文章摘要
改良胶原酶消化法分离培养人原代髓核细胞<sub><sup>△</sup></sub>
In vitro isolation and culture of human degenerative nucleus pulposus cells by improved enzymatic digestions
投稿时间:2019-10-23  修订日期:2019-10-23
DOI:
中文关键词: 髓核细胞  椎间盘退变  Ⅱ型胶原酶
英文关键词: nucleus pulposus cells  intervertebral disc degeneration  type Ⅱ collagenase
基金项目:
作者单位E-mail
李玲慧 中国中医科学院望京医院骨伤综合科 270641352@qq.com 
杨少锋 湖南中医药大学第一附属医院  
陈明 中国中医科学院望京医院骨伤综合科  
展嘉文 中国中医科学院望京医院骨伤综合科  
李凯明 中国中医科学院望京医院脊柱二科  
朱立国 中医正骨技术北京市重点实验室 likaiming2017568@163.com 
王尚全 中国中医科学院望京医院骨伤综合科  
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中文摘要:
      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48h内贴壁,培养第8天左右细胞融合度可达90%,第三代细胞12h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。
英文摘要:
      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 proteoglycanSand type Ⅱ collagen were detected by toluidineSblueSstaining 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 proteoglycanSand type Ⅱ collagen. 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.
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