摘要
目的:建立能够稳定表达BMP-2及bFGF的骨髓基质干细胞,优化骨组织工程种子细胞。方法:通过RT-PCR方法获取全长BMP-2及bFGF基因,克隆入真核表达载体pcDNA3.0,鉴定正确后,经脂质体介导导入分离培养的大鼠骨髓基质干细胞,G418筛选稳定表达株。利用RT-PCR、Westernblot、ELISA、免疫组织化学染色方法分析转染细胞外源基因表达情况。结果:成功构建了全长BMP-2及bFGF的真核表达载体,转染大鼠骨髓基质干细胞后经G418筛选获得稳定表达株。RT-PCR结果显示稳定转染细胞中具有大量目的基因mRNA转录,ELISA检测培养上清中目的蛋白表达阳性,Westernblot、免疫组化结果显示胞质中有目的蛋白的表达。结论:获得了可以稳定表达BMP-2及bFGF的骨髓基质干细胞,有利于组织工程骨缺损修复中的应用。
AIM： To establish modified bone marrow stromal cells（BMSCs） which can express BMP-2 and bFGF stably. METHODS： BMP-2 and bFGF gene were amplified by RT-PCR, and then cloned into the expression vector pcDNA3.0. After being confirmed by DNA sequencing, pcD- NA3.0-BMP-2 and pcDNA3.0-bFGF were co-transfected into rat BMSCs with Lipofectamine 2000 reagent. The expression of BMP-2 and bFGF gene in rat BMSCs was detected by RT-PCR, Westem blot, immunohistochemical staining and ELISA. RESULTS： BMP-2 and bFGF gene were cloned, and their sequences were identical with those in GenBank. The expression plasmids, pcDNA3.0-BMP-2 and pcDNA3.0- bFGF, were constructed and co-transfected into rMSCs successfully. RT-PCR showed the mass transcriptfon of BMP-2 and bFGF mRNA in transfected BMSCs. Western blot, immunohistochemical staining and ELISA confirmed the expression of BMP-2 and bFGF genes in transfected cells and in the supernatant. CONCLUSION： We have con- structed the optimal rat BMSCs which can be used in bone tissue engineering.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期133-136,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家高技术研究发展计划(863)资助(No.2002AA326080)
