【摘要】 目的探讨经肌肉注射转染pEGFP-C1/Akt的鼠骨髓间充质干细胞(MSCs)对后肢缺血大鼠血管生成的影响。方法Wistar大鼠30只,制成双后肢缺血模型,双盲法随机分为基因治疗组(肌注经pEGFP-C1/Akt转染的MSCs)、非基因治疗组(肌注MSCs)及对照组(肌注PBS液)。造模前、造膜后即刻及MSCs移植后1~7d内,每天用红外线皮温仪测定大鼠后肢皮温变化。28d时经动脉造影观察后肢血管生成情况;免疫组化染色检测后肢毛细血管密度;逆转录-多聚酶链反应(RT-PCR)和Western blot法检测后肢肌肉组织中Akt及血管内皮细胞生长因子(VEGF)的mRNA和蛋白的表达。结果移植3d后基因治疗组大鼠后肢皮温升高明显。28d时经动脉造影观察基因治疗组后肢侧支血管生成明显;荧光显微镜观察有绿色荧光细胞在基因治疗组的内收肌和半膜肌分布。毛细血管密度:基因治疗组为(7.1±0.3)个/高倍镜,非基因治疗组为(4.2±0.4)个/高倍镜,对照组为(1.3±0.2)个/高倍镜,各组间差异均有统计学意义(P<0.01)。Akt及VEGF的mRNA和蛋白的表达分析:基因治疗组Akt mRNA(2.44±0.14)和蛋白(1.12±0.13)及VEGF mRNA(1.11±0.11)和蛋白(0.97±0.13)表达水平均明显高于非基因治疗组Akt mRNA(1.58±0.13)和蛋白(0.78±0.12)及VEGF mRNA(0.78±0.14)和蛋白(0.67±0.11)以及对照组Akt mRNA(0.64±0.11)和蛋白(0.36±0.12)及VEGF mRNA(0.56±0.11)和蛋白(0.33±0.13)的表达水平(P<0.01),后2组间比较差异亦均有统计学意义(P<0.01)。结论pEGFP-C1/Akt体外转染骨髓MSCs促进后肢缺血大鼠血管生成的效果优于单纯MSCs治疗,为基因转染MSCs治疗缺血性疾病提供可能。更多还原

【Abstract】 Objective To investigate the effect of bone mesenchymal stem cells(MSCs)transfected with pEGFP-C1/Akt after intramuscular injection on angiogenesis in ischemic hindlimb of mice.Methods A total of 30 Wistar mice used in the present experiments were divided into gene therapy group(injected MSCs transfected with pEGFP-C1/Akt),non-gene therapy group(injected MSCs)and control group(injected PBS).Mice skin temperature in left hindlimbs was measured by a infrared ray temperature meter before and right after ischemia and 1-7 days after transfection,respectively.Digital subtraction angiography was carried out to observe angiogenesis in hindlimb of mice after 28 days.The capillary density was determined with immunohistochemical analysis in the resected specimen.The expressions of Akt mRNA and protein,VEGF mRNA and protein were measured via RT-PCR and Western blot.Results Skin temperature increased significantly on 3 d after transfection in gene therapy group.On 28 d after transfection,angiography showed that the number of collateral vessels markedly increased in gene therapy group.Green fluorescence cells were observed in ischemic tissue from the adductor and semimembranous muscles of gene therapy group under fluorescent microscope.The result of immunohistochemical analysis of capillary density showed that the capillary density was significantly higher in gene therapy group 〔(7.1±0.3)/high power〕 than that in non-gene therapy group 〔(4.2±0.4)/high power〕 and control group 〔(1.3±0.2)/high power〕,with significantly statistical differences among three groups(P<0.01).Akt mRNA(2.44±0.14)and protein(1.1±0.13),VEGF mRNA(1.1±0.11)and protein(0.97±0.13)of gene therapy group markedly increased compared with Akt mRNA(1.58±0.13)and protein(0.78±0.12),VEGF mRNA(0.78±0.14)and protein(0.67±0.11)of non-gene therapy group as well as Akt mRNA(0.64±0.11)and protein(0.36±0.12),VEGF mRNA(0.56±0.11)and protein(0.33±0.13)of control group(P<0.01).Conclusion The effect of MSCs transfected with pEGFP-C1/Akt on angiogenesis is significantly better than only MSCs therapy,which may offer a new way for the ischemic diseases.更多还原