hfNCSCs-sEVs, centrifuge the resuspended solution at 12,000g for 2 min at 4 ℃, transfer the supernatant to the upper chamber of the Exosome Purification Filter column, centrifuge at 3,000g for 10 min at 4 ℃, collect the liquid at the bottom of the column as the hfNCSCs-sEVs suspension, aliquot and store at -80 ℃ for later use.

[0108] 1.4 Western blot assay

[0109] (1) Sample preparation: Rats sciatic nerve regeneration tissue (tissue grinding and then lysis) or cultured nerve perimysium cells were lysed on ice for 20 min using protein lysis buffer (containing protease inhibitor and phosphatase inhibitor). After that, the samples were centrifuged at 8,000g at 4 ℃ for 15 min. The supernatant was collected and the protein concentration was detected by BCA method. The protein concentration was adjusted to 2 μg / μL. Then, 1 / 4 volume of loading buffer (5× loading buffer) was added, mixed well, and boiled in water at 95 ℃ for 10 min.

[0110] (2) Prepare SDS-PAGE adhesive: After rinsing and drying the glass plate, place it into the mold as required, prepare SDS-PAGE adhesive and pour it into the glass plate, and then insert the comb teeth.

[0111] (3) Sample loading and electrophoresis: After the gel solidifies, place it in the electrophoresis tank, pour in Tris / glycine / SDS electrophoresis buffer, remove the comb, clean the sample wells with electrophoresis buffer, load the prepared samples, take the same total protein amount for each sample, slowly add it into the sample wells, start electrophoresis, change the voltage to 120V after half an hour of electrophoresis at 80V.

[0112] (4) Immunoblotting: After electrophoresis, pour an appropriate amount of transfer buffer into the tray, remove the electrophoresis gel, and place the sponge pad, gel, methanol-activated PVDF membrane and sponge pad in the transfer apparatus from negative to positive electrode in sequence. Then transfer to the transfer tank and add an appropriate amount of transfer buffer.

[0113] (5) Blocking: After the PVDF membrane is transferred, it is washed with TBST and then blocked with blocking solution at room temperature for 30 min.

[0114] (6) Primary antibody incubation: After discarding the blocking solution, wash the PVDF membrane with TBST for 8 min × 4 times, and then incubate it overnight at 4 ℃ in primary antibody diluted with universal antibody diluent.

[0115] (7) Secondary antibody incubation: After discarding the primary antibody, wash the PVDF membrane with TBST for 8 min × 4 times, and then incubate it with the secondary antibody of the corresponding species diluted with universal antibody diluent at room temperature for 2 h.

[0116] (8) Development: After discarding the secondary antibody, the PVDF membrane was washed with TBST for 8 min × 4 times, then developed with chemiluminescent developing solution, and exposed and imaged using a chemiluminescent imaging system. The gray values ​​of the bands were detected and statistically analyzed using ImageJ software.

[0117] 1.5 Transmission electron microscopy observations of hfNCSCs-sEVs

[0118] (1) Centrifuge the extracted hfNCSCs-sEVs at 10,000g and 4℃ for 5 min.

[0119] (2) Take 10 μL of hfNCSCs-sEVs supernatant and drop it onto a copper grid to precipitate for 2 min. Then use filter paper to remove the floating liquid.

[0121] (3) Take 10 μL of 3% uranium acetate and drop it onto the copper grid for staining for 2 min, then use filter paper to absorb the floating liquid.

[0122] (4) After drying at room temperature, imaging was performed using a transmission electron microscope at 100 kV.

[0123] 1.6 Nanoparticle Tracking Analysis of hfNCSCs-sEVs

[0124] (1) Rinse the sample pool three times with ultrapure water.

[0125] (2) Instrument calibration was performed using polystyrene microspheres (110 nm).

[0126] (3) Clean the sample pool with PBS.

[0127] (4) Dilute hfNCSCs-sEVs with PBS and load them onto a nanoparticle tracking analyzer for detection.

[0128] result:

[0129] The culture supernatant of fourth-generation hfNCSCs was collected, and hfNCSCs-sEVs were isolated and purified by ultracentrifugation and using a cell supernatant exosome extraction kit (Umobio, UR52121). Western blot analysis showed that the sEVs-specific markers CD9, CD63, and TSG101 were all positive in hfNCSCs-sEVs. Figure 2A) The cell membrane marker Calnexin was negative. Nanoparticle tracking analysis showed that the obtained hfNCSCs-sEVs particle size distribution was unimodal, with a peak particle size of 117 nm and a particle concentration of approximately 1.54 × 10⁻⁶. 10 particles / mL ( Figure 2 B), transmission electron microscopy images show that hfNCSCs-sEVs are circular or elliptical cup-shaped vesicle structures (B). Figure 2 C).

[0130] 1.7 Isolation, culture, and purification of perimeningeal cells

[0131] (1) Take about 80g of male SD rats, anesthetize them with isoflurane, then kill them by cervical dislocation and immerse them in 75% ethanol for 5 min for disinfection.

[0132] (2) Take out the rat, cut open the skin on the back of the right thigh, bluntly separate the muscle and take out the sciatic nerve and place it in a culture dish on an ice box. Wash it with PBS containing 1% double antibiotics at 4 ℃ for 5 min × 3 times. Then, dissect and separate the sheet-like nerve bundle membrane under a stereomicroscope and wash it with PBS for 5 min × 3 times.

[0133] (3) The perimembranes were attached to 12-well plates and incubated in a saturated humidity, 5% CO2, 37 ℃ incubator for 20 min. Then, 200 μL of perimembranes cell culture medium [DMEM / F12, containing 10% (v / v) fetal bovine serum, 2 μmol / L Forskolin, 12.5 ng / mL Heregulin-β1 and 1% (v / v) Penicillin Streptomycin] was added. After 3 days, another 200 μL was added. The medium was changed in half every 3 days. The cells were passaged at about 10 days. The cells were passaged to the 4th generation using the "time-limited digestion-differential adhesion-chemical drug" method for subsequent in vitro experiments (see the invention patent "A method for in vitro culture and purification of perimembranes cells", patent number: ZL202011522398.7).

[0134] result:

[0135] To obtain high-purity perimeningal cells, the "time-limited digestion-differential adhesion-chemical drug" method previously invented by our research group was used for primary culture of perimeningal cells. After purification and passage to the fourth generation, the perimeningal cells exhibited a large and flattened morphology. Figure 3A). Immunofluorescence staining results showed high expression of the specific markers Claudin1 and Zonula Occludens 1 (ZO1) and glucose transporter 1 (GLUT1) on perineurium cells, but no expression of the Schwann cell-specific marker S100. Figure 3 B). To investigate whether hfNCSCs-sEVs can be taken up by perineurial cells in vitro, hfNCSCs-sEVs labeled with red PKH26 dye were added to the culture medium of perineurial cells. After co-culturing for 2 days, immunofluorescence staining revealed that ZO1-positive (green) perineurial cells contained PKH26-labeled hfNCSCs-sEVs. Figure 4 This indicates that nerve perimysial cells can take up hfNCSCs-sEVs in vitro.

