内皮祖细胞移植治疗慢性深静脉血栓形成的实验研究

目的 研究内皮祖细胞(endothelial progenitor cells,EPCs)移植对慢性深静脉血栓的治疗作用. 方法 Ficoll法分离大鼠骨髓单个核细胞(bone marrow-derived mononuclear cells,BMMNCs),采用内皮祖细胞培养基(EGM-2MV)诱导分化为骨髓源性EPCs.建立大鼠慢性深静脉血栓模型,饲养至10 d,并分三组:A组(25只):单纯EPCs组,移植1 ml含有10~6 个EPCs细胞悬液;B组(25只):EGM-2MV培养基对照组,移植1 ml EGM-2MV培养基;C组(25只):空白对照组.移植后第14天取出血栓段下腔静脉及血栓,应用HE染色及vWF免疫组化染色观察血栓机化再通情况,高倍镜下计数血栓内毛细血管数目;Western blot检测血管内皮生长凶子(VEGF)、碱性成纤维细胞生长因子(bFGF)的蛋白表达变化;实验数据采用SPSS13.0软件进行分析.结果 体外成功培养了骨髓源性EPCs和构建了大鼠慢性深静脉血栓模型,EPCs移植后A组VEGF、bFGF的表达有明显的上调(P＜0.05).B组和C组之间差异无统计学意义(P＞0.05).移植后A组血栓再通毛细血管数目明显高于B、C组.免疫组化染色vWF确定再通管道为新牛血管,管道为内皮细胞组成.结论 EPCs为内皮细胞的前体细胞,移植到深静脉血栓中能够促进血管新生,加快血栓的机化和再通.

Effects of EPCs transplantation on chronic deep vein thrombosis in rats

Objective To study the effect of endothelial progenitor cells(EPCs) transplantation on chronic deep venous thrombosis.Methods Bone marrow-derived mouonuclear cells (BMMNCs) were isolated from rat bone marrow by ficoll and cultured with EGM-2MV medium.A rat model of chronic deep vein thrombosis was established by partial ligation of the inferior vena cava and intravenous injection of thrombosin.Model rats were randomly divided into three groups:A(n =25),EPCs group,1 ml 10~6 EPCs transplantation;B(n = 25),EGM-2MV medium group,1 ml EGM-2MV medium transplantation;C (n =25),control group,without any treatment.After transplantation,HE staining and immunohistochemical staining was conducted to detect recanalization of the inferior vena cava.Western blotting of inferior vena cava thrombosis was used to detect VEGF,bFGF protein expression changes.SPSS13.0 software was used for analysis.Results Compared with group B and C,VEGF,bFGF protein significantly increased in group A.The recanalization capillary density was significantly higher in group A than that in group B,and C (P ＜0.05).The neovascularization was identified by immunohistochemical staining using vWF antibody,as endothelial cells.Conclusions EPCs were the precursor of endothelial cells,when transplanted into the deep vein thrombos,initiating angiogenesis and accelerating organization and recanalization of vein thrombus.