摘要
目的 筛选大鼠缺血心肌微血管内皮细胞（myocardial microvascular endothelial cells,CMEC）血管新生过程中的核心微小RNA（microRNA,miRNA）,并探讨其调控机制.方法 健康雄性SD大鼠,6周龄,SPF级.结扎法建立大鼠心肌缺血模型,植块法培养CMEC.免疫细胞化学法鉴定CMEC.大鼠缺血CMEC作为缺血组,正常大鼠CMEC作为正常组,观察缺血CMEC血管新生的生物学特征,确定其血管新生过程中迁移、增殖和成管的窗口期.miRNA芯片检测miRNA的动态表达变化,筛选差异表达显著的miRNA并应用实时聚合酶链反应验证,拟定出缺血CMEC血管新生过程中核心miRNA.生物信息学方法预测核心miRNA靶基因,并用实时聚合酶链反应验证.同时,蛋白印迹法检测靶基因及血管新生相关基因p38丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇3-激酶（PI3K）、丝氨酸-苏氨酸激酶（Akt）、血管内皮生长因子（VEGF）的蛋白表达水平.结果 培养的CMEC具备典型微血管内皮细胞特征,第Ⅷ因子、血小板-内皮细胞黏附分子（CD31）相关抗原免疫染色鉴定均为阳性.正常组和缺血组迁移窗口期均为第1天、成管窗口期均为第2天,而他们的增殖窗口期分别为第3天和第6天.根据表达量差异的显著性以及与血管生成的关系,最终确定miRNA-223-3p为缺血CMEC血管新生的核心miRNA,实时聚合酶链反应验证miRNA-223-3p表达与芯片结果一致.生物信息学方法预测Rps6kb1为miRNA-223-3p的靶基因,Pathway分析Rps6kb1能够参与低氧诱导因子-1α（HIF-1α）信号通路调控血管生成.缺血CMEC的迁移期和增殖期,Rps6kb1/HIF-1 α信号通路下游分子VEGF、p38MAPK、PI3K、Akt的基因和蛋白表达均明显下调.结论 miRNA-223-3p是缺血CMEC血管新生的核心miRNA,miRNA-223-3p可通过调节Rps6kb1/HIF-1α信号通路抑制缺血CMEC血管新生的迁移和增殖过程,从而降低血管新生能力.
Objective To explore the role of microRNA on the myocardial microvascular endothelial cells （CMECs） of ischemic heart rats in the process of angiogenesis and related regulation mechanism.Methods Myocardial ischemic rats model was established by coronary ligation.Seven days after operation,the ischemic CMECs were cultured by the method of planting myocardium tissue and identified by immunocytochemistry to observe the biological characteristics of ischemic CMECs angiogenesis,to determine the window period of migration,proliferation,tube formation in the process of its angiogenesis.Dynamic expression changes of microRNA in the process of ischemic CMECs angiogenesis was detected using microRNA chip and further verified by real-time PCR,the core microRNA of the ischemic CMECs was defined and the predicted target genes of core microRNA were determined by bioinformatics methods and real-time PCR.At the same time,the protein expression of target gene and angiogenesis related genes of p38MAPK,PI3K,Akt,VEGF were measured by Western blot.Results The CMECs of rats presented typical characteristics of microvascular endothelial cells,and factor Ⅷ,CD3 1 related antigens were all positively stained by immunocytochemical analysis.The migration window period was on the first day,and the tube formation window period was on the second day of both control and ischemic groups,while the proliferation window period was on the third day for the normal group,and the sixth day for ischemic group.According to the expressional difference and their relationship with angiogenesis,miRNA-223-3p was ultimately determined as the core microRNA in the process of ischemic CMECs angiogenesis,real-time PCR verified this hypothesis.Bioinformatics methods predicted that Rps6kb1 is the target genes of miRNA-223-3p,the pathway analysis showed that Rps6kb1 could regulate angiogenesis via HIF-1α signal pathway.Moreover,the mRNA and protein expression of VEGF,p38MAPK,PI3K,Akt,which were the downstream molecules of Rps6kb1/HIF-1 α signal pathway,were also significantly downregulated in ischemic CMECs from migration and proliferation stage.Conclusion Our results show that the miRNA-223-3p is the core microRNA of ischemic CMECs angiogenesis.MiRNA-223-3p could regulate Rps6kb1/HIF-1α signal pathway,inhibit the process of migration and proliferation of ischemic CMECs angiogenesis.MiRNA-223-3p is thus likely to be a core target for enhancing angiogenesis of ischemic heart disease.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2014年第12期1039-1047,共9页
Chinese Journal of Cardiology
基金
国家自然科学基金面上项目（81173441）
关键词
心肌缺血
微RNAS
内皮细胞
新生血管化
病理性
Myocardial ischemia
MicroRNAs
Endothelial cells
Neovascularization,pathologic
