家兔髓核间充质干细胞的体外培养与鉴定

Research of in vitro cell culture and identification of nucleus pulposus mesenchymal stem cell in rabbit

(1)目的建立家兔腰椎髓核间充质干细胞(NPMSCs)体外培养模型并对其进行鉴定,为进一步研究椎间盘退变提供理论依据。(2)方法首先用酶消化法得到家兔原代髓核细胞,再将其加入间充质干细胞完全培养基中分离出NPMSCs并进行传代培养,观察NPMSCs的细胞学及生物学特性。最后进行细胞鉴定:运用流式细胞术检测家兔NPMSCs的细胞表面免疫表型CD105、CD90、CD34、CD45的表达情况;运用反转录-聚合酶链反应(RT-PCR)检测间充质干细胞基因SOX2、Nanog的表达。(3)结果家兔髓核组织来源细胞在原代培养4~6天后即可见部分细胞贴壁生长,形态为短梭形或多角形。传代后细胞增殖速度明显加快,到第三代可见细胞大多为长梭形,待细胞达到90%融合时,可见细胞呈漩涡状生长。通过流式细胞术检测所培养细胞的表面免疫分子标志:CD105、CD90阳性表达,CD34、CD45阴性表达;运用RT-PCR检测所培养细胞可表达干细胞基因SOX2、Nanog。(4)结论成功在体外培养出家兔NPMSCs,为研究椎间盘退变的发生发展机制打下基础。

Objective To establish the vitro culture model of the rabbit intervertebral disc nucleus pulposus mesenchymal stem cells（NPMSCs）and identfy it in order to provide a theoretical basis for further study of intervertebral disc degeneration.Methods Firstly,we used enzyme digestion method to obtain the original generation nucleus pulposus cells.Next,the NPMSCs was isolated from the complete medium of mesenchymal stem cells and subcultured.Then we observed cytology characteristics and biological characteristics of the NPMSCs.Finally,we did cell identification.The analysis of NP-derived MSCs surface-antigen CD105、CD90、CD34 and CD45 were done by flow cytometry.Additionally,the expression of classic and novel NP-derived MSCs marker genes SOX2 and Nanog were detected by real-time polymerase chain reaction（RT-PCR）.Results After 4~6 days of primary culture,the cells of the nucleus pulposus of rabbit’s nucleus pulposus could be seen to grow on the wall,and the form was short shuttle or polygon.After subculture,the cell proliferation was accelerated,and most of the cells were long spindle shaped in the third generation.When reached 90% fusion,the cells showed vortex shaped growth.The markers of cell surface antigens were identified by flow cytometry,the results showed that it was positive for CD105 and CD90,negative for CD34 and CD45 in the cells of experimental group.Additionally,the expression of classic and novel NP-derived MSCs marker genes SOX2 and Nanog were detected by real-time polymerase chain reaction（RTPCR）.Conclusion We have successfully cultured the rabbit intervertebral disc NPMSCs in vitro which lay the foundation for the study of the pathogenesis of disc degeneration.