Preparation method and application of autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration

Abstract

The application provides a preparation method of autologous cell source nanovesicle loaded miRNA in intervertebral disc degeneration, and steps include: (1) adding miRNA mimics into PBS resuspension of nucleus pulposus cells; (2) using polycarbonate membrane with gradient pore size reduction and NV preparation instrument to extrude the nucleus pulposus cells, and obtaining autologous cell source NV loaded miRNA after sterilization and centrifugation; the autologous cell source nanovesicle loaded miRNA is used in preparation of drugs and complex drugs for treating intervertebral disc degeneration. The application provides a method of autologous cell source nanovesicle loaded miRNA, stress response key miRNA in intervertebral disc degeneration and biological functions thereof are recognized, and autologous nucleus pulposus cell source NV is used to load the stress response key miRNA, effective delivery of miRNA in intervertebral disc is realized, and finally the function of delaying intervertebral disc degeneration under abnormal stress load is played.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of autologous cell-derived nanovesicle loaded with miRNA in intervertebral disc degeneration. BACKGROUND

[0002] Intervertebral disc degeneration is an important basic pathological change causing clinical diseases such as lower back pain, intervertebral disc herniation and spinal stenosis. However, the current clinical treatment for intervertebral disc degeneration aims to relieve clinical symptoms rather than intervene in its progression. Therefore, exploring methods to relieve the progression of intervertebral disc degeneration is a research hotspot.

[0003] miRNA is a kind of non-coding RNA, which can regulate various biological behaviors in intervertebral disc degeneration by regulating the expression of downstream mRNA. Regulating the expression of miRNA and then regulating the biological function of intervertebral disc cells is a promising method to delay intervertebral disc degeneration. Intervertebral disc degeneration is a process involving multiple factors, and abnormal stress load is an important factor causing the progression of intervertebral disc. Some miRNAs have the characteristics of responding to mechanical signals and are involved in the biological changes caused by abnormal stress load in intervertebral disc degeneration. Further identification of stress-responsive miRNAs and their biological functions is of great significance to delay intervertebral disc degeneration.

[0004] Due to the problems of easy degradation and instability of naked miRNA in vivo, the effective delivery of miRNA in the intervertebral disc is a key step for its function. Extracellular vesicles can carry various RNAs, proteins and lipids to realize cell-to-cell communication and regulate biological functions. However, the production of extracellular vesicles is low and the cost is high, which cannot meet the large-scale application in clinic. A kind of extracellular vesicle mimics, nanovesicles (NV), is a promising delivery carrier. NVs can be obtained by extruding cells through nano-filtration membranes with gradient pore size reduction, and have similar physicochemical characteristics, biological distribution behavior and targeting effect as extracellular vesicles. More importantly, compared with extracellular vesicles, NVs can be produced on a large scale and are a promising delivery carrier. At the same time, NVs can exhibit effective internalization through homologous targeting. The above shows that autologous cell-derived NVs can be an effective delivery carrier for intervertebral disc tissue. Based on this, the application provides a preparation method and application of autologous cell-derived nanovesicle loaded with miRNA in intervertebral disc degeneration. SUMMARY

[0005] The application provides a preparation method and application of autologous cell-derived nanovesicle loaded with miRNA in intervertebral disc degeneration, which realizes the effective delivery of miRNA in the intervertebral disc and finally plays the function of delaying the intervertebral disc degeneration caused by abnormal stress load.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a preparation method of autologous cell-derived nanovesicle loaded with miRNA in intervertebral disc degeneration, comprising the following steps: (1) adding miRNA mimics into PBS resuspension; (2) using polycarbonate membranes with gradient pore size reduction and NV preparation instruments to extrude the nucleus pulposus cells, sterilize, centrifuge, add PBS buffer to resuspend the precipitate, and finally obtain autologous cell-derived NV loaded with miRNA.

