摘要
目的 探讨Ad-hTGF-B1转染骨髓间充质干细胞（BMSCs）并诱导其向成软骨细胞分化的可行性及其相关转录凶子TAZmRNA表达的变化．方法全骨髓法获取和培养大鼠骨髓间充质干细胞，取第三代细胞接种于细胞培养板。实验分成三组：Ad－hTGF．B1转染组、Ad－EGFP转染组、空白对照组，前两组分别使用含Ad－hTGF－B1与Ad－EGFP的无札清培养基，空白对照组使用不做任何处理的完全培养基。7d后，运用实时荧光定量PCR与Westernblot检测TGF－βl的表达，利用免疫组织化学方法与Westernblot检测Collagen1I的表达．采用实时荧光定量PCR检测TAZmRNA的表达。结果转染后7d，实时荧光定量PCR结果显示TGF—β1表达量的平均相对比值分别为：Ad．hTGF－β1转染组O．863、Ad—EGFP转染组0．183、空白对照组0．180；TAZ表达量的平均相对比值分别为：Ad－hTGF－β1转染组0．810、Ad—EGFP转染组O．416、空白对照组0．366。TGF－β1和TAZ的表达差异有统计学意义（P〈0．05）。Westernblot和免疫组化发现Ad－hTGF－β1转染组的细胞内Collagen11表达呈强阳性，而Ad－EGFP转染组及空白对照组细胞内的CollagenII表达很弱。结论Ad－hTGF－β1能够成功且稳定的诱导骨髓间充质干细胞向软骨细胞表型分化．同时．在骨髓间充质干细胞向软骨细胞表型分化过程中，转录增强因子TAZ的mRNA水平随之上调，认为TGF－β1可能通过TAZ的作用促进骨髓间充质干细胞向软骨细胞表型分化。
Objective To investigate the feasibility of Ad-hTGF-β1 transfected bone marrow mesenchymal stem celI（BMSCs） into chondrocytes differentiation and the change of TAZ mRNA. Methods Rats BMSCs were obtained and cultured by whole bone marrow method, and then the third-generation cells were seeded into cell culture plate, and divided into three groups：Ad-hTGF-β1 transfected group,Ad-EGFP transreeled group and the control group. The control group was added in common medium without any treatment while the other two groups were respectively added in serum-free medium containing Ad-hTGF-β1 or that con- taining Ad-EGFP. Seven days later, real-time fluorescent quantitation PCR and Western blot were employed for detecting the expression of TGF-β1 ,while immunohistochemical and Western blot for the expression of collagen 11 , and real-lime fluorescent quantitation PCR for the expression of TAZ mRNA. Results Seven days after the transfection, real-time fluorescent quantitation PCR revealed that the average relative expression of TGF-[31 was：Ad-hTGF-β1 group 0.863, Ad-EGFP group 0.183, and the control group 0.180; The average relative expression of TAZ was：Ad-hTGF-β1 group 0.810, Ad-EGFP group 0.416,and the control group 0.366. The expression difference of TGF-β1 and TAZ were statistically significant （P 〈 0.05）. Western blot and immunobistochemical proved strong collagen II expression in Ad-hTGF-I31 group while it was detected a little in the other two groups. Conclusion BMSCs could be successfully and stably induced into chondrocytes differ- entiation by Ad-hTGF-β1. Meanwhile, the mRNA of TAZ is up regulate during the differentiation,so it is suppose that TGF-β1 improve BMSCs into chondrocytes differentiation by TAZ.
出处
《中华显微外科杂志》
CSCD
北大核心
2010年第6期465-468,534,共5页
Chinese Journal of Microsurgery
基金
国家自然科学基金资助项目（30770574）
湖北省卫生厅科技基金资助项目（JX3821）