[0136] Example 2: Detection of the effects of hfNCSCs-sEVs on the proliferation and migration of perineurium cells in vitro

[0137] 2.1 CCK-8 cell proliferation experiment

[0138] (1) Seeding and drug administration: 2,000 in vitro cultured perineurium cells per well and 100 μL of cell suspension were seeded into 96-well plates. After the cells adhered, the corresponding concentration (the concentration should be specified) of hfNCSCs-sEVs or si-Has2 transfection was added to the corresponding group. There were 6 replicates per group and the cells were cultured.

[0139] (2) Add CCK-8 working solution: After 3, 5 and 7 days of culture, add CCK-8 working solution to each well in the dark at a ratio of 10 μL of CCK-8 working solution per 100 μL of culture medium. After gently shaking to mix, put it back into the incubator and continue incubation for 1 h.

[0140] (3) Detection: The microplate reader was set to a detection wavelength of 450 nm, and the OD values ​​of each well were measured and recorded. Cell viability was calculated according to the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0141] 2.2 Transwell cell migration assay

[0142] (1) Placement of the chamber: Place the Transwell (8μm) chamber into the corresponding well of the 24-well plate in the clean bench.

[0143] (2) Cell seeding: Perimyeloid cells starved for 24 h were resuspended in FBS-free cell culture medium and seeded at a rate of 1.5 × 10⁶ cells per well. 4One cell and 200 μL of suspension were seeded into the upper chamber of the cell culture chamber. 800 μL of 10% exosome-free FBS-free culture medium for each group was added to the lower chamber, and three replicates were set up for each group for cell culture.

[0144] (3) Fixation: The chambers were removed at 6, 18 and 24 h of culture, and the unmigrated cells on the upper chamber membrane surface were wiped off with a cotton swab. The chambers were then fixed in 4% paraformaldehyde at room temperature for 15 min.

[0145] (4) Staining: Discard the fixative, rinse with PBS for 5 min × 3 times, then place in crystal violet staining solution and stain at room temperature for 5 min, then rinse with PBS for 5 min × 3 times and air dry.

[0146] (5) Observation and image acquisition: Place the chamber under an inverted microscope and randomly select 5 fields of view to take pictures and record the number of migrating cells.

[0147] 2.3 The Western blot (WB) experimental procedure is the same as in 1.4.

[0148] result:

[0149] To investigate whether hfNCSCs-sEVs can promote the proliferation and migration of perimysial cells in vitro, the proliferation and migration abilities of perimysial cells were first detected by CCK-8 and Transwell assays, respectively. The CCK-8 assay results showed that, compared with the control group (0 particles / mL), different concentrations (2×10⁻⁶ particles / mL) significantly increased the proliferation and migration of perimysial cells. 8 5 × 10 8 10 × 10 8 The viability of perimysial cells cultured in the hfNCSCs-sEVs group (particles / mL) was significantly increased on days 3, 5, and 7, with the most significant increase on day 5 (5×10⁶). 8 particles / mL and 10×10 8 The particle / mL group was significantly higher than 2×10. 8 particles / mL, and there was no significant difference between the two high-concentration groups (particles / mL). Figure 5 A). Transwell assay results showed that the number of migrating perineurial cells gradually increased within 18 hours in each group, and different concentrations of hfNCSCs-sEVs significantly increased the number of migrating perineurial cells, with 5×10⁻⁶ cells showing the highest increase. 8 particles / mL and 10×10 8 particles / mL group compared to 2×10 8 The particles / mL group increased significantly, while there was no significant difference between the two higher concentration groups. Figure 5B). Therefore, the experimental concentration of hfNCSCs-sEVs used in subsequent experiments was 5 × 10⁻⁶. 8 particles / mL. Western blotting was used to detect protein expression levels in perimysial cells cultured for 5 days. The results showed that, compared to the PBS group, proliferating cell nuclear antigen (PCNA), which is positively correlated with cell proliferation, and vimentin, which is positively correlated with cell migration, were both significantly upregulated in the hfNCSCs-sEVs group of perimysial cells. Figure 5 (C, D) indicates that hfNCSCs-sEVs can promote the proliferation and migration of nerve perithelial cells in vitro.

[0150] Example 3: Detection of the effects of hfNCSCs-sEVs on the tube-forming ability, barrier function, and tight junction protein expression of nerve perimysial cells in vitro.

[0151] 3.1 Cell Tube Formation Experiment

[0152] (1) Matrigel preparation and plating: The pre-cooled Matrigel was slowly thawed on ice, diluted with serum-free culture medium at a ratio of 1:1, and then quickly and evenly plated at 50 μL per well onto the bottom of a 96-well plate. The plate with the Matrigel plated was placed in a cell culture incubator and incubated for 40 min to allow the Matrigel to solidify.

[0153] (2) Inoculation and grouping: Pericyneal cells in the logarithmic growth phase were collected, and the cells were divided into groups of 0 and 1 × 10⁻⁶ cells respectively. 8 2×10 8 5×10 8 10×10 8 For the hfNCSC-sEVs group with particles / mL, prepare the corresponding culture medium and apply 3 × 10⁶ particles / mL per well. 4 Cells were seeded into wells pre-coated with matrix gel, with three replicates per group. After gentle mixing, the cells were placed in a cell culture incubator for incubation.

[0154] (3) Observation and image acquisition: The cell tube formation was observed and photographed under an inverted microscope at 0, 6, 12, 18 and 24 h respectively.

[0155] (4) Data analysis: The number of branching points and capillary length of tubules at 18 h were analyzed using ImageJ software and the reference (Carpentier G, Berndt S, Ferratge S, et al. Angiogenesis analyzer for ImageJ- a comparative morphometric analysis of “endothelial tube formation assay” and “fibrin bead assay”[J].Scientific Reports, 2020, 10(1): 11568).

[0156] result:

[0157] The perineurium, surrounding the nerve, possesses the tubular characteristics of endothelial cells and the function of forming a tight junction barrier. Therefore, a series of tests were conducted on its related functions. First, a tubular formation experiment was performed on perineurium cells to assess their tubular capacity. Results showed that the lumen had formed and gradually expanded 6 h after cell seeding. At 18 h post-seeding, compared to the PBS group, the hfNCSCs-sEVs group had more branching points among perineurium cells and a longer total lumen length. By 24 h post-seeding, the outer diameter of the perineurium network in both groups had decreased, and the entire network began to atrophy. Figure 6 AC). This indicates that hfNCSCs-sEVs can enhance the tube-forming ability of nerve perimysial cells.

