摘要
目的观察骨形成蛋白（BMP）-2、转化生长因子（TGF）-β1双基因真核表达载体在骨髓基质细胞中的共表达。方法以pGEM—T—BMP-2及pGEM—T—TGF—β1为模板，分别用引入新的酶切位点的引物，聚合酶链反应（PCR）扩增出1188bp长度的BMP-2和1173bp长度的TGF-β1两个目的基因片段；依次将其定向克隆入真核双基因表达载体pIRES；酶切、分析及序列测定重组子；然后，用脂质体包裹转染骨髓基质细胞；荧光定量逆转录（RT）-PCR方法检测BMP-2及TGF-β1基因的共表达情况。结果双酶切可见1188bp的BMP-2条带及核酸序列测定证实重组质粒plRES—BMP-2-TGF—β1构建正确，并在骨髓基质细胞中能同时高效表达BMP-2和TGF—β1 mRNA。结论BMP-2和TGF—β1双基因真核载体能够在骨髓基质细胞中实现BMP-2和TGF-β1基因共表达。
Objective To investigate co-expression of BMP-2 and TGF-β1 genes of the bicistronic enkaryotic expression plasmid pIRES-BMP-2-TGF-β1 in rabbit marrow stromal cells （MSCs）. Methods Using PCR technique, the DNA fragments containing two new enzyme digestion sites of BMP-2 （ 1188 bp） and TGF-151 （ 1173 bp） genes were obtained from pGEM-T-BMP-2 and pGEM-T-TGF-β1 plasmids, then inserted into bicistronic eukaryotic expression plasmid pIRES. The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing analysis. MSCs were transfected with this bicistronic eukaryotic expression plasmid using lipofecarmin reagent. The expression of BMP-2 and TGF-β1 were detected by RT-PCR. Results The directions and sequences of BMP-2 （ 1188 bp） and TGF-β1 （1173 bp） genes in plasmid pIRES-BMP-2-TGF-β1 were correct and BMP-2 and TGF-β1 mRNAs could be detected in MSCs. Conclusion BMP-2 and TGF-β1 genes of the bicistronic cukaryotic expression plasmid pIRES-BMP-2-TGF-β1 can be co-expressed successfully in MSCs,which can be used for further investigation of co-gene therapy of articular cartilage injury.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第1期98-101,共4页
Chinese Journal of Experimental Surgery
基金
基金项目：河南省医学高新技术发展扶持项目（2006027）
