摘要
目的构建含有人抗肌萎缩蛋白小基因的逆转录病毒载体并检测经逆转录病毒介导基因转移的假肥大型肌营养不良模型小鼠(mdx)骨髓间充质干细胞(MSCs)中目的基因的表达。方法构建含有抗肌萎缩蛋白小基因的逆转录病毒载体,利用脂质体介导的基因转移技术转染包装细胞PA317,制备含有目的基因的重组逆转录病毒并感染mdx鼠的MSCs,利用免疫荧光、RT-PCR法检测目的基因的表达。结果成功构建了抗肌萎缩蛋白小基因的逆转录病毒载体。含抗肌萎缩蛋白小基因的逆转录病毒感染mdx鼠MSCs后48h,RT-PCR产物经琼脂糖凝胶电泳出现319bp DNA片断;免疫荧光检测显示感染组mdx鼠MSCs中可见红色颗粒,深浅不一。结论采用逆转录病毒介导的方法可以将抗肌萎缩蛋白小基因转移至mdx鼠MSCs并表达。
Objective To construct the retroviral vector containing human micro-dystrophin gene and detect the expression of human micro-dys in mdx mice bone marrow-derived mesenchymal stem ceils （MSCs） after retrovirus infection. Methods Retroviral vector for micro-dystrophin gene was constructed and transferred into the packing cell PA317 mediated by LipofectamineTM 2000. The retroviral supematant containing the target genes were subsequently used to infect mdx mice MSCs. Micro-dystrophin expression was examined by methods of immunofluorescence staining and reverse transcriptase-polymerase chain reaction. Results Micro-dystrophin retroviral vector was successfully constructed and transferred into PA317 cells, and 48 h after infection with the recombinant retrovirus in mdx mice MSCs, 319 bp fragment could be detected by electrophoresis in the RT-PCR products. The red particles could be detected in some infected mdx mice MSCs with immunofluorescence staining. Conclusion mdx mice MSCs infected with retrovirus containing micro-dystrophin gene can express micro-dystrophin protein.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2006年第4期558-561,i0002,共5页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金(30370510)~~
关键词
抗肌萎缩蛋白小基因
骨髓间充质干细胞
基因转染
micro-dystrophin
bone marrow-derived mesenchymal stem cells
gene transfection