[0158] 3.2 Transmembrane resistance measurement experiment

[0159] (1) Placement of the chamber: Place the Transwell (0.4μm) chamber into the corresponding well of the 24-well plate in the clean bench.

[0161] (2) Preparation of cell suspension: Take nerve perimysial cells in the logarithmic growth phase, wash with serum-free culture medium, and then...

[0162] Digest with trypsin, stop digestion with serum-containing culture medium, pipette to prepare a single-cell suspension, count the cells, and adjust to the appropriate concentration with serum-free culture medium.

[0163] (3) Inoculation and grouping: Inoculate 2.5 × 10⁶ cells per well. 4 One cell and 200 μL of suspension were seeded into the upper chamber of the cell culture chamber. PBS and hfNCSCs-sEVs groups were also included, with 800 μL of the corresponding culture medium added to the lower chamber. Each group was cultured in triplicate.

[0164] (4) Resistance measurement: The transmembrane resistance of the upper chamber was measured at 1, 2, 3, 4, 5, 6 and 7 days of culture.

[0165] Resistance. Calculate the resistance per unit area using the following formula: TEER (resistance per unit area, Ω·cm) 2 = (Rexperiment - Rblank) (resistance measurement, Ω) × effective film area (cm²) 2 In this experiment, the effective membrane area was 1.12 cm². 2 .

[0166] 3.3 Cell monolayer permeability detection experiment

[0167] (1) Inoculation and grouping: The inoculation procedure for nerve perimysial cells is the same as that for the transmembrane resistance value detection experiment.

[0168] (2) Incubation: On day 7 of culture, 1 mg / mL of FITC-labeled dextran was added to the top of each chamber and incubated for 30 min.

[0169] (3) Permeability test: 100 μL of the lower chamber solution was collected from each well, and the fluorescence value was detected using a multi-functional microplate reader. The fluorescence was measured using the Gen5 program at an excitation wavelength of 492 nm and an emission wavelength of 520 nm.

[0170] result:

[0171] The role of hfNCSCs-sEVs in the barrier function of perimysial cells after membrane formation was investigated by measuring transmembrane resistance. Perimembranous cells were seeded and cultured in the upper chamber of a Transwell with a pore size of 0.4 μm. Transmembrane resistance was measured every 24 h; higher resistance values ​​indicated lower permeability. The results showed that the transmembrane resistance of perimysial cells in the hfNCSCs-sEVs group was significantly higher than that in the PBS group after 3 days of seeding, and this difference persisted until day 7. Figure 7 A) indicates that hfNCSCs-sEVs can enhance the barrier function between nerve perimysium cells in vitro, and its upper limit of barrier function is higher than that of the PBS group. To further verify this, on day 7 after inoculation, FITC-dextran was added to the upper chamber medium of Transwell and co-incubated for 30 min before a cell monolayer permeability experiment was performed. The results showed that the fluorescence intensity in the lower chamber of the hfNCSCs-sEVs group was significantly lower than that in the PBS group (A). Figure 7 (B) indicates that the permeability of the nerve perineurium cells in the hfNCSCs-sEVs group was lower after membrane formation, and their ability to form a barrier was stronger. These results demonstrate that hfNCSCs-sEVs can improve the barrier function of the nerve perineurium in vitro.

[0172] 3.4 The Western blot (WB) experimental procedure is the same as in 1.4.

[0173] result:

[0174] The barrier function of the perineurium is closely related to the formation of tight junctions between cells. To further investigate the role of hfNCSCs-sEVs in the formation of tight junctions between perineurium cells, relevant experiments were performed on perineurium cells cultured in vitro for 7 days. Immunofluorescence staining results showed that the cumulative optical density of ZO1 between perineurium cells in the hfNCSCs-sEVs group was significantly higher than that in the PBS group. Figure 8 The results of Western blot analysis (WB) also showed that the expression levels of tight junction proteins (ZO1 and Claudin1) in the hfNCSCs-sEVs group were significantly higher than those in the PBS group. Figure 8 (C, D) The above results demonstrate that hfNCSCs-sEVs can promote the formation of tight junction proteins between nerve perimysial cells in vitro, which helps to enhance the tube-forming ability and barrier function of nerve perimysial cells.

[0175] Example 4: Detection of the effect of hfNCSCs-sEVs on the expression of tight junction proteins in nerve perimysial cells in vivo

[0176] 4.1 Construction of a rat sciatic nerve injury model

[0177] (1) Take male SD rats weighing about 220g~240g and anesthetize them with isoflurane. First, induce anesthesia with a concentration of 4%, and then maintain anesthesia with a concentration of 2.5% after moving them to the operating table. At the same time, monitor vital signs such as respiration and heart rate.

[0178] (2) The skin of the rat's right hind limb was prepared and disinfected. A 2 cm incision was made along the course of the sciatic nerve, and the muscles were bluntly dissected to expose the main trunk of the sciatic nerve.

[0179] (3) Cut the sciatic nerve about 1 cm away from the ischial tuberosity to create a 5 mm defect and ensure that the cut ends are neat.

[0180] (4) The nerve stump was sutured into a silicone tube (7 mm long, 1.5 mm inner diameter, 2 mm outer diameter) with 9-0 sutures for 1 mm. The PBS group, hfNCSCs-sEVs group, and hfNCSCs-sEVs+miR-21-5p group were filled with the corresponding substances. The Autograft group had a 5 mm sciatic nerve cut off at the same position and then flipped and sutured. The sciatic nerve in the Sham group was not treated.

[0181] (5) Suture the muscles and skin layer by layer, and administer antibiotic treatment for 5 days after the operation.

[0182] 4.2 Material Selection and Paraffin Slicing

[0183] (1) Fixation: Rats were anesthetized and perfused with physiological saline and 4% paraformaldehyde in sequence. Regenerated tissue and bilateral gastrocnemius muscles were obtained in silicone tubes. After weighing, photographing and trimming the tissues, they were fixed in 4% paraformaldehyde at 4°C for 48 h.

[0184] (2) Dehydration: The fixed tissue was removed and dehydrated for 1.5 h each with 70%, 80%, 90% and 100% graded ethanol.

[0185] (3) Transparency: Xylene was used to transparentize the tissue (5 min × 2 times for regenerated tissue; 30 min × 2 times for gastrocnemius muscle).

[0186] (4) Wax immersion: The tissue was immersed in paraffin in layers I, II and III at 60 °C for 2 h each.