[0008] Further, the miRNA includes at least one of rno-miR-10a-5p, rno-miR-30a-3p, rno-miR-16-5p, rno-miR-1249 and rno-miR-370-3p.

[0009] Further, the PBS resuspension is a PBS resuspension of 2.7 million nucleus pulposus cells.

[0010] Further, the gradient pore size includes 1.0 μm, 0.4 μm or 0.2 μm.

[0011] Further, the NV preparation instrument is a liposome extruder.

[0012] The extrusion step includes: installing the NV preparation instrument and the 1.0 μm polycarbonate membrane, adding the PBS resuspension in the syringe, and pushing the push rod on both sides 11 times, the cells are extruded and loaded with miRNA mimics when passing through the polycarbonate membrane, and then the 0.4 μm and 0.2 μm polycarbonate membranes are replaced according to the above steps to continue extruding the cells.

[0013] Further, the centrifugation step includes: after 1.5 g, 15 min, 4℃ centrifugation, the precipitate is discarded and the supernatant is retained, and the supernatant is centrifuged at 15 g for 60 min at 4℃, and the supernatant is discarded.

[0014] In a second aspect, the autologous cell-derived nanovesicle loaded with miRNA is applied in the preparation of a drug or a complex drug for delaying intervertebral disc degeneration.

[0015] In a third aspect, the autologous cell-derived nanovesicle loaded with miRNA is applied in the preparation of a drug or a complex drug for treating intervertebral disc degeneration.

[0016] Further, the dosage form of the drug is aerosol, tablet, capsule, drop pill, pill, powder, solution, suspension, emulsion, granule, lipid, transdermal, buccal, suppository or lyophilized powder injection.

[0017] The application has the beneficial effects that the application designs a strategy of autologous nucleus pulposus cell-derived NV loading miRNA, recognizes key miRNA in intervertebral disc degeneration and its biological function through stress response, and loads autologous nucleus pulposus cell-derived NV to achieve effective delivery of miRNA in the intervertebral disc, and finally plays a function of delaying intervertebral disc degeneration caused by abnormal stress load. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1-1 The establishment of an in vivo model of intervertebral disc degeneration caused by abnormal stress load, and intervertebral disc magnetic resonance detection and X-ray detection; wherein (a) a successful model of intervertebral disc degeneration caused by stress load is established, and the compression device loads stress on the intervertebral disc Co8-9 of the caudal vertebra; (b) representative T2 magnetic resonance images of the Sham group and the IDD group; (c) T2 signal intensity statistics of the T2 magnetic resonance images of the caudal vertebra intervertebral disc Co8-9 of the Sham group and the IDD group; (d) representative X-ray images of the Sham group and the IDD group; (e) statistics of the change of the intervertebral disc height index of the caudal vertebra intervertebral disc Co8-9 of the Sham group and the IDD group. **** P<0.0001, n=7;

[0019] Figure 1-2 It is an in vitro model schematic diagram of the intervertebral disc degeneration model caused by abnormal stress load;

[0020] Figure 1-3 It is the extracellular matrix synthesis and metabolism, and catabolism gene expression after the nucleus pulposus cells are loaded with stress; wherein (a-b) after the nucleus pulposus cells are loaded with stress for 12 hours, the extracellular matrix synthesis and metabolism genes Col2 and Acan are significantly down-regulated in the Compression 12h group compared with the Sham 12h group; (c-d) after the nucleus pulposus cells are loaded with stress for 12 hours, the extracellular matrix catabolism genes Mmp13 and Adamts5 are significantly up-regulated in the Compression 12h group compared with the Sham 12h group. * P<0.05, ** P<0.01, *** P<0.001, n=3;

[0021] Figure 1-4 It is the detection of the extracellular matrix COL2 and MMP13 protein level expression after the nucleus pulposus cells are loaded with stress; wherein (a) the Western Blot development diagram of the extracellular matrix COL2 and MMP13 protein level expression after the nucleus pulposus cells are loaded with stress; (b) statistical analysis of the extracellular matrix COL2 protein level expression after the nucleus pulposus cells are loaded with stress for 12 and 24 hours, respectively; (c) statistical analysis of the extracellular matrix MMP13 protein level expression after the nucleus pulposus cells are loaded with stress for 12 and 24 hours, respectively. *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, n=3.

