mHCN2基因修饰的大鼠骨髓间充质干细胞内向电流的记录及检测

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。

To record and detect the inward currents of mouse mesenchymal stem cells modified by mHCN2 gene

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 ]