慢病毒介导hVEGF-165基因转染骨髓间充质干细胞治疗大鼠后肢缺血实验研究

发布时间：2018-08-24 17:57

【摘要】：目的：检测分析携人血管内皮生长因子-165基因骨髓间充质干细胞对大鼠缺血后肢的治疗效果,为下肢缺血的临床治疗提供新思路。方法：本实验设立三个实验组,分别为转染hVEGF-165基因BMSC组、BMSC组、空白对照组,每组有效样本8例,共48条肢体。采用结扎SD大鼠腹主动脉分叉处上方建立缺血模型,并通过术后观察、DSA血管造影、测股动脉血流速度及阻力检测鉴定；通过慢病毒介导hVEGF-165基因转染大鼠骨髓间充质干细胞,GFP荧光蛋白标记,荧光显微镜下检测hVEGF-165基因转染率；再采用大鼠后肢肌肉多点注射方法将1X106/ml浓度的干细胞移植到缺血的肢体内,一个月后通过观察、DSA血管造影、测股动脉血流速度及血流阻力检测疗效。结果：1、荧光显微镜下检测慢病毒介导hVEGF-165基因转染BMSCs阳性率可达85%以上；2、采用结扎腹主动脉法制作缺血模型,术后30天所有大鼠出现跛行,足趾坏死,绝大部分大鼠肢体出现缺血性溃疡。彩色多普勒超声、后肢血管造影等检查显示结扎术后实验大鼠后肢血流、阻力指数显著下降,血管密度显著减少。3、移植术后30天检测疗效,大鼠后肢血流速度、阻力指数与微血管密度转基因BMSC组、BMSC组均明显高于空白对照组,而转基因BMSC组高于BMSC组(p0.01)。结论：慢病毒载体介导hVEGF-165基因转染BMSC阳性率高；采用SD大鼠腹主动脉分叉处上方结扎法制作后肢缺血模型缺血效果明显,持续时间长；相同移植条件下的携hVEGF-165基因BMSCs、单纯BMSCs均能改善缺血后肢的血供,但转染hVEGF165基因BMSCs促血管新生能力更有优势。
[Abstract]:Aim: to investigate the therapeutic effect of bone marrow mesenchymal stem cells carrying human vascular endothelial growth factor-165 gene on hindlimb ischemia in rats. Methods: three experimental groups were set up in this experiment. They were hVEGF-165 gene transfected BMSC group and blank control group. There were 8 effective samples in each group with 48 limbs in each group. The ischemia model was established by ligating the abdominal aortic bifurcation of SD rats. The blood flow velocity and resistance of femoral artery were measured and identified by observing DSA angiography after operation. The hVEGF-165 gene was transfected into rat bone marrow mesenchymal stem cells (BMSCs) by lentivirus-mediated transfection with GFP fluorescent protein. The transfection rate of hVEGF-165 gene was detected by fluorescence microscope. The stem cells with 1X106/ml concentration were transplanted into ischemic limbs by multi-point injection of rat hindlimb muscle. One month later, the blood flow velocity and resistance of femoral artery were measured by observing DSA angiography. Results under the fluorescence microscope, the positive rate of lentivirus mediated hVEGF-165 gene transfection BMSCs was over 85%. The ischemic model was made by ligation of abdominal aorta. 30 days after operation, all rats showed claudication and toe necrosis. Most rats had ischemic ulcers in their limbs. Color Doppler ultrasound, hindlimb angiography and other examinations showed that the blood flow of the hind limb of the experimental rats after ligation was significantly decreased, the resistance index was significantly decreased, the blood vessel density was significantly decreased by 0.3, the curative effect was detected 30 days after transplantation, and the blood flow velocity of the hind limb of the rats was measured. The resistance index and microvessel density in BMSC group were significantly higher than those in control group, while those in transgenic BMSC group were higher than those in BMSC group (p0.01). Conclusion: the positive rate of hVEGF-165 gene transfection with lentivirus vector is high, and the ischemic effect of hind limb ischemia model made by ligation above the abdominal aorta bifurcation of SD rats is obvious and lasting for a long time. BMSCs, carrying hVEGF-165 gene could improve the blood supply of hind limb after ischemia, but transfection of hVEGF165 gene BMSCs had more advantages in promoting angiogenesis.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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