Use of id1 / id3 in inducing fibroblast reprogramming into schwann cells to promote nerve regeneration

Abstract

The application belongs to the field of biomedical technology, and particularly relates to application of ID1 and ID3 in reprogramming of fibroblasts into Schwann cells and in promoting peripheral nerve regeneration, which uses ID1 or ID3 reprogrammed Schwann cells or ID1 or ID3 overexpressed viral vectors, can promote nerve regeneration after trauma, promote trauma healing, and improve trauma healing quality, and is an effective way to solve difficult-to-heal trauma caused by local nerve regeneration after trauma or peripheral neuropathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ID1 / ID3 in inducing fibroblast reprogramming into Schwann cells to promote nerve regeneration. Background Technology

[0002] Wound repair is a complex biological process involving various ordered cellular biological processes such as reepithelialization and angiogenesis. Nerve innervation has a significant impact on wound healing; previous studies have shown that pathological or traumatic impairment of nerve innervation can lead to delayed or even non-healing wounds. Furthermore, the restoration of pain, temperature, and tactile sensation in the local tissues after trauma is crucial for improving patients' quality of life. Therefore, improving local nerve repair after trauma has significant clinical implications for treating refractory wounds and improving the quality of wound healing.

[0003] Previous studies have shown that Schwann cells play a crucial role in the repair of peripheral nerve injuries. Following nerve injury, Schwann cells interact with various cells at and around the injury site, participating in important processes such as debris clearance, nerve repair, and nerve regeneration. Furthermore, Schwann cells can secrete neurotrophic factors that promote nerve regeneration. Previous research has found that transplanting exogenous Schwann cells after peripheral nerve injury can effectively promote peripheral nerve regeneration. However, the large quantities of Schwann cells required for cell transplantation therapy face limitations in cell supply and matching, potentially leading to immune rejection. Therefore, reprogramming adult somatic cells, such as fibroblasts, to produce Schwann cells can effectively overcome the bottlenecks in immune matching and cell source, making it a promising cell source for clinical use.

[0004] The inhibitor of DNA binding (ID) family is a group of evolutionarily conserved proteins. Four ID proteins, ID1 through ID4, have been identified in mammals. ID proteins exhibit wide but highly variable expression across various cell types. Previous studies have found that ID proteins can promote cell cycle progression, accelerate cell migration, inhibit the differentiation of different progenitor cell types, and reduce cellular senescence. Therefore, ID proteins are involved in various pathophysiological processes in vivo, such as neurogenesis, stem cell maintenance, angiogenesis, organ development, tumorigenesis and metastasis, and energy metabolism. However, the role of ID protein molecules in adult cell reprogramming has not been reported. Summary of the Invention

[0005] One object of the present invention is to provide an application of ID1 / ID3 in inducing fibroblast reprogramming into Schwann cells to promote nerve regeneration. This application uses Schwann cells reprogrammed with ID1 or ID3 or viral vectors overexpressing ID1 or ID3 to promote nerve regeneration after trauma, promote wound healing, and improve the quality of wound healing. It is an effective way to solve refractory trauma caused by local nerve regeneration or peripheral neuropathy after trauma.

[0006] The specific technical solution is as follows:

[0007] The application of differentiation inhibitory factor in the preparation of drugs for treating peripheral nerve injury, wherein the application is that differentiation inhibitory factor promotes nerve regeneration by inducing fibroblast reprogramming into Schwann cells.

[0008] The differentiation inhibitory factor is ID1 or / and ID3.

[0009] The Schwann cells include those induced by ID1 or ID3, or those induced by both ID1 and ID3 simultaneously.

[0010] The fibroblasts are human or murine adult or embryonic fibroblasts.

[0011] The peripheral nerve injury mentioned refers to local nerve injury following trauma.

[0012] The term "post-traumatic local nerve injury" refers to peripheral nerve injury, including full-thickness sciatic nerve transection, full-thickness skin injury leading to local nerve injury, or other types of trauma resulting in local nerve injury to tissues.

[0013] The trauma includes mechanical trauma to the skin and mechanical trauma to other tissues, or burns or combined injuries to the skin or other tissues.

[0014] The ID1 and ID3 involved in this invention include recombinant vectors, recombinant microorganisms, etc., encoding the protein gene sequences of ID1 and ID3. Overexpression of the ID1 or ID3 gene in fibroblasts can increase the expression of Schwann cell markers s100β and Gfap in fibroblasts, and at the same time promote axonal elongation of dorsal root ganglion neurons.

