慢病毒载体介导CGRP基因体外转染及对MSC生物学特性的影响

Lentiviral vector mediated CGRP gene in vitro transfection and its effects on biological properties of MSC

目的探讨慢病毒载体介导降钙素基因相关肽(CGRP)基因体外转染间充质干细胞(MSC)及其对MSC生物学特性的影响。方法大鼠MSC进行分离、培养及鉴定。CGRP重组慢病毒转染MSC后,采用荧光显微镜和流式细胞技术测定其转染率。实时定量PCR、免疫荧光细胞化学及ELISA法检测MSC中CGRP的表达。慢病毒转染后通过MTT、β-半乳糖苷酶染色和诱导分化评价MSC的增殖、衰老及分化能力。结果慢病毒载体介导CGRP基因转染MSC后48h可稳定表达,MOI=30时,转染率达80%以上。与MSC组和空载病毒转染(MSC-EGFP组)比较,CGRP修饰的MSC(MSC-CGRP组)中CGRP的mRNA和蛋白表达水平增高(均P<0.01)。MTT、β-半乳糖苷酶染色和诱导分化结果显示,病毒转染后对MSC增殖、衰老及向内皮分化基本没有影响。结论 MSC是一种理想的基因载体细胞,可作为CGRP基因转染的靶细胞用于基因治疗,为后续体内、体外实验奠定基础。

Objective To explore in vitro mesenchymal stem cell （MSC） transfection of lentiviral vector mediated eatcitonin gene-related peptide（CGRP） gene and its effects on biological properties of MSC. Methods MSC were isolated,cultured and identified. MSC were infected by lentivirus encoding recombinant enhanced green fluorescent protein （EGFP） gene and CGRP （Lv-EGFP-CGRP）. The transfection efficiency was determined by the inverted fluorescence microscope and flow cytometry. The expression levels of CGRP were detected in CGRP-modified MSC by using real-time PCR, immunoeytochemistry and enzyme-linked immunosorbent assay （ELISA）. The proliferation,aging and differentiation ability of MSC were evaluated by MTT,β-gatactosidase staining and inducing differentiation respectively. Results After 48 h of MSC transfection by Lv-EGFP-CGRP, EGFP/CGRP could be expressed stably. When multiplicity of infection （MOI） was 30,the transfection efficiency reached more than 800;. Compared with the MSC group and the MSC-EGFP group, the mRNA and protein expression levels of CGRP in CGRP-modified MSC（ MSC-CGRP group） were markedly increased（all P〈0.01）. The results of MTT,β-galactosidase staining and inducing differentiation assay demonstrated that the transfected CGRP basically had no effect on the proliferation,aging and endotheliocyte differerntiation of MSC. Conclusion MSC is a kind of ideal genetic vector cell,which can serve as the target cell of CGRP gene transduction for the application of gene therapy and lays the foundation for follow-up in vitro and vivo experiments.