[0022] Figure 1-5 The candidate stress response miRNA clustering heat map and the verification of miR-1249 in degenerative nucleus pulposus cells and nucleus pulposus tissue caused by abnormal pressure stress load; wherein (a) the candidate stress response miRNA clustering heat map; (b) the expression level of the candidate stress response miRNA in the nucleus pulposus cell Compression group and the Sham group; (c) the verification of miR-1249 in the nucleus pulposus tissue IDD group and the Sham group. * P<0.05, ** P<0.01, n=3.

[0023] Figure 1-6 The effect of overexpression of miR-1249 on extracellular matrix anabolism of nucleus pulposus cells under abnormal pressure stress load; wherein (a) detection of the expression level of Acan and Col2 genes of extracellular matrix anabolism of nucleus pulposus cells under abnormal pressure stress load by overexpression of miR-1249; (b) Western Blot detection development map of the protein level of ACAN and COL2 of extracellular matrix anabolism of nucleus pulposus cells under abnormal pressure stress load by overexpression of miR-1249; (c) quantitative analysis of the protein level of ACAN and COL2 of extracellular matrix anabolism of nucleus pulposus cells under abnormal pressure stress load by overexpression of miR-1249. ** P<0.01, *** P<0.001, **** P<0.0001, n=3.

[0024] Figure 1-7 The effect of overexpression of miR-1249 on extracellular matrix catabolism of nucleus pulposus cells under abnormal pressure stress load; wherein (a) detection of the expression level of Mmp13 and Adamts5 genes of extracellular matrix catabolism of nucleus pulposus cells under abnormal pressure stress load by overexpression of miR-1249; (b) Western Blot detection development map of the protein level of MMP13 and ADAMTS5 of extracellular matrix catabolism of nucleus pulposus cells under abnormal pressure stress load by overexpression of miR-1249; (c) quantitative analysis of the protein level of MMP13 and ADAMTS5 of extracellular matrix catabolism of nucleus pulposus cells under abnormal pressure stress load by overexpression of miR-1249. * P<0.05, ** P<0.01, **** P<0.0001, n=3.

[0025] Figure 2-1 Characterization of nucleus pulposus cell-derived NVs; wherein, (a) Nucleus pulposus cell-derived NVs nanoparticle size analysis; (b) Transmission electron microscopy observation of nucleus pulposus cell-derived NVs, typical lipid bilayer-like extracellular vesicle structure can be seen; (c) Western Blot detection of extracellular vesicle markers on nucleus pulposus cell-derived NVs, expression imaging map; (d) Quantitative analysis of the protein expression level of extracellular vesicle markers on nucleus pulposus cell-derived NVs, * P < 0.05, *** P < 0.001, **** P < 0.0001, n = 3.

[0026] Figure 2-2 NVs can effectively deliver miRNA to recipient nucleus pulposus cells; wherein, (a) Representative fluorescence microscopy images of NVs, miRNA, and nucleus pulposus cells at 20X and 40X magnification; (b) Fluorescence colocalization analysis of NVs and miRNA at 20X and 40X magnification, both fluorescence have obvious colocalization distribution characteristics; (c) Counting statistics of cells with blue fluorescence, and cells with both blue fluorescence / yellow fluorescence, there is no significant difference between the two. ns P > 0.05, n = 6.

[0027] Figure 3-1 SD rat caudal intervertebral disc Co8-9 magnetic resonance detection; wherein, (a) Representative intervertebral disc Co8-9 magnetic resonance image results of each group; (b) Statistical analysis of intervertebral disc Co8-9 magnetic resonance T2 intensity of each group. # P < 0.0001 compared with the Sham group; + P < 0.0001 compared with the IDD group, **** P < 0.0001, n = 7.

