Slit2／Robo4信号通路对小鼠心肌微血管内皮细胞增殖和迁移的影响  

Myocardial Slit2/Robo4 expression and impact of exogenous Slit2 on proliferation and migration of cardiac microvascular endothelial cells

作　　者：陈桂秀[1] 王浩宇[1] 刘涛[1] 杨明涛 周振宇[1] 冯刚[3] 

机构地区：[1]川北医学院第二临床学院南充市中心医院心内科,637000 [2]河北省石家庄市平山县人民医院心内科 [3]川北医学院第二临床学院组织工程与干细胞研究所

出　　处：《中华心血管病杂志》2013年第12期1034-1039,共6页Chinese Journal of Cardiology

基　　金：四川省科技厅应用基础研究计划（2013JY0129）;南充市立项课题（11A0105）

摘　　要：目的检测分泌型细胞外基质糖蛋白Slit2及其受体Rob04蛋白在小鼠心室肌的血管组织中的表达情况，探讨Slit2／Rob04信号通路对小鼠心肌微血管内皮细胞增殖及迁移的影响。方法免疫组织化学法检测小鼠心室肌的血管组织中Slit2、Rob04蛋白的表达情况。从小鼠心室肌分离出微血管内皮细胞，通过免疫荧光和酶联免疫法分别检测小鼠心肌微血管内皮细胞中Rob04和Slit2蛋白的表达情况。利用CCK-8试剂盒，观察不同浓度外源性的Slit2对小鼠心肌微血管内皮细胞增殖的影响。利用transwell小室，设计对照组、VEGF组、Slit2组（100rig／mlSlit2），Slit2＋VEGF组（100ng／ml Slit2＋10ng／mlVEGF），观察外源性Slit2对小鼠心肌微血管内皮细胞迁移的影响。结果免疫组织化学结果显示小鼠心室肌的血管组织中Slit2、Rob04蛋白均有表达。RT—PCR及免疫荧光检测结果显示小鼠心肌微血管内皮细胞中有Robo4 mRNA及其蛋白的表达。酶联免疫法检测结果显示小鼠心肌微血管内皮细胞中有Slit2蛋白的表达。在细胞增殖实验中，不同浓度的Slit2组450nm处的吸光度值与阴性对照组比较差异均无统计学意义（P均〉0．05）。在细胞迁移实验中，Slit2组小鼠心肌微血管内皮细胞迁移数为（22．1±2．8）与对照组的（23．2±3．8）比较差异无统计学意义（P〉0．05），Slit2组小鼠心肌微血管内皮细胞迁移数为（22．1±2．8），Slit2组小鼠心肌微血管内皮细胞迁移数为（22．1±2．8）与VEGF组的（65．3±3．8），Slit2＋VEGF组的（29．2±3．4）之间比较差异均有统计学意义（P均〈0．05）。结论Slit2、Robo4蛋白均表达于小鼠心室肌组织，Rob04蛋白表达于小鼠心肌微血管内皮细胞，不同浓度的外源性Slit2对小鼠心肌微血管内皮细胞的增殖没有影响。100ng／ml外源性Slit2可抑制VEGF诱导的小鼠心肌微血管内皮细胞迁移。Objective To detect expression of Slit2 and Robo4 in mouse ventricular muscle blood vessel and explore the impact of exogenous Slit）. on proliferation and migrate of mouse cardiac microvascular endothelial cells. Methods Slit2 and Robo4 expression in mouse ventricular muscle blood vessel was detected by immunohistochemistry. Slit2 and Robo4 expression in cardiac microvascular endothelial cells isolated from mouse ventricular muscle were detected by euzymelinked immunosorbent assay and immunofluorescence, respectively. The effects of various concentrations exogenous Slit2 on proliferation of mouse cardiac microvascular endothelial cells was examined by CCK-8 cell proliferation kit. Transwe11 chamber was used to detect migration of mouse cardiac microvascular endothelial cells treated with 800 p,1 M199 culture medium containing 20% FBS （negative control ）, 10 ng/ml VEGF （positive control ）, 100 ng/ml Slit2（Slit2） and 100 ng/ml Slit2 ＋ 10 ng/ml VEGF （Slit2 ＋ VEGF） and incubated for 18 h at 37℃ and 5% CO2. Results Both Slit2 and Robo4 protein expressions were detected in ventricular muscle blood vessel. Slit2 protein expression was detected in mouse microvascular endothelial cells. Protein and mRNA Robo4 expressions were also evidenced in mouse microvascular endothelial cells. Proliferation of mouse cardiac microvascular endothelial ceUs was not affected by exogenous Slit2. Migration of mouse cardiac microvascular endothelial cells was not affected by exogenous Slit2 （22. 1 ±2. 8 vs. 23.2 ±3.8 in negative control, P 〉 0. 05 ） and significantly enhanced by VEGF （65.3 ±3.8, P 〈 0. 05 vs. Slip2 and negative control）, this effect could be blocked by cotreatment with Slip2 （29.2 ±3.4 in Slip2 ＋ VEGF, P 〈 0. 05 vs. VEGF）. Conclusion Slit2 and Robo4 are expressed in mouse ventricular muscle blood vessels and cardiac microvascnlar endothelial cells. Exogenous Slit2 has no impact on the proliferation of mouse cardiac microvascular endothelial cells but could inhibit VEGF-induced

关 键 词：心肌 内皮细胞 细胞运动 细胞增殖 

分 类 号：R542.2[医药卫生—心血管疾病]