CAJ | 학술논문

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.
목적 사선대서결혈심기미혈관내피세포(myocardial microvascular endothelial cells,CMEC)혈관신생과정중적핵심미소RNA(microRNA,miRNA),병탐토기조공궤제.방법 건강웅성SD대서,6주령,SPF급.결찰법건립대서심기결혈모형,식괴법배양CMEC.면역세포화학법감정CMEC.대서결혈CMEC작위결혈조,정상대서CMEC작위정상조,관찰결혈CMEC혈관신생적생물학특정,학정기혈관신생과정중천이、증식화성관적창구기.miRNA심편검측miRNA적동태표체변화,사선차이표체현저적miRNA병응용실시취합매련반응험증,의정출결혈CMEC혈관신생과정중핵심miRNA.생물신식학방법예측핵심miRNA파기인,병용실시취합매련반응험증.동시,단백인적법검측파기인급혈관신생상관기인p38사렬원활화단백격매(MAPK)、린지선기순3-격매(PI3K)、사안산-소안산격매(Akt)、혈관내피생장인자(VEGF)적단백표체수평.결과 배양적CMEC구비전형미혈관내피세포특정,제Ⅷ인자、혈소판-내피세포점부분자(CD31)상관항원면역염색감정균위양성.정상조화결혈조천이창구기균위제1천、성관창구기균위제2천,이타문적증식창구기분별위제3천화제6천.근거표체량차이적현저성이급여혈관생성적관계,최종학정miRNA-223-3p위결혈CMEC혈관신생적핵심miRNA,실시취합매련반응험증miRNA-223-3p표체여심편결과일치.생물신식학방법예측Rps6kb1위miRNA-223-3p적파기인,Pathway분석Rps6kb1능구삼여저양유도인자-1α(HIF-1α)신호통로조공혈관생성.결혈CMEC적천이기화증식기,Rps6kb1/HIF-1 α신호통로하유분자VEGF、p38MAPK、PI3K、Akt적기인화단백표체균명현하조.결론 miRNA-223-3p시결혈CMEC혈관신생적핵심miRNA,miRNA-223-3p가통과조절Rps6kb1/HIF-1α신호통로억제결혈CMEC혈관신생적천이화증식과정,종이강저혈관신생능력.
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.