[0015] The fibroblasts involved in this invention include, but are not limited to, primary fibroblasts isolated from adult tissues. Fibroblasts isolated during the embryonic period are also within the scope of protection of this invention.

[0016] The trauma involved in this invention includes, but is not limited to, mechanical trauma to the skin, mechanical trauma to other tissues, or burns and combined injuries to the skin or other tissues, which are also within the scope of protection of this invention. After ID1 and ID3-induced fibroblasts are reprogrammed into Schwann cells, transplantation of the induced Schwann cells can effectively promote local nerve regeneration after trauma, accelerate wound healing, reduce collagen deposition in the wound, and improve the quality of wound healing.

[0017] The peripheral nerve injuries involved in this invention include, but are not limited to, sciatic nerve injuries and post-traumatic local nerve injuries. After ID1 and ID3-induced fibroblasts were reprogrammed into Schwann cells, transplantation of the induced Schwann cells could effectively promote the regeneration of damaged peripheral nerves and reduce atrophy of the muscles innervated by the nerves.

[0018] This invention also directly uses ID1 and ID3 viral vectors to the wound site, which can promote nerve repair at the wound site, accelerate wound healing, post-traumatic re-epithelialization, and granulation tissue formation.

[0019] Based on the establishment of a full-thickness skin lesion (which can heal through the normal wound repair process) and an in vitro fibroblast scratch injury model, the applicant confirmed through histopathological and molecular biological techniques that the expression of ID1 and ID3 in fibroblasts was significantly enhanced after trauma.

[0020] Overexpression of ID1 or ID3 in fibroblasts revealed that both ID1 and ID3 enhanced fibroblast proliferation and migration. Furthermore, fibroblasts were reprogrammed into Schwann cells expressing Gfap and s100β. Co-culturing the induced Schwann cells with dorsal root ganglion neurons showed that, compared to uninduced fibroblasts, the induced Schwann cells better promoted neuronal axonal growth.

[0021] The applicant's experimental verification showed that local transplantation of ID1 or ID3-induced Schwann cells into wound tissue after full-thickness skin injury promoted local nerve repair, accelerated wound healing, reduced collagen deposition, and improved wound healing quality. Direct injection of ID1 or ID3 viral vectors into ear wounds showed that, compared to control viral vectors, ID1 or ID3 viral vector transplantation promoted local nerve repair, accelerated wound healing, post-traumatic re-epithelialization, and granulation tissue formation. Local transplantation of ID1 or ID3-induced Schwann cells into the sciatic nerve stump model showed that, compared to blank transplantation and control fibroblast transplantation, ID1 or ID3-induced Schwann cells better promoted nerve regeneration and reduced muscle atrophy at the nerve innervation site. Attached Figure Description

[0022] Figure 1 shows the expression of ID1 and ID3 in post-traumatic fibroblasts as described in this invention;

[0023] Figure 2 shows the case where the ID1 and ID3 induced fibroblasts of the present invention are Schwann cells;

[0024] Figure 3 The effect of ID1 or ID3-induced Schwann cells on the axonal growth of dorsal root ganglion neurons described in this invention;

[0025] Figure 4 shows the effect of ID1 or ID3-induced Schwann cells on promoting local nerve regeneration and wound healing as described in this invention.

[0026] Figure 5 shows the effect of ID1 or ID3-induced Schwann cells on the repair of sciatic nerve injury described in this invention.

[0027] Figure 6 The ID1 or ID3 lentiviral vector described in this invention promotes local nerve regeneration and wound healing. Detailed Implementation

[0028] The present invention will be specifically described below through embodiments. It should be noted that these embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above content of the present invention.

[0029] The mice used in this invention were C57 mice and SD rats, both purchased from the Experimental Animal Center of Army Medical University.

[0030] ID1, ID3, Tuj1, and s100β antibodies were purchased from Abcam.

[0031] Gfap antibody was purchased from Wuhan Sanying Company;

[0032] Fluorescent secondary antibody, HRP secondary antibody, double antibody, and crystal violet staining solution were all purchased from Beyotime Biotechnology Co., Ltd.

[0033] B27, L-glutamine, and fetal bovine serum were purchased from Gibco.

[0034] 4% paraformaldehyde and PBS were purchased from Boster Biological Technology Co., Ltd.

[0035] Chloral hydrate was purchased from Shanghai Sangon Biotech Co., Ltd.; type I collagenase was purchased from Worthington Biotech.

[0036] Trypsin and DMEM culture medium were purchased from Hyclone.

[0037] Neurobasal TM The culture medium was purchased from Millpore.