[0187] (5) Embedding and sectioning: After embedding the tissue in the paraffin block, cut 5 μm paraffin sections, attach them to poly-L-lysine-treated glass slides, and dry them at 37 ℃ for later use.

[0188] 4.3 Immunofluorescence staining of tissues

[0189] (1) Dewaxing: Pass the paraffin slices through two xylene channels in sequence, 10 min each time.

[0190] (2) Rehydration: The dewaxed sections were sequentially rehydrated by passing them through a gradient of 100%, 100%, 95%, 90%, 80%, and 70% ethanol for 5 minutes each, and finally rinsed with distilled water for 5 minutes.

[0191] (3) Antigen retrieval: Place the tissue slides in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0), heat in a microwave oven on medium heat for 8 min until almost boiling, turn off the heat for 8 min, turn to medium-low heat for 7 min, and let cool naturally. Then wash the slides in PBS for 5 min × 3 times.

[0192] (4) Draw circles: After shaking off the moisture from the sections, use a histological pen to draw circles around the tissue.

[0193] (5) Closure: Add 3% BSA to the circle and incubate at room temperature for 30 min.

[0194] (6) Add primary antibody: Discard the blocking solution, add the primary antibody diluted in a certain proportion to the tissue, and then place the slide flat in a humidified chamber and incubate overnight at 4 °C.

[0195] (7) Add secondary antibody: discard the primary antibody, wash the slide in PBS for 5 min × 3 times, dry the slide slightly, add the secondary antibody diluted in a certain proportion corresponding to the species of the primary antibody in the circle, and incubate at room temperature for 2 h.

[0196] (8) Nuclear staining and mounting: Discard the secondary antibody, wash with PBS for 5 min × 3 times, and mount with anti-fluorescence quenching mounting medium containing DAPI.

[0197] (9) Microscopic examination and photography: The slides were observed and images were acquired under a fluorescence microscope. The number and diameter of myelinated nerve fibers were detected and counted using ImageJ software.

[0198] result:

[0199] To investigate the role of hfNCSCs-sEVs in the formation of tight junctions between nerve perineurium cells in vivo, a rat model of 5 mm sciatic nerve defect was established. Figure 9 A) Fill silicone tubes with PBS or hfNCSCs-sEVs. Immunofluorescence staining was performed on regenerated tissue 7 days post-surgery. The results showed that Claudin1 expression was relatively continuous and regular among perineurium cells at the severed nerve ends. During the proliferation and migration of regenerated perineurium cells from the nerve ends, tight junctions were gradually formed between cells, and Claudin1 expression was discontinuous and scattered. In the hfNCSCs-sEVs group, Claudin1 extended a longer distance and had a higher expression level in the regenerated tissue. Figure 9 B) suggests that hfNCSCs-sEVs promote the expression of tight junction proteins on nerve perimysial cells in vivo.

[0200] Example 5: Detection of the effects of hfNCSCs-sEVs on nerve fiber regeneration and myelin formation in vivo

[0201] 5.1 The procedure for tissue immunofluorescence staining is the same as in 4.3.

[0202] result:

[0203] Besides perineurium cells, nerve fibers and Schwann cells that form myelin sheaths also play important roles in nerve regeneration and functional recovery. Immunofluorescence staining of regenerated tissue at 3, 5, and 7 days post-surgery showed that S100-positive (green) Schwann cells converged from both ends towards the center, while NF200-positive (red) nerve fibers extended from the proximal end to the distal end. At the same time points, the migration distance of Schwann cells and nerve fibers in the hfNCSCs-sEVs group was significantly greater than that in the PBS group. Figure 10 The results suggest that hfNCSCs-sEVs can enhance the migration rate of Schwann cells and the extension rate of nerve fibers. The results indicate that hfNCSCs-sEVs can promote Schwann cell migration and nerve fiber regeneration in the early stages of sciatic nerve defect repair.

[0204] 5.2 HE staining experiment

[0205] (1) Dewaxing: Pass the paraffin slices through two xylene channels in sequence, 10 min each time.

[0206] (2) Rehydration: The dewaxed sections were sequentially rehydrated by passing them through a gradient of 100%, 100%, 95%, 90%, 80%, and 70% ethanol for 5 minutes each, and finally rinsed with distilled water for 5 minutes.

[0207] (3) Hematoxylin staining: Immerse the rehydrated paraffin sections in hematoxylin staining solution for 5 minutes, and rinse with running water until the sections turn blue-purple.

[0208] (4) Differentiation: Immerse the slices in 1% hydrochloric acid ethanol for 10 seconds until the cell nuclei are clear, then rinse with running water for 5 minutes.

[0209] (5) Blueing: Immerse the slices in 1% ammonia water until the cell nuclei turn dark blue.

[0210] (6) Eosin staining: The sections were immersed in 85% and 95% ethanol for 2 min each, and then immersed in eosin solution for 20 s.

[0211] (7) Dehydration: Immerse the slices in three passes of anhydrous ethanol, each lasting 5 minutes.

[0212] (8) Transparency: Pass the paraffin sections through xylene twice, 5 min each time.

[0213] (9) Sealing: After removing the film and drying it, seal it with neutral resin.

[0214] 5.3 Transmission electron microscopy observation of neural tissue

[0215] (1) Sampling: After sampling the regenerated tissue inside the tube, quickly cut it into small pieces of 1~2mm³.

[0216] (2) Pre-fixation: Place in pre-cooled 2.5% glutaraldehyde fixative, keep at room temperature in the dark for 2 h, then transfer to 4 ℃ overnight.

[0217] (3) Post-fixation: Rinse with PBS for 15 min × 3 times, then immerse in 1% osmium tetroxide fixative at 4 ℃ in the dark for 1.5 h.

[0218] (4) Dehydration: After rinsing with PBS for 15 min × 3 times, the tissue was soaked in 30%, 50%, 70%, 90% and 100% ethanol for 20 min each (100% ethanol was soaked twice) to dehydrate.

[0219] (5) Infiltration: After infiltration with a 1:1 mixture of acetone and epoxy resin for 1 hour, it was then infiltrated with pure epoxy resin overnight.

[0220] (6) Embedding: After the tissue is removed, it is placed in an embedding mold and a new epoxy resin embedding agent is added. The mixture is polymerized at a constant temperature of 60 °C for 48 h to obtain the embedding block.

[0221] (7) Ultrathin sectioning: Cut into 50 nm thin sections using an ultrathin slicer and transfer to a copper mesh.

[0222] (8) Staining: The sample was stained with 2% uranium acetate staining solution and lead citrate staining solution, washed with double-distilled water for 5 min × 3 times, and dried for observation under transmission electron microscopy.