[0028] Figure 3-2 SD rat caudal intervertebral disc Co8-9 X-ray detection; wherein, (a) Representative intervertebral disc Co8-9 X-ray image results of each group; (b) Statistical analysis of intervertebral disc Co8-9 magnetic resonance intervertebral disc height index change of each group. # P < 0.0001 compared with the Sham group; + P < 0.0001 compared with the IDD group, **** P < 0.0001, n = 7.

[0029] Figure 3-3SD rats were used for histological analysis of the tail disc; wherein, (a) representative disc Co8-9 hematoxylin-eosin staining and ponceau O-fast green staining results of each group; (b) statistical analysis of the histological scores of the disc Co8-9 disc of each group. # P < 0.0001 compared with the Sham group; + P < 0.01 compared with the IDD group, * P < 0.05, n = 7. DETAILED DESCRIPTION

[0030] The application will be further described in conjunction with the specific embodiments. The following examples are intended to illustrate the application and are not further limiting. The application will be described in more detail by the following more detailed examples:

[0031] Example 1: Identification and biological function exploration of stress-responsive miRNAs in disc degeneration

[0032] 1.1 Construction of disc degeneration model caused by abnormal stress load

[0033] (1) In vivo model construction: A compression device was installed on the 7th to 10th tail vertebrae (Co7-10) of rats as the disc degeneration group (IDD group, Figure 1-1 (a)) and the Sham group only inserted a Kirschner needle. Four weeks later, imaging detection was performed.

[0034] The results showed that the T2 signal intensity of the IDD group tail disc Co8-9 was significantly lower than that of the Sham group (P < 0.0001, Figure 1-1 (b-c)). The disc height index of the IDD group tail disc Co8-9 was significantly lower than that of the Sham group (P < 0.0001, Figure 1-1 (d-e)). Four weeks of compression stress loading can cause disc degeneration.

[0035] (2) In vitro model construction: A self-made compression stress loading device was used to load compression stress on the nucleus pulposus cells and as the compression group (Compression group), the compression stress size was 2.0 g / cm 2 . Figure 1-2 The Sham group only inserted a glass slide, and the compression stress size was 0.1 g / cm 2 .

[0036] The results showed that, as shown in Figure 1-3 (a-b), the extracellular matrix synthesis and metabolism genes Col2 and Acan in the Compression 12h group were significantly down-regulated compared with the Sham 12h group, and the difference was statistically significant (all P < 0.05). As shown in Figure 1-3As shown in (cd), the extracellular matrix catabolism genes Mmp13 and Adamts5 were significantly upregulated in the Compression 12h group compared to the Sham 12h group, with statistically significant differences (both P < 0.01). Furthermore, the expression levels of extracellular matrix COL2 and MMP13 proteins in nucleus pulposus cells after 12 and 24 hours of compressive stress loading were detected. Figure 1-4 As shown, after 12 hours of compressive stress loading, the expression of COL2 protein in the Compression 12h group was significantly lower than that in the Sham 12h group (P<0.0001), while the expression of MMP13 protein in the Compression 12h group was significantly higher than that in the Sham 12h group (P<0.0001). 12 hours of compressive stress loading can induce degeneration of nucleus pulposus cells.

[0037] 1.2 Recognition of stress-responsive miRNAs

[0038] An in vivo abnormal compressive stress load intervertebral disc degeneration model was constructed. High-throughput miRNA sequencing was performed on the nucleus pulposus tissue. Using standardized read counts, fold change, p-value, and free radiative density (FDR) were calculated. A fold change ≥1.5 and a p-value <0.05 were used as the screening threshold for differentially expressed miRNAs. Based on the identified differentially expressed miRNAs, they were ranked from largest to smallest fold change. Using the miRBase database, the sequences of differentially expressed miRNAs in humans and rats were carefully compared. For upregulated and downregulated genes, the top 5 miRNAs with the largest fold changes in sequence were selected as candidate miRNAs, totaling 10 candidate miRNAs. The expression levels of stress-responsive miRNAs in the in vivo and in vitro abnormal stress intervertebral disc degeneration models were then verified.

