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本文研究大鼠骨髓间充质干细胞分化生成的内皮样细胞(rat bone marrow mesenchymal stem cells-differentiated endothelial like cells,rBMSC-ECs)在血管新生中的作用及Rho激酶(Rho kinase,ROCK)活性抑制的影响。实验建立rBMSC-ECs与主动脉环体外共培养实验模型,设单纯血管环组、血管环与细胞共培养组和HA-1077低、中、高浓度组,HA-1077组在共培养的基础上分别在培养液中加不同浓度(10、30、60mmol/L)的ROCK特异抑制剂HA-1077。结果显示,培养第3天,血管环与细胞共培养组新生微血管数是单纯血管环组的1.3倍(P<0.05);HA-107710、30和60mmol/L组较共培养组分别减少57.70%、64.13%和48.23%(均P<0.01)。第6天,共培养组及HA-1077组rBMSC-ECs数量明显增加,并迁移至血管环周边,新生微血管生长缓慢;HA-1077组新生微血管数较共培养组明显减少。第9天,共培养组新生微血管部分增粗、增厚、延长,部分退化;一些rBMSC-ECs出芽,形成毛细血管样外观;而HA-1077组新生微血管几乎完全退化。Western blot检测血管环ROCKⅠ和ROCKⅡ的蛋白表达,与单纯血管环组比较,血管环与细胞共培养组表达稍增强,HA-1077各浓度组的表达稍降低。罗丹明-鬼笔毒环肽染色,激光共聚焦显微镜下观察显示,血管环新生微血管分布大量的肌动蛋白(F-actin),细胞内呈现大量粗大的应力纤维,细胞前沿向前方伸出富含F-actin的丝状伪足。另一种ROCK特异抑制剂Y-27632(10μmol/L)抑制新生微血管细胞应力纤维形成,F-actin分布于细胞周边,细胞前沿丝状伪足消失。结果提示,rBMSC-ECs自身可分化为新生微血管,与动脉环体外共培养促进血管新生,ROCK抑制剂抑制其促血管新生作用,其机制涉及F-actin细胞骨架蛋白重组。
This study was designed to investigate the role of rat bone marrow mesenchymal stem cell-differentiated endothelial cells (rBMSC-ECs) in angiogenesis and the inhibitory effect of Rho kinase (ROCK) on rat bone marrow mesenchymal stem cells influences. The experimental model of rBMSC-ECs co-cultured with aortic rings was established experimentally, and the co-culture of rCMSC-ECs and aortic rings was established. The co-culture of HA group, HA-1077 group and HA- The ROCK specific inhibitor HA-1077 with different concentrations (10, 30 and 60mmol / L) was added to the culture medium respectively. The results showed that the number of new blood vessels in the co-culture group was 1.3 times higher than that in the pure vascular group (P <0.05) on day 3, and decreased by 57.70% in the HA-107710, 30 and 60 mmol / L groups , 64.13% and 48.23% respectively (all P <0.01). On the 6th day, the number of rBMSC-ECs in co-culture group and HA-1077 group increased significantly and migrated to the perivascular ring. The growth of newborn microvessel was slow. The number of newborn microvessel in HA-1077 group was significantly decreased compared with co-culture group. On the 9th day, some new blood vessels in the co-culture group became thicker, thicker, lengthened and partially degenerated. Some rBMSC-ECs sprouted to form capillary-like appearance. However, the neovascularization of HA-1077 group almost completely degenerated. The protein expressions of ROCK I and ROCK II in the vascular rings were detected by Western blot. Compared with the simple vascular rings group, the expression of the vascular rings and the cell co-culture group slightly increased, and the expression of HA-1077 in each concentration group slightly decreased. Rhodamine-phalloidin staining and confocal laser scanning microscopy showed that a large number of actin (F-actin) were distributed in the capillary vessels of the vascular rings, and a large number of coarse stress fibers were present in the cells. The leading edge of the cells extended forwardly F-actin containing filopodia. Another specific inhibitor of ROCK, Y-27632 (10μmol / L), inhibited the formation of stress fibroblasts in newborn microvascular cells. F-actin was distributed in the periphery of the cells, and the filopodia disappeared in the front of cells. The results suggest that rBMSC-ECs can differentiate into newborn microvessels by themselves, co-culture with arterial rings to promote angiogenesis, and ROCK inhibitors can inhibit angiogenesis. The mechanism involves the reorganization of F-actin cytoskeletal proteins.