[0223] result:

[0224] To evaluate the effects of hfNCSCs-sEVs on nerve fiber and myelin formation in the later stages of sciatic nerve defect repair, immunofluorescence and HE staining were performed on the central cross-section of regenerated tissue in the PBS group, hfNCSCs-sEVs group, autograft group, and sham surgery group 10 weeks postoperatively. The results showed that some NF200-positive (red) nerve fibers were surrounded by MBP-positive (green) myelin sheaths. In the Sham group, most nerve fibers were regularly arranged and encased in myelin sheaths, with relatively uniform nerve fiber diameter and myelin sheath thickness. In the other three groups, the number of regenerated nerve fibers was greater, but the myelin sheath thickness was uneven. Figure 11 A). Statistical results showed that, except for the Sham group, the number of myelinated nerve fibers was the highest in the Autograft group, followed by the hfNCSCs-sEVs group, and the lowest in the PBS group. Figure 11 B); while the myelinated nerve fibers in the Sham group were the thickest, there was no significant difference between the Autograft group and the hfNCSCs-sEVs group, but both were significantly higher than the PBS group (B); Figure 11 C). The results showed that hfNCSCs-sEVs could effectively promote the regeneration of nerve fibers in the later stage of ischial defect repair in rats.

[0225] Transmission electron microscopy images and statistical results at the same locations showed that the Sham group had the highest number of myelin sheath lamellar layers in myelinated nerve fibers. There was no significant difference between the Autograft group and the hfNCSCs-sEVs group, but both were significantly higher than the PBS group. Figure 12 A, B), and the statistical results of G-ratio also showed that there was no significant difference in the promoting effect of hfNCSCs-sEVs group and Autograft group on myelin regeneration, but both were significantly better than PBS group ( Figure 12 C). These results indicate that hfNCSCs-sEVs can effectively promote myelin formation during the later stages of ischial defect repair in rats.

[0226] Example 6: Detection of the in vivo effects of hfNCSCs-sEVs on nerve function recovery and skeletal muscle atrophy relief

[0227] 6.1 Gait analysis and Sciatic Functional Index (SFI) testing

[0228] (1) Marking the ankles: The ankle joints of the hind limbs of the experimental rats were coated with ink so that they would leave a mark on white paper.

[0229] Clear footprints.

[0230] (2) Walking test: Place white paper at the bottom of the wooden trough, put the rat at one end of the trough, and let it walk freely.

[0231] As the rat moves to the other end, it leaves five to six clear pairs of footprints on the white paper.

[0232] (3) Measurement of footprint parameters: The footprints left by the rats were measured, and the normal footprint (N) and the injured footprint were recorded.

[0233] The three indicators of lateral foot (E) are footprint length (PL), toe width (TS), and middle toe width (IT).

[0234] (4) Substitute into the formula to calculate: Substitute the above data into the following formula and calculate the SFI value.

[0235] SFI=-38.3×(EPL-NPL) / NPL+109.5×(ETS-NTS) / NTS+13.3×(EIT-NIT) / NIT-8.8.

[0236] 6.2 Compound Muscle Action Potential (CMAP) Detection

[0237] (1) Animal preparation: 10 weeks after surgery, rats in each group were selected, anesthetized with isoflurane and fixed, hair was removed and the sciatic nerve and related muscle areas were exposed.

[0238] (2) Electrode placement: Insert the recording electrode into the mid-abdomen of the ipsilateral gastrocnemius muscle, and connect the grounding electrode to the tip of the tail. Use a pair of bipolar hook stimulation electrodes to contact the distal and proximal sites of the sciatic nerve injury.

[0239] Signal acquisition: A 10 mA electrical stimulus was applied to elicit a CMAP response. The mean amplitude and latency of the CMAP response were recorded for each rat.

[0240] Parameter measurement: Motor Nerve Conduction Velocity (MNCV) = the ratio of the distance between the proximal and distal stimulating electrodes to the latency difference, while Sensory Nerve Conduction Velocity (SNCV) = the distance between the stimulating electrode and the recording electrode to the corresponding latency.

[0241] result:

[0242] To assess the electrophysiological function of regenerated nerves after sciatic nerve defect, electrophysiological measurements were performed on the gastrocnemius muscle, the target muscle of the sciatic nerve on the operated side, in rats 10 weeks post-surgery, and compound muscle action potentials (CMAPs) were recorded. Figure 14 A) Analysis of CMAP results showed that the peak CMAP amplitude was highest in the Sham group rats, with no significant difference between the hfNCSCs-sEVs group and the Autograft group, and both were significantly higher than the PBS group; correspondingly, the CMAP latency was shortest in the Sham group, shorter in the Autograft group than in the hfNCSCs-sEVs group, and both groups were significantly shorter than the PBS group. Figure 14 B). Motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were calculated based on the conduction distance and duration of electrical signals in nerves and regenerated nerves. Statistical results showed that both nerve conduction velocities were highest in the Sham group. There was no significant difference between the hfNCSCs-sEVs group and the Autograft group, and both were higher than those in the PBS group. Figure 14 B). These results indicate that hfNCSCs-sEVs can improve the signal transduction function of regenerated nerves in the later stages of sciatic nerve defect repair.

[0243] 6.3 Weighing test of gastrocnemius muscle

[0244] (1) Weighing: Rinse the gastrocnemius muscle specimen with physiological saline to remove blood and impurities from the surface. Then, use filter paper to absorb the surface moisture and weigh it on an electronic balance. Record the wet weight value.

[0245] (2) Calculate the wet weight ratio: Wet weight ratio = wet weight of gastrocnemius muscle on the experimental side / wet weight of gastrocnemius muscle on the control side.

[0246] result:

[0247] Peripheral nerve injury often leads to atrophy of its target muscles, and target muscle reinnervation is one of the important indicators for evaluating nerve function recovery. To evaluate the effect of hfNCSCs-sEVs on target muscle reinnervation in the later stages of sciatic nerve defect repair, bilateral gastrocnemius muscles were harvested and weighed from rats in each group 10 weeks post-surgery. The results showed that, except for the Sham group, the gastrocnemius muscles on the operated side of the other three groups were significantly atrophied. There was no significant difference in the wet weight ratio of the gastrocnemius muscles between the hfNCSCs-sEVs group and the Autograft group, but both were significantly higher than those in the PBS group. Figure 15A). Masson staining results of cross-sections at the same location in the gastrocnemius muscle belly also showed that the cross-sectional area of ​​muscle fibers in the PBS group was significantly reduced, but the degree of muscle fiber atrophy was weaker in the hfNCSCs-sEVs group and the Autograft group, and there was no significant difference in the cross-sectional area of ​​muscle fibers between the two groups. Figure 15 B). The above results indicate that hfNCSCs-sEVs can slow down target muscle atrophy in the later stages of sciatic nerve defect repair in rats.

