| 李玲慧,龚 浩,陈 明,展嘉文,李凯明,朱立国,王尚全.改良胶原酶消化法分离培养人原代髓核细胞[J].,2020,(6):1011-1014 |
| 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 |
| 投稿时间：2019-10-23 修订日期：2019-11-18 |
| DOI：10.13241/j.cnki.pmb.2020.06.003 |
| 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: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） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| ABSTRACT 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 type Ⅱ collagen 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 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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