【作者】 徐盛开；

【导师】 江洪；

【作者基本信息】 武汉大学 ， 内科学， 2010， 博士

【摘要】 目的：探讨尾加压素Ⅱ(UⅡ)对大鼠骨髓源内皮祖细胞(EPCs)的作用及其分子机制。方法：选体重110-150g的雄性SD大鼠,分离获取骨髓单核细胞,在内皮细胞培养基中培养诱导,观察细胞生长形态变化,免疫荧光染色鉴定表面标志以及Dil-acLDL摄取试验细胞功能学检测。用RT-PCR、Western Blotting和免疫荧光检测等方法确定GPR14在EPCs上基因和蛋白水平的表达。利用Transwell小室进行趋化实验,观察不同浓度UⅡ对EPCs迁移的影响,检测UⅡ作用EPCs后,RhoA和GTP结合RhoA、MLC磷酸化水平,以及GPR14受体拮抗剂Urantide和Rho激酶抑制剂Y-27632对UⅡ诱导EPCs迁移的影响。CCK-8增殖实验观察不同浓度UⅡ对EPCs增殖的影响,检测UⅡ作用EPCs后,MAPK的激活状态,以及GPR14受体拮抗剂Urantide和MAPK抑制剂对UⅡ诱导EPCs增殖和MAPK磷酸化水平的影响。结果：从细胞贴壁,伪足样突起,“集落”形成,到条索样结构以及呈典型铺路石样外观,表现内皮祖细胞生长特性。免疫荧光染色鉴定提示CD133、VEGFR-2、vWF表达阳性。摄取DiI-acLDL结合FITC-UEA-1试验,FITC-UEA-1和DiI-acLDL双染色阳性。RT-PCR、Western Blotting和免疫荧光染色等方法检测显示,在分子和蛋白水平EPCs均有GPR14表达,免疫荧光检测GPR14在EPCs膜上染色阳性。UⅡ诱导的EPCs迁移呈典型的剂量依赖型,Urantide和Rho激酶抑制剂Y-27632抑制UⅡ对EPCs的趋化作用(p<0.05,vs.UⅡ组)。UⅡ诱导GTP结合RhoA增加,和对照组相比,UⅡ(10-8M)诱导RhoA和GTP结合RhoA增加约3倍(p<0.05,UⅡ组vscontrol组),GPR14抑制剂Urantide可以显著抑制这种效应(p<0.01 vs.UⅡ组),Rho激酶抑制剂Y-27632抑制作用不明显(p>0.05 vs.the 10-8M UⅡgroup)。UⅡ诱导增加MLC磷酸化增加,和对照组相比,UⅡ诱导增加MLC磷酸化增加约2.5倍(p<0.05,vscontrol组),GPR14抑制剂Urantide和Rho激酶抑制剂Y-27632均可显著抑制这种效应(p<0.05,vs.UⅡ组)。UⅡ对EPCs促增殖作用呈剂量依赖型,Urantide、SB203580和PD98059对UⅡ诱导的促增殖效应有显著抑制作用,抑制程度分别为62.4%、54.3%、和49.2%(all p<0.05 vs.the10-8M UⅡ组)。但SAPK/JNK抑制剂SP600125对增殖无抑制作用,且有增强趋势,但无显著差异(p>0.05 vs.the 10-8M UⅡ组)。和对照组相比,UⅡ组p38MAPK磷酸化程度显著增高(p<0.05 vscontrol组),这种效应可以被GPR14受体拮抗剂Urantide完全阻断(p<0.05 vs.UⅡ组),被p38MAPK抑制剂SB203580部分阻断(p<0.05 vs.UⅡ组)。和对照组相比,UⅡ组p44/42MAPK磷酸化程度显著增高(p<0.05 vscontrol组),这种效应可以被Urantide完全阻断(p<0.05 vs.UⅡ组),被p44/42MAPK抑制剂PD98059部分阻断(p<0.05 vs.UⅡ组)结论：1、所培养的细胞证实为内皮祖细胞,EPCs表达UⅡ特异性受体GPR14受体。2、UⅡ是一种新的内皮祖细胞趋化肽,趋化作用呈剂量依赖性,其机制与RhoA/Rho激酶信号通路相关。3、尾加压素Ⅱ促进骨髓源EPCs增殖,其机制与ERK1/2和P38MAPK的活化有关。更多还原