[0248] Example 7: Detection of the effect of hfNCSCs-sEVs on Has2 gene expression in vivo

[0249] 7.1 Eukaryotic transcriptome sequencing and bioinformatics analysis of regenerated tissues

[0250] (1) Tissue collection: Regenerated nerve tissue from the PBS group and hfNCSCs-sEVs group was collected 7 days after surgery, collected with sterile instruments, and then quickly frozen in liquid nitrogen.

[0251] (2) Sample processing: Place the tissue in 1 mL TRIzol and grind it with a cryo-grinder until no visible tissue fragments are visible.

[0252] (3) RNA extraction: Add 200 μL of chloroform to TRIzol lysis buffer and shake vigorously for 2 min. After standing for 8 min, centrifuge at 12,000g at 4℃ for 10 min. Transfer the uppermost aqueous phase to a new centrifuge tube and add an equal volume of isopropanol. Gently invert and mix 10 times, then let stand for 15 min. After centrifugation at 12,000g at 4℃ for 30 min, discard the liquid. Wash the precipitate twice with pre-cooled 75% ethanol by centrifugation and discard the supernatant. After the precipitate is completely dry and transparent, add 30 μL of RNAse-free water.

[0253] (4) Quality detection: RNA concentration was detected using a spectrophotometer, and the A260 / A280 and A260 / A230 ratios were recorded to assess RNA quality.

[0254] (5) Integrity test: RNA integrity is tested using a bioanalyzer. Once it passes the test (RIN≥7.0), it proceeds to the library construction process.

[0255] (6) Library construction: Transcriptome libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep kit.

[0256] (7) Library quality control: Detect library concentration, fragment size distribution, and concentration. The fragment size distribution must meet expectations and have no tailing.

[0257] (8) Sequencing: Sequencing was performed using the Illumina Novaseq 6000 platform.

[0258] (9) Data preprocessing: Filter the raw data, remove low quality, connectors and sequences to obtain clean reads for subsequent data analysis.

[0259] (10) Alignment Quantification: Alignment Quantification: HISAT2 was used to align clean reads with the rat Rnor_6.0 genome to determine the location and count gene expression levels (FPKM). The read counts for each gene were obtained by HTSeq-count.

[0260] (11) Differential analysis: Compare gene expression levels among samples to screen differentially expressed genes and correct for P-values.

[0261] (12) Functional analysis: GO and KEGG enrichment analysis was performed on differentially expressed genes on the Ouyi Cloud platform.

[0262] result:

[0263] To explore the molecular mechanism by which hfNCSCs-sEVs promote perineurium cell regeneration in the early stage of nerve defect repair, eukaryotic transcriptome sequencing was performed on nerve regeneration tissues from rats in the PBS group and the hfNCSCs-sEVs group 7 days after surgery. Volcano plots showed that compared with the PBS group, the hfNCSCs-sEVs group had 253 significantly upregulated and 398 significantly downregulated genes. Figure 16 A), select genes that are significantly related to endothelial cell proliferation and migration and perform clustering heatmaps ( Figure 16 B). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were performed on the screened differentially expressed genes. The results showed that in the hfNCSCs-sEVs group, functions related to the positive regulation of cell proliferation and migration were significantly enriched. Figure 16 C), the cellular biological processes of tight junction formation and the TGF-β signaling pathway were also significantly enriched. Figure 16 D). GO enrichment analysis showed that Has2 was the most significantly associated with both cell proliferation and migration functions. Figure 16 C). Therefore, we hypothesize that hfNCSCs-sEVs may promote the proliferation and migration of nerve perithelial cells by activating the TGF-β pathway and upregulating HAS2 expression.

[0264] Example 8: Detection of the effects of miR-21-5p on the TGF-β / Smad / HAS2 axis in perimysial cells and its influence on perimysial cell proliferation and migration in vitro.

[0265] 8.1 miRNA sequencing and bioinformatics analysis in hfNCSCs-sEVs

[0266] (1) miRNA extraction: miRNA was extracted using the mirVana miRNA Isolation Kit.

[0267] (2) Library construction: Small RNA libraries were constructed using the Truseq Small RNA sample prep Kit, purified using 6% NovexTBE PAGEge, and quantified using a TBS380 Picogreen before being loaded onto the instrument.

[0268] (3) Sequencing: The sequencing was completed with the assistance of Shanghai Meiji Biotechnology Co., Ltd. The constructed library was sequenced using the Ilumina NovaSeo 6000 sequencing platform with a read length of SE75.

[0269] (4) Ranking of the top 10 miRNAs: The expression levels of miRNAs in hfNCSCs-sEVs were statistically analyzed using the miRBase database (miRBase21, http: / / www.mirbase.org / ) and miRDeep2 software to obtain information on the known and unknown target genes of the top 10 miRNAs.

[0270] (5) Functional and pathway analysis of target genes: GO enrichment analysis and KEGG enrichment analysis of miRNA target genes in hfNCSCs-sEVs were performed using the DAVID database (http: / / david.abcc.ncifcrf.gov / ) to analyze the biological functions and related signaling pathways of possible target genes during the process of hfNCSCs-sEVs acting on nerve perimysial cells.

[0271] 8.2 miRNA dual-luciferase verification experiment

[0272] (1) Based on the predicted binding sites of the target genes, Smad7 WT and Smad7 MUT sequences were designed and synthesized.

[0273] (2) The Smad7 WT and Smad7 MUT sequences were ligated to the pMIR-REPORT Luciferase vector by restriction endonuclease and T4 ligase, respectively, and transformed into competent Escherichia coli for LB plate culture and single-clone identification. After confirming that there were no errors, subsequent experiments were carried out.

[0274] (3) Cell seeding: 293T cells were seeded in 96-well plates and transfected when the confluence reached 70%.

[0275] (4) Preparation of DNA and transfection reagents: The amount of reagents used per well is as follows:

[0276]

[0277] (5) Reagent incubation: Mix the diluted DNA and miRNA thoroughly, then mix with the transfection reagent and incubate at room temperature for 20 min.

[0278] (6) Transfection: Discard 50 μL of culture medium from each well and add 50 μL of DNA-miRNA transfection mixture to each well of cell sample. Replace with complete culture medium after 6 h. After co-transfection of plasmids for 48 h, discard the culture medium and wash with PBS.

[0279] (7) Lysis: Add 50 μL of 1×PLB (Passive Lysis Buffer) to each well and lyse on a shaker at room temperature for 15 min.

[0280] (8) Detection: Add 5 μL of supernatant to each well of a white, opaque 96-well microplate, then add 50 μL of pre-mixed LAR II. Detect data after 2 seconds. Add 50 μL of pre-mixed Stop&Glo Reagent to each well, let stand for 2 seconds, and then detect data.

