摘要
目的研究促红细胞生成素(erythropoietin, EPO)对慢性肾衰竭(chronic renal failure, CRF)大鼠外周血内皮祖细胞(endothelial progenitor cells, EPCs)的数目和功能的影响,以及对肾脏的修复作用。
方法采用分阶段5/6肾切除术制备大鼠慢性肾功能衰竭动物模型。实验动物随机分为4组:假手术组(Control),慢性肾衰竭组(模型组)及rHuEPO干预的两个剂量亚组(小剂量组和大剂量组)。慢性肾衰竭大鼠皮下注射rHuEPO 6周后,密度梯度离心法从大鼠心脏血中分离单个核细胞,接种于铺有纤维连接蛋白的培养瓶内,给予含有血管内皮生长因子(VEGF)等的M199培养基。通过流式检测细胞表面标记CD34,CD133和KDR以及免疫荧光检测细胞内吞UEA-1和结合acLDL来鉴定EPCs,并检测EPCs增殖、粘附及形成血管结构的能力。用免疫组化检测肾组织CD34+细胞数目、肾小球CAP内皮细胞密度,用RT-PCR检测肾组织eNOS、ET1、TGF-β、VEGF、COL IVmRNA的表达,检测Scr、BUN、尿蛋白指标并观察肾组织病理改变。
结果内皮祖细胞的鉴定用激光共聚焦显微镜观察,显示细胞DiI-acLDL和FITC-UEA-1呈免疫双荧光阳性。应用流式细胞仪检测体外培养的细胞表达EPCs公认的表面标记CD133、CD34、VEGFR-2的阳性率分别为4.14%、79.42%、55.7%。应用rHuEPO治疗能显著增加残肾模型大鼠EPC增殖、粘附及形成血管结构的能力(P<0.05),显著增加肾组织CD34+细胞的数目以及肾小球毛细血管内皮细胞密度(P<0.05),上调肾组织VEGF、eNOS mRNA的表达(P<0.05),下调肾组织ET1、TGF-β、COL IV mRNA的表达(P<0.05),改善肾小球毛细血管内皮功能。尿蛋白、血肌酐及尿素氮水平明显降低(P<0.05),肾小球系膜增生及间质纤维化程度明显减轻(P<0.05)。
结论EPO可能通过促进EPC的动员、增殖、粘附以及形成血管样结构的能力,从而修复和改善肾小球毛细血管内皮功能、改善肾功能、减轻肾脏的病理改变。
Objective To investigate the influence of erythropoietin (EPO) to the number and ability of endothelial progenitor cells (EPCs) and renal renovation in chronic renal failure (CRF) rats.
Methods The model of chronic renal failure was established by a two stage 5/6 nephrectomy proeedure in rats. Experimental animals were randomly divided into four groups: Control group, CRF group, CRF rats treated with low-dosage EPO and with high-dosage EPO. CRF rats were given EPO by hypodermic injection for 6 weeks, then density gradient centrifugation from isolated rat heart blood mononuclear cells, vaccination in the shop were fibronectin culture bottle,to contain the vascular endothelial growth factor (VEGF), such as the M199 medium. Flow cytometry was used to detect the expression of CD34, CD133 and KDR of the cultured cells, and immunofluorescence was performed to display the character of endocytosing UEA-1 and combining acLDL, and the capacity of the cells’proliferation, adhesion and invitro vasculogenesis were further ovserved. The number of CD34 cells in nephridial tissue and the density of glomerulus CAP endothelial cell were detected by Elivision. The expression of mRNA of eNOS, ET1, TGF-β,VEGF and IV collagen in nephridial tissue were detected by RT-PCR. The Scr, BUN and urine protein were measured and renal pathological changes were observed.
Results The cells could take up DiI-acLDL, and bind to FITC-UEA-1, showed double positive fluorescence under LSCM. Cultured EPCs at the passage 1 were positive for CD133, CD34 and VEGFR-2 (4.14%, 79.42% and 55.7% respectively).After treatment of EPO, the capacity of the cells’proliferation, adhesion and invitro vasculogenesis were promoted significantly (P<0.05), the number of CD34 cells in nephridial tissue and the density of glomerulus CAP endothelial cell were increased significantly (P<0.05), the expression of mRNA of VEGF and eNOS in nephridial tissue were raised significantly (P<0.05), the expression of mRNA of ET-1, TGF-βand IV collagen were falled significantly (P<0.05), the urinary protein, Scr and BUN of erythropoietin group were reduced significantly, and the glomerular mesangial proliferation and interstitial fibrosis were ameliorated significantly (P<0.05).
Conclusion It might be through improving the ability of EPCs’mobilization, proliferation, adhesion and invitro vasculogenesis that EPO can ameliorate the ability of glomerulus CAP endothelial cell, renal function and pathological changes.
引文
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