摘要
目的椎间盘退行性变的生物学治疗方法是骨科学研究热点,寻找一种有效诱导BMSCs向椎间盘细胞分化的方法成为应用细胞移植治疗椎间盘退变的必要前提。探讨重组质粒pcDNA3.1IE-SOX9 Flag体外转染诱导兔BMSCs向类髓核细胞分化的影响。方法构建真核表达载体pcDNA3.1IE-SOX9 Flag。取1月龄新西兰大白兔骨髓分离培养BMSCs,体外诱导BMSCs向成骨细胞分化并鉴定;流式细胞仪检测细胞表面标记。将第3代BMSCs随机分为转染组、阴性对照组和空白对照组。转染组转染pcDNA3.1IE-SOX9Flag重组质粒,阴性对照组转染质粒pcDNA3.1,空白对照组不转染任何质粒。将构建好的重组质粒pcDNA3.1IE-SOX9Flag转染第3代BMSCs,72h后加入浓度为500mg/L的G418筛选7d后,改为浓度200mg/L的G418维持筛选压力并培养转染细胞。取各组BMSCs采用RT-PCR检测SOX9和Ⅱ型胶原基因(Col2al)的mRNA表达,Western blot检测SOX9蛋白的表达,免疫组织化学染色观察Ⅱ型胶原的表达。结果 BMSCs成骨诱导7d后ALP染色呈阳性;CD44表达阳性,CD34和CD45表达阴性。成功构建了真核表达载体pcDNA3.1IE-SOX9Flag。将重组质粒转染兔BMSCs,72h后转染效率为34.32%±1.75%;转染2周后BMSCs形态改变,呈多角形或椭圆形,细胞体积增大,增殖速度减缓,与椎间盘髓核细胞生长特点十分接近。RT-PCR鉴定发现转染组细胞SOX9 mRNA和Col2al mRNA表达阳性,对照组均为阴性。Westernblot检测发现转染组SOX9基因蛋白表达阳性,对照组未见表达。转染组BMSCs的Ⅱ型胶原免疫组织化学染色呈阳性。结论 SOX9基因真核表达载体成功转染兔BMSCs,被转染的干细胞向类髓核细胞分化,为进一步研究应用BMSCs移植治疗椎间盘退行性变奠定了理论基础。
Objective The biological treatment ofintervertebral 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 ofintervertebral disc degeneration.To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit BMSCs into nucleus pulposus-like cells.Methods 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 ？ ow 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.Results 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.Conclusion 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 ofintervertebral disc degeneration with BMSCs transplantation.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2010年第7期811-816,共6页
Chinese Journal of Reparative and Reconstructive Surgery
关键词
BMSCS
SOX9基因
转染
诱导分化
髓核细胞
椎间盘退行性变
Bone marrow mesenchymal stem cells SOX9 gene Transfection Induction and differentiation Nucleus pulposus cells Intervertebral disc degeneration