[0281] result:

[0282] To explore miRNAs that may play a key role in the proliferation and migration of perimysial cells (hfNCSCs-sEVs), miRNA sequencing was performed on hfNCSCs-sEVs. The top ten known miRNAs by abundance were screened, and target genes and functions of the miRNAs were predicted using TargetScan and miRDB databases. It was found that miR-21-5p may play a key role in this process. Figure 17 A). Target gene prediction results show that miR-21-5p may target and inhibit the translation of Smad7 mRNA ( Figure 17B). Therefore, we hypothesize that miR-21-5p in hfNCSCs-sEVs may activate the TGF-β / smad pathway by targeting and inhibiting Smad7 mRNA translation, thereby upregulating HAS2 expression in perimysial cells and promoting proliferation and migration during EMT. qRT-PCR results also showed that the level of miR-21-5p in perimysial cells cultured in vitro for 3 days in the hfNCSCs-sEVs group was significantly higher than that in the PBS group ( Figure 17 C). This indicates that hfNCSCs-sEVs can increase the content of miR-21-5p in nerve perimysial cells in vitro.

[0283] To verify the effect of miR-21-5p on Smad7, a dual-luciferase reporter gene assay was designed based on the base sequence predicted by Targetscan. Figure 18 (A) This study verified the inhibitory effect of miR-21-5p on its potential target gene Smad7. The results showed that overexpression of miR-21-5p reduced the activity of the Smad7-3' UTR-WT reporter gene, but there was no significant change in activity within the Smad7-3' UTR-Mut reporter gene, indicating that miR-21-5p can target and inhibit the translation of Smad7. Figure 18 B).

[0284] 8.3 Processing and transfection of nerve perimysial cells

[0285] (1) Cell plating: Perineuronal cells were resuspended in complete culture medium and then plated with 1×10⁻⁶ cells. 5 Cells were seeded per well in 6-well plates and cultured at 37 °C until the cell density reached approximately 50%.

[0286] (2) Change the culture medium: Remove the original culture medium and add 1.5 mL of culture medium without PS antibiotics.

[0287] (3) Preparation of transfection mixture: Transfection was performed using the Lipofectamine RNAiMAX kit. The transfection steps for si-NC, si-Has2, and miR-21-5p mimics were the same. First, si-NC, si-Has2, and miR-21-5p mimics were diluted with Opti-MEM (centrifuge tube A) and gently mixed. Lipofectamine was diluted with Opti-MEM in centrifuge tube B. After gently mixing the two tubes, they were allowed to stand for 5 min.

[0288] (4) Transfection: Add the corresponding transfection mixture to each well of the cell culture plate and gently shake to mix.

[0289] (5) TGF-β intervention: TGF-β was administered to nerve perimysial cells at a final concentration of 5 ng / mL according to the group requirements.

[0290] 8.4 The procedure for Western blotting (WB) is the same as in 1.4.

[0291] result:

[0292] To further investigate the molecular mechanism by which miR-21-5p regulates HAS2 expression in perimysial cells, Western blot analysis was performed on cultured perimysial cells. The results showed that after 5 days of culture, exogenous TGF-β increased the phosphorylation levels of Smad2 and Smad3 in perimysial cells. Adding miR-21-5p mimics significantly downregulated Smad7 protein expression and further promoted the phosphorylation levels of Smad2 and Smad3, while HAS2 expression also significantly increased. Figure 19 (A, B). These results indicate that miR-21-5p can further upregulate HAS2 expression by targeting and inhibiting Smad7 to activate the TGF-β / Smad pathway.

[0293] 8.5 Cell Scratch Assay

[0294] (1) Cell seeding: The nerve perimysial cells starved for 24 h were resuspended in the corresponding culture medium of each group and seeded with 5×10⁻⁶ cells. 5 10 cells / well were seeded into 6-well plates.

[0295] (2) Cell intervention: Once the cells adhere to the wall, the cells are transfected with siRNA or hNCSCs-sEVs are added according to the group.

[0296] (3) Scratching: After 3 days, the cells were manually scratched on the monolayer cells using the tip of a sterile pipette.

[0297] (4) Observation and image acquisition: At 0, 12 and 24 h after scratching, cell migration was observed and photographed using a microscope.

[0298] (5) Data analysis: ImageJ software was used to analyze the scratch area. Cell migration rate (%) = change in scratch area / initial scratch area × 100%.

[0299] 8.6 The experimental procedure for CCK8 is the same as in 2.1.

[0300] result:

[0301] To verify the role of HAS2 expression levels in the proliferation and migration of perineurium cells, Western blot analysis was performed on cultured perineurium cells. The results showed that after 5 days of in vitro culture, compared to the control group, the expression of PCNA and Vimentin in the hfNCSCs-sEVs group perineurium cells was significantly upregulated with HAS2 upregulation. Conversely, when si-Has2 was used to downregulate HAS2 expression in both the control and hfNCSCs-sEVs groups perineurium cells, the expression of PCNA and Vimentin was also downregulated. Figure 20 (A, B) suggests that HAS2 plays an important role in the proliferation and migration of perineurium cells. In addition, the results of the cell scratch assay also showed that, at the same time point, compared with the control group, the scratch gap area between perineurium cells in the hfNCSCs-sEVs group was smaller. However, when HAS2 expression in perineurium cells of the control group and hfNCSCs-sEVs group was downregulated using si-Has2, the scratch area in both groups increased accordingly at the same time point. Based on the initial scratch area, the cell migration rate at different time points in each group was calculated. The results showed that at 12 h and 24 h after the start of the scratch assay, the migration rates of perineurium cells from high to low were hfNCSCs-sEVs / - group, hfNCSCs-sEVs / si-Has2 group, - / - group, and - / si-Has2 group (…). Figure 20 C, D). The CCK-8 experiment results also showed that after 5 days of in vitro culture, the absorbance values ​​representing the viability of perineurium cells, from highest to lowest, were hfNCSCs-sEVs group, hfNCSCs-sEVs+si-Has2 group, PBS group, and si-Has2 group (…). Figure 20 E). These results indicate that hfNCSCs-sEVs promote the proliferation and migration of nerve perineurium cells in vitro by upregulating the expression of HAS2 in nerve perineurium cells, and that inhibiting the expression of HAS2 in nerve perineurium cells can significantly attenuate these promoting effects.

[0302] Example 9: Detection of the effect of miR-21-5p in hfNCSCs-sEVs on HAS2 expression in perimysial cells and its effect on perimysial cell proliferation, migration and tight junction protein expression.

[0303] 9.1 Loading of miR-21-5p inhibitor in hfNCSCs-sEVs

[0304] (1) Reagent preparation: Add 1 ml of ETP solution to the transport peptide, dissolve it completely, and store it at 4 °C.

