TrkB-shiRNA质粒转染心脏微血管内皮细胞  

Transfection of cardiac microvascular endothelial cells using TrkB-shiRNA plasmid

作　　者：陈斯韵[1,2,3,4] 曹亮[1,2,3,4] 李震[1,2,3,4] 沈晓涛[1,2,3,4,5] 郑馨[1,2,3,4] 梁永佳[1,2,3,4] 蔡冬青[1,2,3,4,5] 

机构地区：[1]暨南大学再生医学教育部重点实验室,广东省广州市510632 [2]暨南大学再生医学 [3]香港中文大学-暨南大学联合实验室,广东省广州市510632 [4]暨南大学广东省科技厅国际科技合作基地,广东省广州市510632 [5]暨南大学生物医学工程系,广东省广州市510632

出　　处：《中国组织工程研究与临床康复》2010年第20期3655-3659,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：863项目(2007AA02Z105);国家自然科学基金(30770886,30570369,30340038,30973158);广东省自然科学基金重点项目(04105826);广东省科技攻关项目(2004B30601007)~~

摘　　要：背景:脑源性神经营养因子与其受体TrkB在心脏与骨骼肌内皮细胞存在表达,在心脏血管系统的发育中及骨骼肌缺血时能有效促进血管新生,但其促血管新生的细胞与分子机制尚不清楚。目的:应用针对脑源性神经营养因子受体TrkB的shiRNA质粒对心脏微血管内皮细胞进行转染,观察TrkB 3种亚型的表达及心脏微血管内皮细胞的生长状况和形态,初步探讨脑源性神经营养因子-TrkB通路在心脏微血管内皮细胞中的调控作用。方法:使用TrkB-shiRNA质粒转染大鼠心脏微血管内皮细胞,应用荧光实时定量PCR检测TrkB的3个亚型,TrkB-FL、TrkB-T1及TrkB-T2 mRNA的表达,并观察经转染的心脏微血管内皮细胞的增殖情况。结果与结论:应用TrkB-shiRNA质粒转染心脏微血管内皮细胞后,TrkB 3种亚型在转染后的4d内mRNA表达均下降(P<0.05或P<0.01),且经转染的心脏微血管内皮细胞的生长变慢。说明TrkB-shiRNA质粒可沉默心脏微血管内皮细胞的TrkB-FL、TrkB-T1及TrkB-T2的表达,且TrkB表达被抑制可能会影响心脏微血管内皮细胞的增殖。BACKGROUND： Recent studies have shown that endothelial cells （ECs） in heart and skeletal muscle express brain derived neurotrophic factor （BDNF） and its receptor tyrosine receptor kinase B （TrkB）. BDNF-TrkB pathway might play an important role in cardiovascular system development, angiogenesis and ischemic limb regeneration. However, the molecular mechanism regarding to BDNF-induced angiogenesis is still unknown. OBJECTIVE： TrkB-shiRNA vector was transfected into cardiac microvascular endothelial cells （CMECs） to investigate the expression of 3 TrkB isoforms, the growth and cell morphology, in addition, to explore the regulation of TrkB pathway on CMECs. METHODS： TrkB-shiRNA vector was used. The expressions of TrkB isoforms （TrkB-FL, TrkB-T1 and TrkB-T2） in CMECs was analyzed by real-time PCR. The silencing effect of TrkBs in CMECs proliferation was analyzed by cell culture and cell counting. RESULTS AND CONCLUSION： It was found that the expression of TrkB isoforms, TrkB-FL, TrkB-T1 and TrkB-T2, was decreased at 4 days after CMECs transfected with TrkB-shiRNA vector （P 0.05 or P 0.01）. In addition, the proliferation of transfected CMECs was decreased. It demonstrated that transfection of CMECs with TrkB-shiRNA vector was able to decrease expression of TrkB-FL, TrkB-T1 and TrkB-T2 and decrease the proliferation of CMECs.

关 键 词：心脏微血管内皮细胞 脑源性神经营养因子 TRKB shiRNA TrkB-FL TrkB-T1 TrkB-T2 细胞增殖 

分 类 号：R318[医药卫生—生物医学工程]