[0039] The results showed that among the 10 candidate stress-responsive miRNAs, rno-miR-125a-5p, rno-miR-28-5p, rno-miR-21-5p, rno-miR-22-5p, and rno-miR-335 were upregulated in the IDD group, while rno-miR-10a-5p, rno-miR-30a-3p, rno-miR-16-5p, rno-miR-1249, and rno-miR-370-3p were downregulated in the IDD group. Figure 1-5 (a)).

[0040] The expression of the above candidate miRNAs was detected in nucleus pulposus cells that had degenerated due to abnormal compressive stress loading. Figure 1-5 (b)). Data showed that miR-1249 was a miRNA that met the screening criteria. The expression level of miR-1249 was detected in degenerated nucleus pulposus tissue induced by abnormal compressive stress.Figure 1-5 (c)), which is consistent with the sequencing results and in vitro experimental results, miR-1249 is significantly down-regulated in IDD group relative to Sham group (P<0.01). Therefore, miR-1249 is a key stress-responsive miRNA in intervertebral disc degeneration.

[0041] 1.3 Biological function of stress-responsive miRNA

[0042] This example transiently transfected nucleus pulposus cells with miR-1249 overexpression, to explore its effect on the expression of extracellular matrix metabolism molecules in nucleus pulposus cells.

[0043] The results show that: as shown in Figure 1-6 at the gene level, the Compression group caused significant down-regulation of anabolic genes Acan (P<0.01) and Col2 (P<0.0001) relative to the Sham group. After overexpression of miR-1249 mimics, the Compression+mimics group can significantly up-regulate the down-regulation of Acan (P<0.001) and Col2 (P<0.0001) genes caused by stress. At the protein level, the Compression group caused significant down-regulation of anabolic proteins ACAN and COL2 (both P<0.0001) relative to the Sham group, and after overexpression of miR-1249 mimics, the Compression+mimics group can significantly up-regulate the down-regulation of ACAN (P<0.01) and COL2 (P<0.01) protein levels caused by stress.

[0044] As shown in Figure 1-7As shown, at the gene level, the Compression group caused significant upregulation of catabolic genes Mmp13 (P<0.01) and Adamts5 (P<0.05) relative to the Sham group. After overexpression of miR-1249 mimics, the Compression+mimics group could significantly downregulate the upregulation of Mmp13 (P<0.05) and Adamts5 (P<0.0001) genes caused by compression stress relative to the Compression+NC group. At the protein level, the Compression group caused significant upregulation of catabolic proteins MMP13 (P<0.01) and ADAMTS5 (P<0.01) relative to the Sham group, and after overexpression of miR-1249 mimics, the Compression+mimics group could significantly downregulate the upregulation of MMP13 (P<0.05) and ADAMTS5 (P<0.05) at the protein level caused by compression stress relative to the Compression+NC group. The miR-1249 mimic rescued the extracellular matrix metabolic imbalance of the nucleus pulposus cells caused by compression stress.

[0045] Example 2: Characterization of nucleus pulposus cell-derived NVs and identification of their miRNA delivery ability

[0046] 2.1 Characterization of nucleus pulposus cell-derived NVs

[0047] The nucleus pulposus cells were extruded using polycarbonate membranes with gradient pore size reduction (pore sizes of 1.0, 0.4, and 0.2 μm, respectively) and NV preparation equipment (liposome extruder), and NVs were obtained after sterilization and centrifugation.

[0048] The nucleus pulposus cell-derived NVs were characterized, and the results showed that, as shown in Figure 2-1 The nanoparticle size distribution of the NVs was analyzed, and the mode value of the particle size of the NVs was 166.9 nm, and the median particle size was 200.8 nm. Through transmission electron microscopy observation, a typical lipid bilayer cell extracellular vesicle structure could be seen.

