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].现代生物医学进展英文版,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 |
Received:October 23, 2019 Revised:November 18, 2019 |
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) |
Author Name | Affiliation | LI Ling-hui | 1 Department of Orthopaedics and Traumatology, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing, 100102, China 2 Beijing Key Laboratory of Bone-setting Technology of Traditional Chinese Medicine, Beijing, 100102, China | GONG Hao | Beijing Changping Hospital of Intergrated Chinese And Western Medicine, Beijing, 102208, China | CHEN Ming | Department of Orthopaedics and Traumatology, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing, 100102, China | ZHAN Jia-wen | Department of Orthopaedics and Traumatology, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing, 100102, China | LI Kai-ming | Department of Second Spinal Orthopedics, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing, 100102, China | ZHU Li-guo | Beijing Key Laboratory of Bone-setting Technology of Traditional Chinese Medicine, Beijing, 100102, China Department of Second Spinal Orthopedics, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing, 100102, China | WANG Shang-quan | Department of Orthopaedics and Traumatology, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing, 100102, China |
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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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