[0305] (2) Nucleic acid loading: Add reagents in the following order and dosage, and incubate at 37 °C in the dark for 2 h, with continuous shaking at 150 rpm during the period.

[0306]

[0307] (3) Removal of free small nucleic acids: After incubation, transfer the sample to a 100kD ultrafiltration tube, add Wash Buffer to a final volume of 4 mL, and centrifuge at 4000g for approximately 20-30 min to a final volume of approximately 100 μL. Add Wash Buffer again to a final volume of 4 mL and perform a second wash, centrifuging at 4000g to the target volume. Gently rinse the filter membrane repeatedly with hfNCSCs-sEVs, then transfer the samples to an empty EP tube to obtain hfNCSCs-sEVs loaded with miR-21-5p inhibitor.

[0308] 9.2 The Western blot (WB) experimental procedure is the same as in 1.4.

[0309] 9.3 Cell immunofluorescence staining experiment is the same as 1.2

[0310] 9.4 Immunofluorescence staining of tissues is the same as in 4.3.

[0311] 9.5 EdU cell proliferation experiment

[0312] (1) Inoculation and drug administration were performed in accordance with the CCK-8 cell proliferation experiment.

[0313] (2) Add EdU working solution: Add 2×EdU incubation working solution in half per well, shake gently to mix, and then put back into the incubator to continue incubation for 2 h.

[0314] (3) Fixation and permeation reference immunofluorescence cytochemical staining experiment.

[0315] (4) EdU click reaction: After discarding PBS, add click buffer, shake to mix, and incubate in the dark for 30 min.

[0316] (5) Nuclear staining: Discard the click reaction solution, rinse with PBS for 5 min × 3 times, and then incubate with DAPI staining solution in the dark for 8 min.

[0317] (6) Observation and image acquisition: Observe and acquire images under a fluorescence microscope. Cell proliferation rate (%) = (Number of EdU-positive cell nuclei / Number of DAPI-positive cell nuclei) × 100%.

[0318] result:

[0319] To investigate the role of miR-21-5p in regulating HAS2 protein expression and its proliferation and migration in hfNCSCs-sEVs in vitro, we first designed and synthesized a miR-21-5p inhibitor, which was then loaded onto hfNCSCs-sEVs using an exosome nucleic acid loading kit. Concentration analysis showed that the concentration of miR-21-5p inhibitor in hfNCSCs-sEVs was approximately 249 nM. Western blot analysis showed that after 5 days of in vitro culture, compared with the PBS group, the protein expression levels of HAS2, PCNA, and Vimentin in the hfNCSCs-sEVs group were significantly upregulated, while these promoting effects were significantly inhibited in the hfNCSCs-sEVs + miR-21-5p inhibitor group. Figure 21 A, B). Immunofluorescence staining of perineurial cells cultured for 5 days also showed similar results: the cumulative fluorescence density of HAS2 in perineurial cells of the hfNCSCs-sEVs group was significantly higher than that of the PBS group; the proportion of EdU-labeled nuclei to total nuclei was calculated to indicate cell proliferation rate, and the results showed that the proliferation capacity of perineurial cells in the hfNCSCs-sEVs group was significantly higher than that in the PBS group, while these promoting effects were significantly inhibited in the hfNCSCs-sEVs+miR-21-5p inhibitor group. Figure 21 (C, D, E). These results indicate that miR-21-5p in hfNCSCs-sEVs can promote the expression of HAS2 in perineurium cells in vitro and promote the expression of its proliferation and migration-related proteins.

[0320] To investigate the role of miR-21-5p in hfNCSCs-sEVs in the formation of tight junction proteins between perimysial cells in vitro, immunofluorescence staining and Western blotting were performed on perimysial cells cultured for 7 days. The results showed that at the edges of the Tubulin-labeled (red) cytoskeleton, the cumulative fluorescence density of ZO1 (green) between perimysial cells in the hfNCSCs-sEVs group was significantly higher than that in the PBS group. Figure 22 (A, B) Western blot results also showed that the expression levels of ZO1 and Claudin1 in the perineurium cells of the hfNCSCs-sEVs group were significantly higher than those in the PBS group, and these promoting effects were significantly weakened in the hfNCSCs-sEVs + miR-21-5p inhibitor group. Figure 22 (C, D). The results showed that miR-21-5p in hfNCSCs-sEVs could promote the expression of tight junction proteins in nerve perithelial cells in vitro, and the inhibition of miR-21-5p could weaken the promoting effect of hfNCSCs-sEVs.

[0321] To further verify the role of miR-21-5p in hfNCSCs-sEVs in the repair of peripheral nerve defects in rats, Western blot analysis was performed on the regenerated tissue of rats in each group 5 days after surgery. The results showed that the expression levels of HAS2, PCNA, and Vimentin in the regenerated tissue of the hfNCSCs-sEVs group were significantly higher than those in the PBS group. These promoting effects were significantly inhibited in the hfNCSCs-sEVs + miR-21-5p inhibitor group. Figure 23 A, B).

[0322] Western blot analysis of regenerated tissue 7 days post-surgery revealed that during sciatic nerve defect repair, the promoting effect of hfNCSCs-sEVs on the tight junction proteins ZO1 and Claudin1 of the perineural cells was significantly inhibited in the hfNCSCs-sEVs + miR-21-5p inhibitor group. Figure 24 A, B). Immunofluorescence staining results also showed that, compared with the PBS group, the regenerated perineurial cells in the hfNCSCs-sEVs group had a significantly increased migration distance and a significantly increased Claudin1 expression level, while these promoting effects were decreased in the hfNCSCs-sEVs+miR-21-5p inhibitor group. Figure 24 C). These results indicate that miR-21-5p in hfNCSCs-sEVs can promote the expression of cell proliferation, migration-related proteins, and tight junction proteins between nerve perineurium cells in the repair of sciatic nerve defects in rats.

[0323] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of reagents that upregulate HAS2 expression in perineurium cells in the preparation of drugs or reagents that promote perineurium formation.

2. Application of miR-21-5p, a small extracellular vesicle derived from hair follicle neural crest stem cells, in the preparation of drugs or reagents that promote the formation of the nerve perithelial membrane and accelerate the repair of nerve damage.

3. A method for promoting the formation of a nerve perimysium, characterized in that, It involves adding a reagent to the culture medium of perineurium cells to upregulate HAS2 expression within the perineurium cells.

4. Application of reagents that upregulate HAS2 expression in nerve perineurium cells in the preparation of drugs or reagents that promote the repair of sciatic nerve injury.

5. The application according to claim 4, characterized in that, The reagent that upregulates HAS2 expression in nerve perineurium cells promotes tissue repair and functional recovery after sciatic nerve defects.

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