改良胶原酶消化法分离培养人原代髓核细胞  

In vitro Isolation and Culture of Human Degenerative Nucleus Pulposus Cells by Improved Enzymatic Digestions

作　　者：李玲慧[1,2] 龚浩 陈明 展嘉文[1] 李凯明[4] 朱立国 王尚全 LI Ling-hui;GONG Hao;CHEN Ming;ZHAN Jia-wen;LI Kai-ming;ZHU Li-guo;WANG Shang-quan(Department of Orthopaedics and Traumatology,Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing,100102,China;Beijing Key Laboratory of Bone-setting Technology of Traditional Chinese Medicine,Beijing,100102,China;Beijing Changping Hospital of Intergrated Chinese And Western Medicine,Beijing,102208,China;Department of Second Spinal Orthopedics,Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing,100102,China)

机构地区：[1]中国中医科学院望京医院骨伤综合科,北京100102 [2]中医正骨技术北京市重点实验室,北京100102 [3]北京市昌平区中西医结合医院,北京102208 [4]中国中医科学院望京医院脊柱二科,北京100102

出　　处：《现代生物医学进展》2020年第6期1011-1014,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81904230,81674005,81804120,81302992);中央本级重大增减支项目(2060302);中央级公益性科研院所基本科研业务费专项资金资助(ZZ10-015);北京市自然科学基金项目(7164313);中国博士后科学基金项目(2017M611125,2016M591364)。

摘　　要：目的:优化人原代髓核细胞的体外分离培养方法,为椎间盘退变的防治研究提供种子细胞。方法:无菌环境中摘取人椎间盘髓核组织,采用多次胶原酶消化法分离提取原代人髓核细胞,置于5%CO2培养箱中37℃恒温培养,倒置相差显微镜中观察细胞形态,采用MTT法绘制细胞生长曲线,甲苯胺蓝染色法检测髓核细胞内蛋白多糖的表达情况,细胞免疫荧光染色法检测Ⅱ型胶原蛋白表达情况。结果:本研究中获得的细胞形态不规则,呈梭形或多角形,原代细胞48 h内贴壁,培养第8天左右细胞融合度可达90%,第三代细胞12 h内即可贴壁,生长至融合90%约需5d。甲苯胺蓝染色及细胞免疫荧光染色均阳性,提示所得细胞具有分泌蛋白多糖及Ⅱ型胶原蛋白的功能。结论:改良胶原酶消化法可获得大量纯净的人髓核细胞,提高培养效率,原代及传代细胞具备类软骨细胞表型,且活性及功能均较为稳定,可作为椎间盘组织工程研究的种子细胞。Objective:To optimization the method of isolating and culturing human degenerative nucleus pulposus cells in vitro,and to provide seed cells for the prevention and cure investigation of intervertebral disc degeneration.Methods:Nucleus pulposus tissue was collected in a sterile environment.Multiple enzymatic digestions were adopted to extract primary human nucleus pulposus cells.The cells were cultured in thermostat incubator with 5%CO2 under the condition of 37℃.Cell morphous was observed under inverted phase contrast microscope.The cell growth curve was plotted by MTT assay.The expression of proteoglycan and typeⅡcollagen were detected by toluidine blue staining and cellular immunofluorescence staining respectively.Results:The cells had spindle or polygonal shapes.The primary cells had an average adherence time of 48 hours and took nearly 8 days to reach 90%confluence.Cells at passage 3 had an average adherence time of 12 hours and took nearly 5 days to reach 90%confluence.The results of toluidine blue staining and cellular immunofluorescence staining showed that the cultured cells secreted proteoglycan and typeⅡcollagen.Conclusion:Multiple enzymatic digestions of nucleus pulposus tissue could release a large amount of pure human nucleus pulposus cells.The culture efficiency was improved.The primary and passage cells could maintain stable chondrocyte-like phenotype and could be used as seed cells for intervertebal disc tissue engineering.

关 键 词：髓核细胞 椎间盘退变 Ⅱ型胶原酶 

分 类 号：R-33[医药卫生] R681.53