[0049] The expression of the classical markers of extracellular vesicles (ALIX, TSG101, CD63, and CD9) on the NVs was detected, and the nucleus pulposus cells were used as a control.

[0050] The results showed that, relative to the Cell group, the NV group highly expressed the TSG101 (P<0.05), CD63 (P<0.05), and CD9 (P<0.001) markers, while the ALIX marker was lowly expressed in the NV group relative to the Cell group (P<0.0001); the NVs exhibited similar characteristics to extracellular vesicles.

[0051] 2.2 Identification of the miRNA delivery ability of the nucleus pulposus cell-derived NVs

[0052] Preparation of nucleus pulposus cell-derived NV loaded with FAM fluorescent miRNA mimics NC: 40 μL of FAM fluorescent miRNA mimics NC (20 μM) was added to the PBS resuspension of 2.7 million nucleus pulposus cells; the nucleus pulposus cells were extruded using a gradient pore size reducing polycarbonate membrane (1.0 μm, 0.4 μm, and 0.2 μm, respectively) and an NV preparation instrument. The specific steps were as follows: the NV preparation instrument and the 1.0 μm polycarbonate membrane were installed, the PBS resuspension of the nucleus pulposus cells was added to a syringe, and the push rods on both sides were pushed 11 times alternately. The cells were extruded and loaded with FAM fluorescent miRNA mimics NC when passing through the polycarbonate membrane. Then, the 0.4 μm and 0.2 μm polycarbonate membranes were replaced, and the cells were extruded according to the above steps. The cells were sterilized using a 0.22 μm filter, and the supernatant was obtained after 1.5 g, 15 min, and 4 °C centrifugation. The supernatant was discarded after 15 g, 60 min, and 4 °C centrifugation, and the precipitate was resuspended with PBS buffer. Finally, the autologous cell-derived NV loaded with FAM fluorescent miRNA mimics NC was obtained, and the NV was stained with DiI working solution (25 μM). The nucleus pulposus cells were incubated with the NV at a concentration of 100 μg / mL for 12 hours. After 12 hours, the nucleus pulposus cells were stained with DAPI, and observed under a fluorescence microscope.

[0053] The results showed that, as shown in Figure 2-2 different magnifications (20X and 40X), the nucleus pulposus cells (blue fluorescence) were clearly seen to be enriched with miRNA (green fluorescence) and NV (red fluorescence). Meanwhile, the colocalization analysis of the miRNA fluorescence and NV fluorescence showed that the green fluorescence and red fluorescence presented obvious fluorescence colocalization distribution at 20X and 40X magnifications. Through the fusion of the three kinds of fluorescence, the aggregation of yellow fluorescence and blue fluorescence could be seen. Further, the cells with blue fluorescence (Blue group) and the cells with both blue fluorescence and yellow fluorescence (Blue / Yellow group) were counted, and the counting results showed that there was no significant difference between the two groups. The above proved that the nucleus pulposus-derived NV could effectively carry miRNA to deliver to the recipient nucleus pulposus cells after 12 hours of incubation.

[0054] Example 3: Autologous nucleus pulposus cell-derived NV loaded with stress response miRNA delays the progression of intervertebral disc degeneration