【Abstract】 Objective:To explore regulating effects of UrotensinⅡ(UII) on endothelial progenitor cells (EPCs) derived from rat bone marrow and its potential intracellular singal pathways.Method:Bone marrow from the femurs of the SD rats was removed and mononuclear cells were isolated by density gradient centrifugation. Cells were resuspended by EGM-MV BulletKit system and incubated at 37℃in 100% humidified air with 5% CO2. Morphology and growth characteristics of cells were observed by microscopy. Immunohistochemistry staining were performed to detect for the expressoion of von Willebrand factor (vWF) and CD133 and VEGFR-2. Experiment of uptaking DIL-ac-LDL and combining FITC-Lectin-UEA-1 was observed by laser sanning confocal microscope to identify endothelial cells characteristic. To better understand whether UII can regulate EPC function via a receptor-dependent pathway, RT-PCR and Western blot were performed to detect expression of the GPR14 in EPCs at mRNA and protein levels. Chemotaxis assays were performed using Transwell cell-culture chambers with UII (10-10-10-6 M). The activation of RhoA and the phosphorylation of myosin light chain (MLC) in EPCs were analyzed. The effets of Rho-kinase inhibitor Y-27632 and GPR14 receptor blocker Urantide to UII-induced migration of EPCs and the phosphorylation of MLC were observated. Proliferation assays were performed using WST-8 with UII (10-10-10-6M). The activation the phosphorylation of MAPK in EPCs were analyzed. The effets of MAPK inhibitor and GPR14 receptor blocker Urantide to UII-induced proliferation of EPCs and the phosphorylation of MAPK were observated.Result:Parts of cells start attaching after 48 hours. The morphology formed the shuttle, triangle, the spindle or irregular form. The attached cells were able to form cells "colonies" and line up in the core-like structure from 4 to 8 days, and get close to coalesce and exhibite the typical cobble stone morphology. The cells were positively stained for CD133, VEGFR-2 and vWF. Fluorescence microscopy showed that adherent cells took up DiI-acLDL and bound FITC-Lectin-UEA-1. EPCs express GPR14 at mRNA and protein levels. Fluorescence microscopy showed that cells were positively stained for GPR14. UII induces migration of EPCs in a concen- tration-dependent manner in a certain extent. The cell count assay revealed that 1.5-, 2.1-,2.9-,2.3-and 1.6-fold increase was detected in migration of EPCs induced by UII at the concentration of 10-10 M,10-9 M,10-8 M,10-7 M and 10-6 M, respectively (all p＜0.05 vs. control group). The Rho-kinase inhibitor, Y-27632 (10μM), abolished UII (10-8 M)-induced migration of EPCs (p＜0.05 vs. control group, p＜0.05 vs. UII group). UII (10-8 M) induced 3-fold increase in the amount of active GTP-bound RhoA in EPCs (p＜0.05, UII group vs. control group), and Urantide (10μM) abolished the effect of UII (p＜0.01 vs. UII group). UII induced 2.5-fold increase in MLC phosphorylation(p＜0.05, UII group vs. control group) which was blocked by Y-27632 (10μM) and Urantide(10μM) (p＜0.05, Y-27632 group vs. UII group). UII promotes proliferation of EPCs in a concentration-dependent manner in a certain range, and treatment with a GPR14 inhibitor or MAPK inhibitors suppressed UII-induced EPC proliferation. Results of the WST-8 assay showed 14.2%,48.8%,69.2%,46.3%, and 27.3% increases in proliferation in response to treatment with UII at concentrations of 10-10M,10-9M, 10-8M,10-7 M, and 10-6 M, respectively (all p＜0.05 vs. control group). The proliferative effect of UII on EPCs was inhibited by 62.4%,54.3%, and 49.2% in response to Urantide, SB203580, and PD98059, respectively. But the proliferative effect of UII on EPCs wasn’t inhibited by SP600125(p＞0.05 vs. the 10-8 M UII group). Treatment with UII (10-8 M) increased the phosphorylation of p38MAPK (p＜0.05 vs. control group). This effect was inhibited by the inhibitors SB203580 (10μM) (p＜0.05 vs. UII group). Treatment with UII (10-8M) increased the phosphorylation of p44/42MAPK (p＜0.05 vs. control group). This effect was inhibited by the inhibitors PD98059 (10μM) (p＜0.05 vs. UII group).Conclusion:1.Cultured cells were considered EPCs which express specific receptor GPR14 of UII.2. UII induces migration of EPCs in a concentration-dependent manner. UII-induced migration is mediated by activation of the RhoA/Rho kinase pathway. 3.UII induces migration of EPCs in a concentration-dependent manner. UII-induced migration is mediated by activation of the RhoA/Rho kinase pathway.更多还原