超极化激活环化核苷酸门控通道基因转染猪骨髓间充质干细胞  

Transfecting hyperpolarization-activated cyclic nucleotide-gated channel 2 gene into porcine bone marrow mesenchymal stem cells

作　　者：姚颖龙[1] 张浩[1] 龚德军[1] 宋智刚[1] 徐志云[1] 

机构地区：[1]解放军第二军医大学长海医院胸心外科,上海市200433

出　　处：《中国组织工程研究与临床康复》2009年第49期9673-9676,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金面上项目(30672075;30700157)~~

摘　　要：背景:研究证实超极化激活环化核苷酸门控通道(hyperpolarization-activated cyclic nucleotide-gated,HCN)电流在调控心脏的自发搏动中起着非常重要的作用。目的:观察HCN2基因重组腺病毒转染后猪骨髓间充质干细胞目的基因的表达及电生理特征。设计、时间及地点:细胞-基因学体外观察,于2007-07/2008-03在解放军第二军医大学胸心外科实验室完成。材料:Yorkshire猪由解放军第二军医大学动物所提供,HCN2质粒由意大利Dario DiFrancesco教授惠赠,重组腺病毒Ad.HCN2由本实验室采用Ad5系统构建并保存。方法:Percoll密度梯度离心+贴壁法体外分离纯化猪骨髓间充质干细胞,以感染复数=50进行Ad.HCN2转染,同时设立未转染组和转染Ad.Null组。主要观察指标:通过RT-PCR、免疫荧光染色检测各组细胞HCN2 mRNA和蛋白的表达,用全细胞膜片钳检测各组细胞电生理学变化。结果:未转染组和转染Ad.Null组均未见扩增片段,而Ad.HCN2扩增后在250～500bp可见扩增片段,与携带HCN2基因的质粒扩增出的片段位置相同。转染后细胞核染色强度明显弱于胞膜和胞浆,与HCN2蛋白的分布相符合,未转染组及转染Ad.Null组无HCN2蛋白阳性表达。全细胞膜片钳可记录到起搏电流,其激活电位约为-60mV,完全激活电位-140mV,呈电压依赖性,当给予4mmol/LCsCl后,内向的起搏电流即被明显抑制;未转染组及转染Ad.Null组细胞均无起搏电流。结论:通过起搏基因HCN2重组腺病毒载体可成功转染猪骨髓间充质干细胞,使其能够表达HCN2通道蛋白,全细胞膜片钳可检测到起搏电流。BACKGROUND：Hyperpolarization-activated cyclic nucleotide-gated （HCN） current plays an important role in regulating heart spontaneous pulsation.OBJECTIVE：To observe target gene expression and electrophysiological characteristics of pig bone marrow mesenchymal stem cells （BMSCs） transfected with hyperpolarization-activated cyclic nucleotide-gated channel 2 （HCN2） gene recombinant adenovirus.DESIGN,TIME AND SETTING：Cell-gene in vitro study was performed at the Laboratory of Thoracic and Cardiovascular Surgery,Second Military Medical University of Chinese PLA from July 2007 to March 2008.MATERIALS：Yorkshire pig was supplied by Animal Institute,Second Military Medical University of Chinese PLA.HCN2 plasmid was presented by Professor Dario DiFrancesco from Italy.Recombinant adenovirus Ad.HCN2 was constructed and stored using Ad5 in this laboratory.METHODS：Pig BMSCs were isolated with combination of gradient centrifugation of Percoll and adherent treatment in vitro.Ad.HCN2 was transfected at multiplicity of infection=50.We also set non-transfection and transfected Ad.Null groups.MAIN OUTCOME MEASURES：Expression of HCN2 mRNA was detected with RT-PCR,and expression of HCN2 channel protein was examined with immunofluorescent staining.Electrophysiology of HCN2 channel protein was measured with whole-cell patch clamp.RESULTS：No amplified fragments were found in the non-transfection and transfected Ad.Null groups,but amplified fragments were determined at 250-500 bp following Ad.HCN2 amplification,which was the same as plasmid carrying HCN2 gene.Staining strength of cell nuclei following transfection was significantly weaken compared with cell membrane and plasma,which showed identical distribution as HCN2 protein.No HCN2 protein was detected in the non-transfection and transfected Ad.Null groups.Pacemaker current could be recorded with a whole-cell patch clamp.It was fully activated around-140 mV with an activation threshold of-60 mV,presenting voltage dependence.CsCl （4 mmol/L） reversibly block

关 键 词：HCN2 骨髓间充质干细胞 重组腺病毒 基因 起搏电流 

分 类 号：R394.2[医药卫生—医学遗传学]