[0055] In order to detect the intervention effect of the stress response miRNA of the nucleus pulposus cell source NV on the intervertebral disc degeneration in vivo, a rat model of intervertebral disc degeneration caused by abnormal pressure stress was constructed, and NV loaded with miR-1249 mimics (NV-mimics) and NV loaded with miRNA mimics NC (NV-NC) were prepared. 40 μL of miR-1249 mimics or miRNA mimics NC (20 μM) was added to the PBS resuspension of 2.7 million nucleus pulposus cells; the nucleus pulposus cells were extruded using a gradient pore size reducing polycarbonate membrane (1.0 μm, 0.4 μm, and 0.2 μm, respectively) and a NV preparation instrument. The specific steps are as follows: install the NV preparation instrument and the 1.0 μm polycarbonate membrane, add the nucleus pulposus cell PBS resuspension into the syringe, and push the two push rods 11 times alternately. The cells are extruded and loaded with miR-1249 mimics or miRNA mimics NC when passing through the polycarbonate membrane. Then, replace the 0.4 μm and 0.2 μm polycarbonate membranes and continue to extrude the cells according to the above steps. Use a 0.22 μm filter to sterilize. The filter is a disposable needle filter of the Millipore brand. After 1.5 g, 15 min, and 4°C centrifugation, discard the precipitate and retain the supernatant. Centrifuge the supernatant at 15 g for 60 min at 4°C, discard the supernatant, resuspend the precipitate with PBS buffer, and finally obtain the autologous cell source NV loaded with miR-1249 mimics or miRNA mimics NC. Inject the rats Co8-9 at the third and fourth weeks. The concentration of NV-mimics and NV-NC is 100 μg / mL. After four weeks, detect the intervertebral disc tissue.

[0056] 3.1 Intervertebral disc magnetic resonance analysis

[0057] The Co8-9 intervertebral discs of the SD rats in each group were detected by magnetic resonance. The results are shown in Figure 3-1 As can be seen from the representative magnetic resonance image results, the Co8-9 intervertebral discs of the Sham group showed the best signal intensity. The Co8-9 intervertebral discs of the IDD group and the PBS group showed significantly darkened magnetic resonance signal intensity. The Co8-9 intervertebral discs of the NV-NC group showed a certain improvement in magnetic resonance signal intensity compared with the IDD group. The Co8-9 intervertebral discs of the NV-mimics group showed the best enhancement in magnetic resonance signal intensity compared with the IDD group.

[0058] In addition, the magnetic resonance signal intensity of the Co8-9 intervertebral disc of the tail vertebra was counted. Relative to the Sham group, the IDD group was subjected to four weeks of pressure stress loading, and the magnetic resonance T2 signal intensity was significantly reduced (P < 0.0001). Compared with the IDD group, the PBS group had no significant difference in T2 signal intensity. Compared with the IDD group, the T2 signal intensity of the NV-NC group and the NV-mimics group was significantly higher (all P < 0.0001). At the same time, the T2 signal intensity in the NV-mimics group was significantly higher than that in the NV-NC group (P < 0.0001).

[0059] 3.2 X-ray analysis of intervertebral disc

[0060] The Co8-9 intervertebral disc of the tail vertebra of each group of SD rats was detected by X-ray, and the results are shown in Figure 3-2 From the representative results of the Co8-9 X-ray of the tail vertebra, it can be seen that the Sham group showed the best intervertebral disc height, the IDD group and the PBS group had significantly reduced intervertebral disc height, and the NV-NC group had a certain improvement in intervertebral disc height relative to the IDD group. The NV-mimics group showed the best improvement in intervertebral disc height relative to the IDD group.

[0061] In addition, the change in the height index of the Co8-9 intervertebral disc of the tail vertebra was counted. Relative to the Sham group, the IDD group was subjected to four weeks of pressure stress loading, and the change in the height index of the intervertebral disc was significantly reduced (P < 0.0001). Compared with the IDD group, the PBS group had no significant difference in the change in the height index of the intervertebral disc. Compared with the IDD group, the change in the height index of the intervertebral disc of the NV-NC group and the NV-mimics group was significantly higher (all P < 0.0001). At the same time, the change in the height index of the intervertebral disc in the NV-mimics group was significantly higher than that in the NV-NC group (P < 0.0001).

