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作 者:马金[1] 张存泰[1] 黄深[1] 王国强[1] 全小庆[1]
机构地区:[1]华中科技大学同济医学院附属同济医院综合科,湖北武汉430030
出 处:《中国心脏起搏与心电生理杂志》2009年第1期51-54,共4页Chinese Journal of Cardiac Pacing and Electrophysiology
摘 要:目的构建超极化激活环的环核苷酸门控通道亚型2(mHCN2)基因修饰大鼠骨髓间充质干细胞(mMSCs),采用膜片钳技术记录细胞的内向电流并检测其电生理特征。方法采用密度梯度离心法和贴壁分离法分离获得mMSCs。EcoRⅠ和BamHⅠ双酶切质粒pGH-mHCN2和pIRES2-EGFP,T4连接酶连接,构建质粒pIRES2-EGFP-mHCN2。脂质体将质粒转染至mMSCs,荧光显微镜下鉴定。全细胞膜片钳记录IHCN2并加入起搏电流特异性阻断剂Cs+检测其电流变化。结果酶切鉴定及测序检测证明质粒pIRES2-EGFP-mHCN2构建成功。荧光显微镜下可见转染成功的mMSCs发出绿色荧光。膜片钳记录到转染mHCN2基因的mMSCs的电压依赖性内向电流,其半最大激活电位为-95.1±0.9 mV,阈电位为-60 mV,最大激活电位为-140 mV。电极外液中加Cs+,内向电流几乎被完全抑制。未转染mHCN2的mMSCs未记录到内向电流。结论mHCN2基因在mMSCs中表达具有生理性起搏电流特征的电流,基因修饰的mMSCs有可能替代窦房结起搏细胞在自动除极过程中发挥重要作用。Objective To record and detect the inward currents of mouse mesenchymal stem cells (mMSCs) which were transfected with mouse hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) by patch clamp. Methods mMSCs were obtained by density gradient centrifugation method and adherence separation. The plasmid pGH and plasmid pIRES2-EGFP were digested by EcoR I and BamH I ~ The objective fragments were linked by T4 DNA Ligase to construct the plasmid pIRES2-EGFP-EGFP. The objective gene was transfected with Lipofectamine 2000 into mMSCs and the results were observed by fluorescence microscope. IHCNS2 was recorded by whole-cefl patch clamp. The effect of Cs* which was the specific blocker of pacemaker current on IHCN2 was detected. Results The identification using restriction enzyme and se- quencing indicated that the mHCN2 was inserted to the pIRES2-EGFP. The green fluorescence in transfected mMSCs after 24 to 48 hours was observed: Non-transfected mMSCs demonstrated having no significant voltage-dependent currents, mHCN2-transfected mMSCs expressed a large voltage-dependent inward current activating hyperpolarization. IHCN2 was fully ac- tivated around -140 mV with an activation threshold of -60 inV. The midpoint(V50) was -95.1 ±0.9 mV(n =9). Conclusion mHCN2-transfected mMSCs expresses the currents with physiological pacemaker current character. It may substitute the sino-atrial node pacemaker cells and have important effects on depolarization. [ Chinese Journal of Cardiac Pacing and Electrophysiology,2009,23 (1) :51 -54 ]
关 键 词:电生理学 间充质干细胞 超极化激活环核苷酸门控离子通道 膜片钳 起搏电流
分 类 号:R331.38[医药卫生—人体生理学] R318.11[医药卫生—基础医学]
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