Thbs4基因编辑BMSCs移植对糖尿病后肢缺血大鼠VEGF和Ang-1的影响及相关作用机制

Effect of Thbs4 gene editing BMSCs transplantation on VEGF and Ang-1 in diabetic hind limb ischemia rats and related mechanism

目的探讨血小板反应素4(Thbs4)基因编辑骨髓间充质干细胞(BMSCs)移植对糖尿病后肢缺血大鼠血管内皮生长因子(VEGF)和促血管生成素-1(Ang-1)的影响及相关作用机制.方法将30只Sprague-Dawley大鼠随机分为模型组、模型+BMSCs治疗组及模型+Thbs4-BMSCs治疗组,每组10只.各组大鼠在构建Ⅱ型糖尿病后肢缺血模型后,分别于缺血损伤区局部注射100μl的生理盐水、BMSCs悬液和Thbs4-BMSCs悬液(细胞数为2×10^6个).干细胞移植后第14天处死大鼠,取缺血区域附近的肌肉组织,分别采用蛋白质印迹法(Western Blot)检测VEGF及磷酸化Smad2/3(p-Smad2/3)蛋白的表达,免疫荧光染色检测Ang-1蛋白的表达,实时荧光定量PCR检测VEGF和Ang-1基因的mRNA表达,免疫组化染色检测血管性血友病因子(vWF)蛋白的表达.结果模型+Thbs4-BMSCs治疗组的VEGF、Ang-1及vWF蛋白表达均明显高于模型组和模型+BMSCs治疗组(VEGF蛋白:P<0.01,P<0.05);VEGF和Ang-1基因的mRNA表达较模型组和模型+BMSCs治疗组均明显上调,差异均具有统计学意义(VEGF mRNA:均P<0.01;Ang-1 mRNA:P<0.01,P<0.05);p-Smad2/3蛋白表达也明显高于模型组和模型+BMSCs治疗组(均P<0.01),且加入p-Smad2/3通路抑制剂后p-Smad2/3蛋白表达明显下降,差异具有统计学意义(P<0.05).结论 Thbs4-BMSCs移植治疗糖尿病后肢缺血大鼠可有效促进缺血损伤区域促血管生成因子VEGF和Ang-1的表达,其促血管生成作用可能与激活Smad2/3信号通路有关.

Objective To investigate the effects and the mechanism of thrombospondin 4 (Thbs4) gene-edited bone marrow mesenchymal stem cells (BMSCs) transplantation on vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in diabetic rats with hind limb ischemia. Methods Thirty Sprague-Dawley rats were randomly divided into the model group, BMSCs treatment group and Thbs4-BMSCs treatment group on average. After constructing the typeⅡdiabetic rat model with hind limb ischemia, 100μl normal saline, BMSCs suspension and Thbs4-BMSCs suspension (cell number: 2×10^6) were locally injected into the ischemic injury area of rats for the model group, BMSCs group and Thbs4-BMSCs group, respectively. The rats were sacrificed on the 14th day after stem cell transplantation, and the muscle tissues near the ischemic area were collected. The relative expression of VEGF and p-Smad2/3 protein was detected by Western Blot. The Ang-1 protein expression was detected by immunofluorescence staining. The levels of related genes were detected by qRT-PCR, and the von Willebrand Factor (vWF) protein expression was detected by immunohistochemistry staining. Results The relative expression levels of VEGF, Ang-1 and vWF protein in the Thbs4-BMSCs group were significantly higher than those in the model group and BMSCs group (VEGF protein:P<0.01 and P<0.05). The mRNA expression of VEGF and Ang-1 were significantly up-regulated, the differences were statistically significant(VEGF mRNA:all P<0.01;Ang-1:P<0.01 and P<0.05). The expression of p-Smad2/3 protein in the Thbs4-BMSCs group was significantly higher than that in the model group and the BMSCs treatment group (all P<0.01). The expression of p-Smad2/3 protein was significantly decreased after the addition of p-Smad2/3 pathway inhibitor, the differences were statistically significant (P<0.05). Conclusions Thbs4-BMSCs transplantation can effectively promote angiogenesis in diabetic rats with hind limb ischemia, and the effect of angiogenesis may be related to the activation of Smad2/3 signaling pathway.