摘要
目的:构建含增强绿色荧光蛋白(EGFP)及胰岛素融合基因的重组质粒,转染小鼠骨髓间充质干细胞(BMSCs),观察转染融合基因后BMSCs移植治疗糖尿病模型小鼠的治疗效果。方法:应用重叠延伸PCR技术合成insulin-IRES-EGFP和IRES-EGFP两段序列,分别克隆至逆转录病毒载体pMSCV中,经过包装细胞PT67的包装后转染小鼠BMSCs。荧光显微镜观察转染后EGFP的表达;RT-PCR法检测转染后胰岛素基因的表达。将转染了两种质粒的BMSCs培养后分别植入两组糖尿病模型小鼠体内,观察各组受体鼠血糖及体质量等指标的变化,并以正常对照组小鼠作对照。结果:成功构建重组质粒pM-SCV-insulin-IRES-EGFP、pMSCV-IRES-EGFP;倒置荧光显微镜下观察到转染后的BMSCs发出稳定的绿色荧光信号。转染了pMSCV-insulin-IRES-EGFP的BMSCs能稳定表达外源性胰岛素基因,转染后小鼠血糖明显低于转染pMSCV-IRES-EGFP组(P<0.05),但仍高于正常对照组(P<0.05);转染后35d体质量明显高于转染pMSCV-IRES-EGFP组(P<0.05)。结论:重构的insulin-EGFP载体能够起到良好的示踪作用,且EGFP的表达不影响胰岛素基因的表达;转染融合基因的小鼠BMSCs移植治疗糖尿病小鼠可以部分缓解小鼠糖尿病症状。
Objective： To construct a retroviral vector carrying both enhanced green fluorescent protein（EGFP） and human insulin gene for transfecting the bone marrow mesenchymal stem cells （BMSCs） ,and to observe the treatment effect of transfected BMSCs after transplanted into diabetes mice. Methods：Two gene segments of insulin-IRES-EGFP and IRESEGFP were obtained by overlap extension PCR technology, and then the 2 segments were cloned into retroviral vector （pMSCV）. The vectors were used to transfect BMSCs after packaged with PT67 cells. The expression of EGFP was observed by inverted fluorescence microscope and the expression of insulin gene was examined by RT-PCR in the infected BMSCs. The transfected BMSCs with 2 viruses were transplanted into diabetic mice separately. The blood glucose levels and body weights of mice were examined in 2 groups, and the results were compared with those of normal control group. Results： The recombinant retroviral vectors pMSCV-insutin-IRES-EGFP and pMSCV-IRES-EGFP were successfully constructed. The BMSCs infected by both vector stably gave out green fluorescence under microscope; the cells infected with pMSCV-insulin IRES-EGFP also stably expressed human insulin gene. The blood glucose level of the mice transplanted with BMSCs transfected with pMSCV-insulin-IRES-EGFP was significantly lower than that transplanted with BMSCs transfected with pMSCV IRES-EFGP （P〈0. 05）, but was still higher than that of the control group（P〈0.05）. Thirty-five days after transplantation, the body weight of the pMSCV-insulin-IRES-EGFP was significantly higher than that of the pMSCV-IRES-EGFP group（P〈0.05）. Conclusion： The insulin-EGFP recombinant vector can be traced easily, and the expression of EGFP does not affect the expression of insulin. Transplanting the BMSCs transfected with insulin fusion gene can partially relieve the symptoms of the diabetes.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2008年第12期1455-1459,共5页
Academic Journal of Second Military Medical University
基金
江苏省卫生厅资助课题( H200109)
南通市科技局资助课题(S2001)~~
关键词
骨髓间充质干细胞
增强绿色荧光蛋白
转染
糖尿病
胰岛素
bone marrow mesenchymal stem cells
enhanced green fluorescent protein
transfection
diabetes
insulin
