人端粒酶逆转录酶基因修饰内皮祖细胞移植对大鼠肢体缺血的实验研究

Experimental Study on the Effect of Human Telomerase Reverse Transcriptase Gene Modified Endothelial Progenitor Cells Transplantation on Limb Ischemia in Rats

目的:探讨人端粒酶反转录酶(hTERT)修饰的内皮祖细胞(EPCs)移植对SD大鼠缺血肢体治疗机制和规律。方法:EPCs原代培养及鉴定后,脂质体法将PLXSN-hTERT稳定转染EPCs,Western blot鉴定外源hTERT基因在内皮祖细胞中的表达。64只雄性SD大鼠随机单位组设计分为4组:缺血对照组(A组)、hTERT治疗组(B组)、EPCs治疗组(C组)、转染hTERT基因的EPCs治疗组(D组),每组16只。结扎大鼠左股动脉及分支,构建后肢缺血模型。于内收肌和腓肠肌共注射5点,每点注射60μL,A组注射300μL培养液,B组注射DNA-脂质体复合物,C组注射4×10~6个EPCs,D组注射4×10~6个含hTERT基因的EPCs。于移植后4周行动脉造影,采用RT-PCR检测hTERT基因的体内表达,评价血管新生情况;荧光显微镜观察PKH-26标记的EPCs存活情况。结果:64只大鼠均造模成功。Western blot结果显示转染人hTERT基因的人内皮祖细胞体外能表达hTERT。移植后4周动脉造影显示,A组大鼠股动脉及其分支均未显影,B组和C组可见中等量新生血管,两组效果相似,C组可见有大量新生血管及丰富的侧枝循环建立。B组和C组均较A组新生血管数量增加,D组可进一步增强疗效PKH-26阳性细胞数:ABCD四组分别为[(0±0.00),(0±0.00),(27.46±5.48),(54.37±8.34)]个/HP;P<0.05。RT-PCR检测hTERT基因的体内表达,D组和B组均有扩增,D组hTERT mRNA相对含量高于B组。D组和B组hTERT mRNA相对含量分别为(0.89±0.06,0.29±0.04;P<0.05)。结论:通过人端粒酶反转录酶(hTERT)修饰的内皮祖细胞(EPCs)移植治疗大鼠肢体缺血,可以让hTERT基因稳定有效持续的表达,其效果优于直接基因注射。

Objective To investigate the mechanism and regulation of hTERT modified endothelial progenitor cells （EPCs） transplantation on ischemic limb of SD rats. Methods EPCs were cultured and identified after Lipofectamine stably transfected PLXSN-hTERT EPCs expression by Western blot identification of exogenous human hTERT gene in endothelial progenitor cells. Sixty-four male SD rats were randomly divided into 4 groups： A, B, C and D. Each group had a total of 16 rats. After ligation of the femoral artery and branches of the left hind limbs, the hind limb ischemia model was constructed, the adductor and gastrocnemius muscle were injected 5 points, each point injection 60 L. A group rats were injected with 300 L medium, B group rats were injected with 5 g of PLXSN-hTERT gene DNA- liposome complexes, C group rats were injected with 4×10^6 EPCs, and D group rats were injected with 4 × 10^6 hTERT gene EPCs. RT-PCR angiography was performed to detect the expression of hTERT gene and neovascularization in 4 weeks after transplantation, and the survival of PKH-26 labeled EPCs was evaluated by fluorescence microscope. Results All of the 64 rats were successfully constructed, and the results of Western blot showed that human endothelial progenitor cells transfected with human hTERT gene could express hTERT in vitro 4 weeks after transplantation and angiography. A group rat femoral artery and its branches were not visualized, B group and C group showed moderate neovascularization （effects of the two groups were similar）, D group can further enhance the effect. The number of PKH-26 positive cells were： [A,B,C and D four groups （0±0.00）, （0±0.00）, （27.46±5.48）, （54.37±8.34）/HP, P 〈 0.05]. In vivo expression of hTERT gene in RT-PCR, D group and B group were transfected with hTERT gene amplification, hTERT mRNA relative content of D group was higher in B group （0.89 ± 0.06, 0.29± 0.04, P 〈 0.05）. Conclusion Using Htert modified EPCs transplantation to treat limb ischemia in rats, can make hTERT gene stable and effective expression, and its effect is better than the direct gene injection.