携带人胰岛素基因慢病毒载体转染人脐带间充质干细胞的实验研究

RECOMBINANT HUMAN INSULIN GENE LENTIVIRUS TRANSFECTING HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS IN VITRO

目的构建共表达增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因和人胰岛素(insulin)基因的慢病毒载体,探讨其对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)的转染情况,为今后组织工程脂肪构建与体内回植研究奠定基础。方法采用DNA重组技术,将insulin基因克隆至带EGFP的慢病毒表达载体pLenti6.3-内部核糖体进入位点序列(internal ribosome entry site,IRES)-EGFP中,筛选阳性克隆,并用脂质体介导法将慢病毒包装系统和带目的基因的质粒pLenti6.3-insulin-IRES-EGFP共同转染到293T细胞内包装病毒,荧光倒置相差显微镜观察包装细胞报告基因的表达情况。收集病毒上清,纯化浓缩,测定重组病毒的滴度。取足月儿脐带以组织块培养法分离培养hUCMSCs,以不同感染复数(multiple of infection,MOI,分别为0、1、3、5、7、10、15、20)的重组慢病毒感染hUCMSCs,通过报告基因绿色荧光蛋白的表达筛选最适MOI;以最适MOI重组慢病毒感染hUCMSCs,应用实时荧光定量PCR及Western blot法分别检测细胞中insulin基因及insulin蛋白水平的表达情况。结果成功构建了共表达insulin基因和EGFP基因的重组慢病毒载体pLenti6.3-insulin-IRES-EGFP,并对其成功包装、纯化及浓缩,病毒滴度为1.3×108TU/mL。不同MOI的重组慢病毒感染hUCMSCs后,通过绿色荧光蛋白表达的阳性细胞数筛选其最适MOI为10,此时对细胞的转染效率可达到90%。实时荧光定量PCR检测示转染组insulin mRNA表达阳性,未转染组为阴性;Western blot检测示转染组细胞内及上清液insulin蛋白表达阳性,未转染组均为阴性。结论构建的携带insulin基因的重组慢病毒载体pLenti6.3-insulin-IRES-EGFP可有效转染hUCMSCs,表达insulin蛋白。

Objective To construct the lentiviral vector to co-express enhanced green fluorescent protein（EGFP） gene and human insulin（insulin） gene,and to explore the condition to transfect human umbilical cord mesenchymal stem cells（hUCMSCs） so as to lay a foundation for tissue engineered adipose reconstruction and transplantation in vivo in future.Methods The insulin gene was cloned to lentiviral expression vector with EGFP [pLenti6.3-internal ribosome entry site（IRES）-EGFP] by recombinant DNA technology,the positive clones were screened,and lentiviral packaged systems and target gene plasmid were co-transfected to package virus in 293T cells by lipofectin.The reporter gene expression was observed by fluorescent inverted phase contrast microscope,virus supernatant was collected,purificated and concentrated,and the titer of recombinant viruses was determinated.hUCMSCs from umbilical cord tissue of mature neonates were isolated and cultured by different multiple ofinfection（MOI,0,1,3,5,7,10,15,and 20）.By recombinant lentiviral infected hUCMSCs with reporter gene green fluorescent protein expression,the best MOI was screened;recombinant lentiviral infected hUCMSCs at the best MOI,then real-time PCR and Western blot methods were applied to detect insulin gene and insulin protein expression levels in cells.Results The recombinant lentiviral vector of co-expressing insulin gene and EGFP gene（pLenti6.3-insulin-IRESEGFP） was successfully constructed.Virus could be packaged,purificated and concentrated successfully.The virus titer was 1.3 × 108 TU/mL.The best MOI was 10 and the transfer efficiency was up to 90% in the same time.Real-time PCR results showed that insulin gene expression of transfected group was positive and non-transfected group was negative;Western blot detection confirmed that insulin protein expression of transfected group was positive in cells and supernatant,but that of non-transfected group was both negative.Conclusion Lentiviral vector pLenti6.3-insulin-IRES-EGFP carrying recombinant insulin gene could effectively transfect hUCMSCs and express insulin protein.