Zhang Qi, Qian Jixian, Wen Yanhua, Qiu Xiuchun, Ma Baoan, Fan Qingyu
Center of Orthopedic Surgery, Orthopedic Oncology Institute of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi'an Shaanxi, 710038, P R China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Jul;24(7):811-6.
The biological treatment of intervertebral disc degeneration becomes a research hotspot in recent years. It is necessary to find an effective approach to induce bone marrow mesenchymal stem cells (BMSCs) differentiate to disc cells which could make application of cell transplantation as a treatment of intervertebral disc degeneration. To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit BMSCs into nucleus pulposus-like cells.
The eukaryotic expression vector of pcDNA3.1IE-SOX9Flag was constructed. Rabbit BMSCs were isolated and cultured from one-month-old New Zealand white rabbits and were induced into osteogenetic cells in the osteogenesis supplement medium; and the cell surface markers were detected by flow cytometry. The cells at the 3rd passage were randomly divided into 3 groups: in transfected group, the cells were transfected with recombinant plasmid pcDNA3.1IE-SOX9Flag; in negative control group, the cells were transfected with plasmid pcDNA3.1; and in blank control group, the cells were treated with the media without recombinant plasmid. After selected by G418 for 7 days, the cells were harvested and RT-PCR was employed to assay SOX9 mRNA and collagen type II gene (Col2al) mRNA expressions in BMSCs. The expression of SOX9 protein was assayed by Western blot and collagen type II expression was also observed by immunohistochemical staining.
The SOX9 eukaryotic expression vector was constructed successfully. The BMSCs after 5 days of osteogenetic induction were positive for the alkaline phosphatase staining. What was more, CD44 expression was positive but CD34 and CD45 expressions were negative. The transfection efficiency was 34.32% +/- 1.75% at 72 hours after transfection. After 2 weeks of transfection, BMSCs turned to polygonal and elliptical. And the cell proliferation was gradually slow which was similar to the growth characteristic of nucleus pulposus cells. RT-PCR identification showed that SOX9 mRNA and Col2al mRNA expressions were positive in transfected group, and were negative in 2 control groups. Western blot detection showed that SOX9 protein expressed in transfected group but did not express in the control groups. At 2 weeks after transfection, the result of the immunohistochemical staining for collagen type II was positive in transfected group.
The recombinant plasmid pcDNA3.1IE-SOX9Flag can be successfully transfected into rabbit BMSCs, the transfected BMSCs can differentiate into nucleus pulposus-like cells, which lays a theoretical foundation for treatment of intervertebral disc degeneration with BMSCs transplantation.
椎间盘退变的生物治疗成为近年来的研究热点。寻找一种有效的方法诱导骨髓间充质干细胞（BMSCs）向椎间盘细胞分化，对于将细胞移植应用于椎间盘退变治疗具有重要意义。本研究旨在探讨重组质粒pcDNA3.1IE - SOX9Flag对兔BMSCs向髓核样细胞分化的影响。
构建pcDNA3.1IE - SOX9Flag真核表达载体。从1月龄新西兰白兔分离培养BMSCs，并在成骨诱导培养基中诱导其向成骨细胞分化，采用流式细胞术检测细胞表面标志物。将第3代细胞随机分为3组：转染组，用重组质粒pcDNA3.1IE - SOX9Flag转染细胞；阴性对照组，用质粒pcDNA3.1转染细胞；空白对照组，用不含重组质粒的培养基处理细胞。经G418筛选7天后，收集细胞，采用RT - PCR检测BMSCs中SOX9 mRNA和Ⅱ型胶原基因（Col2al）mRNA的表达。采用Western blot检测SOX9蛋白表达，免疫组织化学染色观察Ⅱ型胶原表达。
成功构建SOX9真核表达载体。成骨诱导5天后的BMSCs碱性磷酸酶染色呈阳性。此外，CD44表达阳性，CD34和CD45表达阴性。转染72小时后转染效率为34.32%±1.75%。转染2周后，BMSCs变为多边形和椭圆形，细胞增殖逐渐减慢，类似髓核细胞的生长特性。RT - PCR鉴定显示，转染组SOX9 mRNA和Col2al mRNA表达阳性，2个对照组均为阴性。Western blot检测显示，转染组有SOX9蛋白表达，对照组未表达。转染2周后，转染组Ⅱ型胶原免疫组织化学染色结果为阳性。
重组质粒pcDNA3.1IE - SOX9Flag可成功转染兔BMSCs，转染后的BMSCs可分化为髓核样细胞，为BMSCs移植治疗椎间盘退变奠定了理论基础。
Zotero 插件