Ad-EGFP修饰的MSCs在糖尿病足溃疡大鼠体内的示踪及疗效观察

Study of diabetic foot ulcer transplanted with bone marrow mesenchymal stem cells transfected with Ad-EGFP in rats

目的:观察自尾静脉移植Ad-EGPF修饰的大鼠骨髓间充质干细胞(BMSCs)在糖尿病足溃疡模型大鼠胰腺、肾脏及溃疡局部的示踪情况,并观察移植后大鼠血糖、体重及溃疡面积的改变情况。方法:贴壁法体外分离培养iW st ar大鼠BMSCs,传代培养后用携带增强型绿色荧光蛋白基因的腺病毒(Ad-EGFP)以感染复数(MOI=150)体外转染。60只iW st ar大鼠,雌雄各半,随机分为3组;糖尿病治疗组(糖尿病足溃疡大鼠细胞移植治疗组)、糖尿病对照组(糖尿病足溃疡大鼠PBS对照组)使用链脲菌素(STZ)诱导糖尿病模型后,连同正常对照组(正常足溃疡大鼠PBS对照组)制作足部溃疡模型。造模成功后,糖尿病治疗组经尾静脉注射转染Ad-EGFP 72h后的BMSCs悬液0.5 ml(细胞数5×106个),糖尿病对照组及正常对照组注射等量PBS溶液。分别于细胞移植后7天、14天、21天测量体重、空腹血糖水平、足部溃疡面积;并于细胞移植后14天,观察BMSCs在胰腺、肾脏及足溃疡局部的分布情况。结果:移植后14天可在糖尿病大鼠胰腺、肾脏及皮肤溃疡面观察到Ad-EGFP标记的BMSCs.糖尿病治疗组大鼠体重明显恢复、血糖显著降低、足溃疡愈合加速,与糖尿病对照组相比,差异均具有统计学意义(P<0.05)。结论:BMSCs具有归巢特性,并可能对糖尿病及足溃疡有一定的治疗效果。

Objective To investigate the distribution of rat bone marrow mesenchymal stem cells（BMSCs） transfected with Ad-EGFP in kidney and pancreatic tissues,and the variation of blood glucose,weight and ulcer area after cell transplantation,in model rats of diabetic foot ulcer.Methods MSCs isolated from Wistar rats were cultured in vitro and were then transfected with green fluorescent protein（Ad-EGFP） at the multiplicities of infection（MOI） of being 150.The BMSCs were obtained for transplantation.Sixty Wistar rats were randomly assigned to 3 groups： diabetic treatment group（diabetic foot ulcer rat treated with the transfected BMSCs）,diabetic control group（diabetic foot ulcer rat treated with PBS） and normal control group（normal ulcer rat treated with PBS）.The two groups of diabetic rat model were achieved by intraperitoneally injection of streptozotocin.Foot ulcer for the three groups was manually scratched.For rats in diabetic treatment group,0.5 ml BMSCs（cell count 5×106） obtained 72 hours after transfected with Ad-EGFP were injected via tail vein,while these in two control groups were injected with PBS of same volume.The variation of blood glucose,weight and ulcer area were observed on the 7th,14th and 21th after cell transplantation,and the distribution of BMSCs in kidney and pancreatic tissues in rats on diabetic treatment group were examined.Results In diabetic treatment group,the BMSCs were detected at pancreas,kidney and foot ulcer after 14th day of transplantation.As compared to control group,therapy using the BMSCs decreased blood glucose level,improved symptom of magersucht and accelerated the healing of ulcer.Conclusion The results indicated BMSCs had the capability of nesting and may have beneficial effects in the management of diabetics and associated foot ulcer.