CAJ | 학술논문

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:细胞一基因学体外观察,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250-500 bp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-60 mV,完全激活电位-140 mV,呈电压依赖性,当给予4 mmol/L CsCI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.
배경:연구증실초겁화격활배화핵감산문공통도(hyperpolarization-activated cyclic nucleotide-gated,HCN)전류재조공심장적자발박동중기착비상중요적작용.목적:관찰HCN2기인중조선병독전염후저골수간충질간세포목적기인적표체급전생리특정.설계、시간급지점:세포일기인학체외관찰,우2007-07/2008-03재해방군제이군의대학흉심외과실험실완성.재료:Yorkshire저유해방군제이군의대학동물소제공,HCN2질립유의대리Dario DiFrancesco교수혜증,중조선병독Ad.HCN2유본실험실채용Ad5계통구건병보존.방법:Percoll밀도제도리심+첩벽법체외분리순화저골수간충질간세포,이감염복수=50진행Ad.HCN2전염,동시설립미전염조화전염Ad.Null조.주요관찰지표:통과RT-PCR、면역형광염색검측각조세포HCN2 mRNA화단백적표체,용전세포막편겸검측각조세포전생이학변화.결과:미전염조화전염Ad.Null조균미견확증편단,이Ad.HCN2확증후재250-500 bp가견확증편단,여휴대HCN2기인적질립확증출적편단위치상동.전염후세포핵염색강도명현약우포막화포장,여HCN2단백적분포상부합,미전염조급전염Ad.Null조무HCN2단백양성표체.전세포막편겸가기록도기박전류,기격활전위약위-60 mV,완전격활전위-140 mV,정전압의뢰성,당급여4 mmol/L CsCI후,내향적기박전류즉피명현억제:미전염조급전염Ad.Null조세포균무기박전류.결론:통과기박기인HCN2중조선병독재체가성공전염저골수간충질간세포,사기능구표체HCN2통도단백,전세포막편겸가검측도기박전류.
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. CsCI (4 mmol/L) reversibly blocked the inward currents. No pacemaker current was detected in the non-transfection and transfected Ad.Null groups.CONCLUSION: The HCN2 recombinant adenovirus carrier was transferred into serial subcultivation porcine bone marrow mesenchymal stem cells. HCN2 channel protein has been expressing. Pacemaker current could be recorded with a whole-cell patch clamp.