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| JIANG Changqing,LAN Weiren,LI Haiyin.Effects of exosome derived from rat bone marrow mesenchymal stem cells on degenerative nucleus pulposus cells[J].Chinese Journal of Spine and Spinal Cord,2019,(2):147-155. |
| Effects of exosome derived from rat bone marrow mesenchymal stem cells on degenerative nucleus pulposus cells |
| Received:September 08, 2018 Revised:November 21, 2018 |
| English Keywords:Nucleus pulposus cells Bone marrow mesenchymal stem cells Exosome Coculture SD rats |
| Fund:国家自然科学基金资助项目（编号：81572208） |
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| English Abstract: |
| 【Abstract】 Objectives: To explore the effect of exosome derived from rat bone marrow mesenchymal stem cells(BMSCs) on degenerative nucleus pulposus cells(NPCs). Methods: The adherent cells were extracted from bone marrow of SD rats by whole bone marrow method. After BMSCs were successfully identified by osteoblast differentiation and flow cytometry, the supernatant of cultured cells was collected. The supernatant was centrifuged by differential centrifugation, and the expressions of CD63, CD81, TSG101, Calnexin protein in the precipitate were determined by Western-blot(WB) method, and whether the precipitate exosome or not was observed by transmission electron microscope. After extracting NPCs from SD rats tail, P6 NPCs were identified whether it was more degenerative than P2 NPCs by senescence β-galactosidase staining. The uptake of BMSCs exosome was observed by P6 NPCs under laser confocal microscope. P6 NPCs were taken as the control group, BMSCs and P6 NPCs were as the co-culture group, and P6 NPCs which were induced by BMSCs exosome were taken as the experimental group. The proteoglycan(ACAN), type Ⅱ collagen(COL2), sex-determining region Y box 9(SOX-9), tissue inhibitor of metalloproteinase 1(TIMP1) and the relative expression of matrix metalloproteinase-1(MMP1) gene mRNA of NPCs in the three groups were detected by RT-PCR method. Relative expressions of ACAN, COL2, SOX-9, TIMP1, MMP1 gene corresponding protein were detected by WB. Results: The adherent cells which were extracted by whole bone marrow method were BMSCs and they were identified by osteogenesis and adipogenic cartilage differentiation method and flow cytometry method. The precipitates extracted from BMSCs supernatant by differential centrifugation were circular or elliptical in diameter of 30-100nm, which were coincided with the classical exosome size recorded in literature. CD63, CD81, TSG101 were highly expressed and Calnexin was not expressed in the supernatant. Identified by cell senescence β-galactosidase staining, P6 NPCs were more degenerative than P2 NPCs, and the exosome could be absorbed by P6 NPCs observed under the confocal laser microscope. The relative expressions of mRNA of ACAN, COL2, SOX-9, TIMP1 gene were significantly higher in the co-culture group than those in the control group on the 3rd day, 7th day, 10th day and 14th day(P<0.05), and those in the experimental group were significantly higher than those in the co-culture group(P<0.05). In the co-culture group and the experimental group, those on the 7th day were significantly higher than those on the 3rd day(P<0.05), and those on the 10th day were significantly higher than those on the 7th day(P<0.05), and those on the 14th day were significantly higher than those on the 10th day(P<0.05). The relative expression of mRNA of MMP1 gene was significantly lower in the co-culture group than that in the control group on the 3rd day, 7th day, 10th day and 14th day(P<0.05), and that in the experimental group was significantly lower than that in the co-culture group(P<0.05). In the co-culture group and experimental group, there was a significant decrease in the 7th day compared with the 3rd day(P<0.05), and decrease also showed in the 10th day in comparison with the 7th day(P<0.05), and the 14th day in comparison with the 10th day(P<0.05). ACAN, COL2, SOX-9, TIMP1, MMP1 gene corresponding protein showed the same trend. Conclusions: In vitro, rat BMSCs can secrete exosome and the exosome can be taken up by degenerative NPCs. Exosome can improve the expression of degenerative NPCs marker gene and gene corresponding protein. It can provide a new idea for the treatment of degenerative diseases of nucleus pulposus cells. |
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