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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 celladhesion 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.“,”Objective To 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.Methods Male and female GIT 1 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.Results Western 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 B 1.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 groups Ⅰ,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).Condusion GIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3,thereby affecting the angiogenesis.