[0062] 3. Histological analysis of intervertebral disc

[0063] The Co8-9 intervertebral disc of the tail vertebra of each group of SD rats was paraffin sectioned and analyzed by histological staining, and the results are shown in Figure 3-3The tissue sections were stained to show that the Sham group exhibited good disc height and nucleus tissue morphology, the IDD group was replaced by disordered fibrous tissue in the nucleus region, the PBS group only remained a small part of the nucleus tissue, the nucleus tissue of the NV-NC group was partially recovered compared with the IDD group, and the nucleus tissue of the NV-mimics group exhibited the most recovery compared with the IDD group. After histological score analysis, compared with the Sham group, the histological score of the IDD group was significantly increased after four weeks of pressure stress loading (P<0.0001). Compared with the IDD group, the histological score of the PBS group had no significant difference. Compared with the IDD group, the histological score of the NV-NC group and the NV-mimics group were significantly decreased (both P<0.01). At the same time, compared with the NV-NC group, the histological score of the NV-mimics group was significantly decreased (P<0.05).

[0064] Through the intervertebral disc magnetic resonance, X-ray, histological analysis, the results confirmed that the nucleus cell-derived NV loaded with miRNA has the effect of delaying the progression of intervertebral disc degeneration caused by abnormal pressure stress loading.

[0065] The applicant states that the product and detailed preparation method of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned product and detailed preparation method, that is, it does not mean that the present application must rely on the above-mentioned product and detailed preparation method to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of each raw material of the product of the present application, addition of excipient components, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0066] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the present application, the technical solutions of the present application can be variously and simply modified. These simple modifications all belong to the protection scope of the present application.

[0067] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable method without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination methods.

[0068] In addition, various different embodiments of the present application can also be combined arbitrarily, as long as it does not deviate from the idea of the present application, it should also be considered as the disclosed content of the present application.

Claims

1. A method for preparing autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration, characterized by the following steps: include: (1) Add miRNA mimics to the PBS resuspension of nucleus pulposus cells; The miRNA mimics are rno-miR-1249; (2) The nucleus pulposus cells were squeezed, sterilized, centrifuged, and then resuspended in PBS buffer to obtain autologous cell-derived nanovesicles loaded with miRNA.

2. The method for preparing autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration according to claim 1, characterized in that, The PBS resuspension of the nucleus pulposus cells is a PBS resuspension of 2.7 million nucleus pulposus cells.

3. The method for preparing autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration according to claim 1, characterized in that, The gradient apertures include 1.0 μm, 0.4 μm, and 0.2 μm.

4. The method for preparing autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration according to claim 1, characterized in that, The instrument used to prepare the nanovesicles is a liposome extruder.

5. The method for preparing autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration according to claim 1, characterized in that, The squeezing step includes: setting up the nanovesicle preparation instrument and a 1.0 μm polycarbonate membrane, adding PBS resuspension of nucleus pulposus cells containing miRNA mimics to the syringe, and pushing the pushers on both sides alternately 11 times. The cells are squeezed and loaded with miRNA mimics as they pass through the polycarbonate membrane. Then, the membranes are replaced with 0.4 μm and 0.2 μm polycarbonate membranes, and the cells are squeezed again following the above steps.

6. The method for preparing autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration according to claim 1, characterized in that, The centrifugation steps include: after centrifugation at 1.5g, 15min, and 4℃, the precipitate is discarded and the supernatant is retained; the supernatant is then centrifuged at 15g, 60min, and 4℃, and the supernatant is discarded.

7. The application of the autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration prepared by the method described in any one of claims 1-6 in the preparation of drugs for delaying intervertebral disc degeneration.

8. The application of the autologous cell-derived nanovesicles loaded with miRNA in intervertebral disc degeneration prepared by the method described in any one of claims 1-6 in the preparation of drugs for treating intervertebral disc degeneration.

9. The application according to claim 7 or 8, characterized in that: The dosage form of the drug is aerosol, tablet, capsule, pill, powder, solution, suspension, emulsion, granule, lipid preparation, transdermal preparation, lozenge, suppository, or lyophilized powder for injection.

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