中国修复重建外科杂志

中国修复重建外科杂志

G 蛋白偶联受体激酶相互作用蛋白 1 对 BMSCs 向内皮细胞分化的影响机制研究

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目的通过比较 G 蛋白偶联受体激酶相互作用蛋白 1(G protein coupled receptor kinase interacting protein 1,GIT1)野生型及 GIT1 基因敲除型小鼠 BMSCs 向内皮细胞分化过程,探讨 GIT1 影响血管形成的机制。方法GIT1 杂合子小鼠雌雄配对饲养,对获得的新生小鼠采用 PCR 法行基因型鉴定。取 GIT1 野生型与 GIT1 基因敲除型小鼠胫骨及股骨,分离培养 BMSCs 并传代。取第 2 代 BMSCs 分为 4 组,分别为野生型对照组(A 组)、野生型实验组(A1 组)、基因敲除型对照组(B 组)、基因敲除型实验组(B1 组);A1、B1 组细胞采用内皮细胞诱导液培养,A、B 组细胞进行常规培养。Western blot 检测各组细胞 VEGF 受体 2(VEGF receptor 2,VEGFR-2)、VEGFR-3、磷酸化 VEGFR-2(phospho-VEGFR-2,pVEGFR-2)、pVEGFR-3 蛋白表达;流式细胞仪检测各组内皮细胞标志物血管性血友病因子(von Willebrand factor,vWF)、血小板内皮细胞黏附因子 1(platelet-endothelial cell adhesion molecule 1,PECAM-1)、血管内皮黏钙蛋白(vascular endothelial cadherin,VE-Cadherin)表达。另取 GIT1 野生型小鼠第 2 代 BMSCs 分为 4 组:Ⅰ组,原细胞培养液培养;Ⅱ组,细胞培养液中加入 VEGFR-3 阻断剂 SAR131675;Ⅲ组,内皮细胞诱导液培养;Ⅳ组,内皮细胞诱导液中加入 SAR131675。流式细胞仪检测各组内皮细胞标志物 vWF、PECAM-1、VE-Cadherin 表达。结果Western blot 检测示,A、A1、B、B1 组细胞的 VEGFR-2、pVEGFR-2 蛋白表达无明显差异,A1 组 VEGFR-3、pVEGFR-3 蛋白表达明显高于其余 3 组。流式细胞仪检测示,A1 组 vWF、PECAM-1 及 VE-Cadherin 表达均显著高于 A、B、B1 组,B1 组显著高于 A、B 组(P<0.05);A、B 组间比较差异无统计学意义(P>0.05)。阻断 VEGFR-3 实验中,Ⅲ组 vWF、PECAM-1 及 VE-Cadherin 表达均显著高于Ⅰ、Ⅱ、Ⅳ组,Ⅳ组高于Ⅰ、Ⅱ组(P<0.05);Ⅰ、Ⅱ组间差异无统计学意义(P>0.05)。结论GIT1 通过 VEGFR-3 影响小鼠 BMSCs 向内皮细胞分化,进而影响血管形成。

ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.

关键词: G 蛋白偶联受体激酶相互作用蛋白 1; BMSCs; 内皮细胞; VEGF 受体 3; 小鼠

Key words: G protein coupled receptor kinase interacting protein 1; bone marrow mesenchymal stem cells; endothelial cells; vascular endothelial growth factor receptor 3; mouse

引用本文: 董献成, 殷建, 恽波, 吕斌, 殷国勇. G 蛋白偶联受体激酶相互作用蛋白 1 对 BMSCs 向内皮细胞分化的影响机制研究. 中国修复重建外科杂志, 2018, 32(3): 257-263. doi: 10.7507/1002-1892.201709090 复制

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