[0038] Flow cytometry was performed using BD's FACS AriaIII.

[0039] Immunoblotting was performed using a Bio-Rad electrophoresis system.

[0040] Image acquisition was performed using a Leica DM3000 microscope;

[0041] Immunoblotting, cell and tissue immunofluorescence staining, colony formation ability assay, HE staining, and other experimental procedures and conditions not explicitly described were performed in accordance with the literature (Chen, Z., et al. (2019). "Fibrogenic fibroblast-selective near-infrared phototherapy to control scarring." Theranostics 9(23):6797-6808).

[0042] In addition to the reagents mentioned above, all other reagents used in this invention are commercially available biological grade reagents.

[0043] Example 1: Expression of ID1 and ID3 in post-traumatic fibroblasts

[0044] Establishment of a mouse model of full-thickness skin resection injury: Select female C57 mice that are 8-12 weeks old and weigh about 20g. The mice were routinely prepared and shaved one day before the injury. They were anesthetized by intraperitoneal injection of 1% chloral hydrate (5ml / kg). The skin of the mouse's back was gently lifted along the midline of the mouse's back, and a full-thickness skin punch with a diameter of about 1cm was used to make the wound.

[0045] Specimens were collected from full-thickness skin defects in mice on days 1, 3, 5, 7, 9, 12, 15, and 20. The specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. The sections were used for immunohistochemical staining of ID1 and ID3 proteins. PBS was used instead of the primary antibody as a negative control. Experimental results are shown below. Figure 1A B, section staining showed that ID1 and ID3 were expressed in fibroblasts of newly formed granulation tissue in the wound starting from day 3.

[0046] In vitro fibroblast scratch wound model preparation: Human dermal fibroblasts (passage 6 or less) isolated and cultured from tissue were seeded into 24-well plates with pre-placed round cell spreaders. The culture conditions were: DNEM + 10% fetal bovine serum + penicillin-dextrose antibody. The medium was changed routinely every 3 days. After the cells reached confluence, they were cultured for another 2 days. A horizontal line was drawn along the diameter of the 24-well plate to create a wound using a 200ul pipette tip.

[0047] Cell smears were collected at 6, 12, 24, 36, 48, 60, and 72 hours post-traumatic injury. Immunofluorescence staining was performed on ID1 and ID3 cells. PBS was used as a negative control group instead of the primary antibody. Experimental results are shown in [Figure number missing]. Figure 1C D staining showed that the expression of ID1 and ID3 in fibroblasts was significantly enhanced 12 hours after the injury.

[0048] Cellular proteins were collected at 24, 36, 48, and 72 hours post-traumatic stress. The expression of ID1 and ID3 was detected by Western blotting. The experimental results are shown below. Figure 1E The results showed that the expression of ID1 and ID3 in fibroblasts was significantly enhanced after trauma, suggesting that the high expression of ID1 and ID3 in fibroblasts plays an important role in trauma repair.

[0049] Example 2: ID1 or ID3 induces fibroblasts in Schwann cells.

[0050] Human and mouse dermal fibroblasts were seeded into 24-well plates. When the cell confluence reached 40%, they were seeded into human and mouse lentiviral vectors Vector-mCherry, ID1-mCherry, and ID3-mCherry, which were constructed and purified by Gemma Gene Biotechnology Co., Ltd. The virus particle number / cell number (MOI) was 100. After transfection, the cells were passaged after complete confluence. After one generation of cell expansion, mCherry control fibroblasts, ID1 overexpressing fibroblasts, and ID3 overexpressing fibroblasts were sorted by flow cytometry.

[0051] Colony formation assay to detect the proliferation capacity of ID1 and ID3 overexpressing cells: Human and mouse controls, ID1 overexpressing cells, and ID3 overexpressing fibroblasts were divided into groups of 1×10⁻⁶ cells. 3 Cells were seeded per well in 6-well plates using DMEM + 10% fetal bovine serum + penicillin-dextrose antibody. The medium was changed every 3 days. After 12 days of culture, cells were fixed with 4% paraformaldehyde for 20 min, stained with crystal violet for 15 min, rinsed with water, and colony counted under a microscope. Experimental results are as follows: Figure 2A The results showed that fibroblasts overexpressing ID1 and ID3 formed significantly more colonies than control cells, suggesting that ID1 and ID3 can enhance the self-renewal capacity of fibroblasts.

