脂质体介导外源性基因在骨髓基质细胞表达  

Expression of exogenous gene in bone marrow stromal cells by liposome

作　　者：孙吉平[1] 贾延劼[1] 罗芳[1] 宋建辉[1] 杨于嘉[1] 

机构地区：[1]中南大学湘雅医院儿科,长沙410008

出　　处：《中南大学学报（医学版）》2005年第2期140-144,共5页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(No. 30200128)

摘　　要：目的:用阳离子脂质体介导真核表达载体pEGFP PDX 1转染大鼠骨髓基质细胞,并对其转染条件进行优化以获得较高效率。方法:构建的重组载体鉴定后,以脂质体介导其转染骨髓基质细胞,改变DNA,脂质体的量,在荧光显微镜下观察荧光并计算转染效率;转染后48h进行细胞免疫组化染色检测目的基因的表达情况。结果:限制性酶切分析证实重组后的载体成功载入PDX 1基因;克隆的目的片断经序列测定与GenBank公布的序列一致;质粒∶脂质体为1∶1或1∶2的转染效率最佳;细胞免疫化学染色检测证实转染后骨髓基质细胞有PDX 1基因表达。结论:成功构建了含有PDX 1基因的真核表达载体;通过优化转染条件提高了pEGFP PDX 1转染大鼠骨髓基质细胞的效率,为成为组织工程的种子细胞提供了实验依据。Objective To transform eukaryotic expression vector pEGFP-PDX-1 into marrow stromal cells by liposome and to optimize the conditions of transformation. Methods The recombinant vector was identified by enzyme digestion analysis and sequencing. The recombinant plasmid was transformed into bone marrow stromal cells and it changed the quantity of DNA or liposome.The expression of PDX-1 gene in the transformed cells was detected by immunocytochemical staining. Results Enzyme digestion analysis and sequencing showed that the interesting gene was integreted into the recombinant vector. We obtained satisfactory efficiency of transfection when the ratio of DNA and liposome was 1∶1 or 1∶2. The PDX-1 in the transformed cells was expressed by immunocytochemical staining. Conclusion The eukaryotic expression vector pEGFP-PDX-1 was constructed for the first time in China. We have enhanced the efficiency of transfection by optimizing the transformation conditions. It is possible to use the bone marrow stromal cells as seed cells in tissue-engineering.

关 键 词：外源性基因 细胞表达 大鼠骨髓基质细胞 阳离子脂质体介导 真核表达载体 限制性酶切分析 显微镜下观察 细胞免疫组化 细胞免疫化学 转染效率 染色检测 PDX-1 重组载体 表达情况 目的基因 序列测定 基因表达 种子细胞 组织工程 

分 类 号：R346[医药卫生—基础医学]