摘要
目的 制备稳定高效表达血管生成素 1(Ang 1)的转基因工程骨髓基质干细胞。 方法 应用基因重组的方法构建Ang 1真核表达质粒 pEGFP/Ang 1,利用脂质体将 pEGFP/Ang 1转入骨髓基质干细胞 ,用流式细胞仪检测转染率 ,用荧光显微镜和免疫细胞化学方法检测蛋白质的表达。结果 质粒酶切电泳显示重组质粒构建成功 ,转染pEGFP/Ang 1质粒的骨髓基质干细胞在荧光显微镜下可见绿色荧光 ;流式细胞仪检测转染率约 5 0 % ;免疫细胞化学检测可见转基因骨髓基质干细胞表达Ang 1蛋白。结论 成功制备表达外源性基因Ang 1的骨髓基质干细胞 ,制备方法是可行的。
Objective To construct a kind of engineered cell in which angiopoietin-1 gene is stably expressed. Methods pBluescriptKS+/Ang-1 was amplified by RT-PCR, and ligated into the pEGFP-C1 vector. Then the recombined plasmid was transferred into mesenchymal stem cells (MSCs). The expression of EGFP and Ang-1 was detected by fluorescence microscopy, immunohistochemistry staining and flow cytometry. Results Restrictive digestion and gel electrophoresis revealed the successful construction of expression plasmid pEGFP/ Ang-1, which was mediated via lipofectamine2000 could delivered both EGFP and Ang-1 gene with high efficiency to MSCs. The ratio of transfection analyzed with flow cytometry was 50 %. The blight green fluorescence could be seen by fluorescence microscopy in pEGFP/Ang-1 -transfected MSC. Immunostaining with Ang-1 in engineered cells was strong positive.Conclusion The recombinant eukaryotic expression vector pEGFP/GDNF has been constructed and stably expressed efficiently in MSCs. The method was practical.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2004年第4期504-506,F003,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