[0052] Gfap and S100β in cells were detected using immunofluorescence staining: Control fibroblasts, ID1-overexpressing fibroblasts, and ID3-overexpressing fibroblasts were seeded onto cell slides and cultured for 24 h. After fixation with 4% paraformaldehyde for 20 min, the cells were stained with immunofluorescence. PBS was used as a negative control instead of the primary antibody. The experimental results are as follows: Figure 2BIt was found that ID1 and ID3 can induce fibroblasts to enhance the expression of Schwann cell markers Gfap and S100β.

[0053] Immunoblotting was used to detect Sox2, Nestin, Gfap, and S100β in cells: After seeding control fibroblasts and fibroblasts overexpressing ID1 and ID3, when the cell confluence reached more than 90%, whole-cell proteins were extracted, and the expression of Sox2, Nestin, Gfap, and S100β in control fibroblasts and fibroblasts overexpressing ID1 and ID3 was detected by immunoblotting. The results showed that ID1 and ID3 could induce the expression of Sox2, Nestin, Gfap, and S100β in fibroblasts. Combined with the results of immunofluorescence staining, it was indicated that ID1 and ID3 could induce the expression of Schwann cells in fibroblasts.

[0054] Example 3: ID1 or ID3-induced Schwann cells promote axonal growth in dorsal root ganglion neurons.

[0055] Isolation of dorsal root ganglion neurons in rats: SD female rats aged 8-12 weeks, weighing approximately 200g, were sacrificed by cervical dislocation. The dorsal root ganglia segments 4-5 were isolated. Cells were incubated with type I collagenase (0.25%) + 0.01% trypsin at 37°C for 40 min using a shaker. After passing through a 75µm cell sieve, the obtained cells were cultured in Neurobasalt. TM Cells culturing with medium + 2% B27 + L-glutamine (2mM) for 1 week that still survived were dorsal root ganglion neurons.

[0056] Co-culture of dorsal root ganglion neurons and induced Schwann cells: using control cells and ID1 and ID3 overexpressing fibroblasts sorted by flow cytometry from Example 2, at a ratio of 1×10⁻⁶ 4 Cells were seeded per well in 24-well plates pre-filled with cell spreaders and cultured under the following conditions: DMEM + 10% fetal bovine serum + penicillin-dextrose antibody. After 24 hours, 5 × 10⁶ dorsal root ganglion neurons were seeded. 2 Cells / well were placed in 24-well plates pre-seeded with fibroblasts, and the culture conditions were changed to Neurobasal. TM After culturing with medium + 2% B27 + L-glutamine (2mM) for 72 h, the cells were fixed with 4% paraformaldehyde for 20 min. Cell immunofluorescence staining was then performed on nerve axons labeled with Tuj1. PBS was used as a negative control instead of the primary antibody. Axons of 6-10 neurons were selected from each group at 20x magnification, and the average axon length for each group was calculated using ImageJ software. Results are as follows: Figure 3The study found that the axonal growth of dorsal root ganglion neurons co-cultured with ID1 or ID3-induced Schwann cells was significantly longer than that of dorsal root ganglion neurons co-cultured with control fibroblasts, suggesting that ID1 or ID3-induced Schwann cells can significantly promote nerve axonal growth.

[0057] Example 4: ID1 or ID3-induced Schwann cells promote nerve repair and wound healing in skin wounds from radiation-induced complex injuries.

[0058] Preparation of a skin wound model for radiation-induced combined injury: Eight to twelve-week-old female C57 mice, weighing approximately 20g, were selected. Routine skin preparation and hair removal were performed the day before the injury. The mice were irradiated with 5 Gy of X-rays. Following irradiation, they were anesthetized with an intraperitoneal injection of 1% chloral hydrate (5 ml / kg). The skin on the mouse's back was gently lifted along the midline, and a full-thickness skin punch with a diameter of approximately 1 cm was used to create the wound.

[0059] The control fibroblasts, ID1 or ID3-induced Schwann cells obtained in Example 2 were used at 2 × 10⁻⁶ 6 One cell / mouse, 500 μL of PBS, control fibroblasts, and ID1 or ID3-induced Schwann cells were transplanted around the wound tissue of traumatic mice, and the mice were fed after trauma. Wound size was photographed at 0, 3, 5, 7, 15, and 21 days post-traumatic injury. Wound healing rate and scar area were calculated using ImageJ software. Results are shown below. Figure 4A B, It was found that ID1 or ID3-induced Schwann cells can significantly promote wound healing and reduce scar area.

[0060] Wound tissue samples were taken 19 days post-traumatically and stained with immunofluorescence to detect the neurofibrillary marker Tuj1. The results are as follows: Figure 4C The study found that the expression of Tuj1 in the wound tissue of the ID1 or ID3-induced Schwann cell transplantation group was significantly higher than that in the PBS transplantation and control fibroblast transplantation groups, suggesting that ID1 or ID3-induced Schwann cells can significantly promote local nerve repair after trauma.

[0061] Example 5: ID1 or ID3-induced Schwann cells promote sciatic nerve injury repair

[0062] Sciatic nerve injury model creation: SD female mice aged 8-12 weeks and weighing about 200g were selected and anesthetized by intraperitoneal injection of 1% chloral hydrate (5ml / kg). The sciatic nerve was completely transected near the upper end of the femoral head, and the two ends were sutured together with a silicone catheter, with a distance of 5mm between the two ends.

[0063] The control fibroblasts, ID1 or ID3-induced Schwann cells obtained in Example 2 were used at 2 × 10⁻⁶ 6One cell / rat, 200 μL of PBS, control fibroblasts, and ID1 or ID3-induced Schwann cells were transplanted into silicone catheters, and muscle and skin were sutured layer by layer. Six weeks post-traumatically, normal sciatic nerve fibers from the silicone catheter and the contralateral side were harvested. Half of the tissue was fixed in 4% paraformaldehyde for 72 h, dehydrated, embedded in paraffin, sectioned at 4 μm, and the diameter of the nerve bundle was measured by HE staining. The diameter and length of the nerve bundle were calculated using ImageJ software. The results are shown below. Figure 5A It was found that ID1 or ID3-induced Schwann cell transplantation significantly increased the diameter of the regenerated sciatic nerve.

[0064] Half of the tissue was frozen section (10 μm thick), immunofluorescence stained with Tuj1, and the relationship between transplanted cells and regenerated nerves was examined. The results are as follows: Figure 5B It was found that more nerve bundles regenerated in the sciatic nerve induced by transplanted ID1 or ID3 Schwann cells, and the contact between the nerve bundles and the transplanted cells was closer.

[0065] The gastrocnemius muscle on the side of the injured sciatic nerve was harvested, fixed in 4% paraformaldehyde for 72 hours, dehydrated, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (HE) to examine the degree of gastrocnemius muscle atrophy. The results are as follows: Figure 5C The study found that gastrocnemius muscle atrophy was significantly reduced in patients with ID1 or ID3-induced Schwann cells compared to the control group. These results suggest that ID1 or ID3-induced Schwann cells can significantly promote the repair of sciatic nerve injury.

[0066] Example 6: ID1 or ID3 lentiviral vectors promote local nerve repair and wound healing after ear injury.

[0067] Ear trauma model creation: Select 8-12 week old, C57 female mice weighing about 20g, anesthetize them with 1% chloral hydrate via intraperitoneal injection (5ml / kg), and use a 2mm diameter ear punch to make a hole in the center of the ear to create an ear trauma model.

[0068] The Vector-mCherry, ID1-mCherry, and ID3-mCherry lentiviral vectors used in Example 2 were prepared at a concentration of 1×10⁻⁶ mCherry per 20 μL. 7 The amount of U virus was measured, and the wound was injected locally around the ear wound. The wound size was photographed at 0, 3, 7, 14, 21, and 35 days after the injury. The wound healing rate was calculated using ImageJ software. The results showed that transplanting ID1 or ID3 lentiviral vectors could accelerate the healing of the ear wound.

[0069] Tissue samples were collected from 35-day-old wounds, fixed in 4% paraformaldehyde for 72 hours, dehydrated, embedded in paraffin, sectioned at 4µm, and immunofluorescence stained for Tuj1 expression. Results are as follows: Figure 6 They found that transplantation of ID1 and ID3 lentiviruses can significantly promote local nerve repair and regeneration.

Claims

1. The use of ID1 or ID3-induced Schwann cells in the preparation of a drug that promotes neuronal axonal growth in peripheral nerve injury, wherein the ID1 or ID3-induced Schwann cells are fibroblasts overexpressing ID1 or ID3.

2. The application according to claim 1, characterized in that: The fibroblasts are primary fibroblasts isolated from human or mouse adult tissues, or embryonic fibroblasts.

3. The application according to claim 1, characterized in that: The peripheral nerve injury referred to is a local nerve injury following trauma.

4. The application according to claim 3, characterized in that: The local nerve injury following the trauma includes full-thickness sciatic nerve transection and local nerve injury caused by full-thickness skin injury.

5. The application according to claim 3, characterized in that: The trauma includes mechanical skin injuries, or burns and combined injuries to the skin.

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