重组慢病毒介导超极化激活的环核苷酸门控阳离子通道4基因转染BMSCs的实验研究  

EXPERIMENTAL RESEARCH OF RECOMBINANT LENTIVIRUS MEDIATED HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CATION CHANNEL 4 GENE TRANSFECTING BONE MESENCHYMAL STEM CELLS

作　　者：于风旭[1] 方易冰[1] 刘炫[1] 赖前成[1] 贾建博[1] 廖斌[1] 

机构地区：[1]泸州医学院附属医院心胸外科,四川泸州646000

出　　处：《中国修复重建外科杂志》2013年第12期1512-1516,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81041048)~~

摘　　要：目的探讨重组慢病毒（1entivirus，LVs）介导超极化激活的环核苷酸门控阳离子通道4（hyperpo。larization．activatedcyclicnucleotide．gatedcationchannel4，HCN4）基因转染大鼠BMSCs构建生物起搏细胞的可行性。方法选取3～5周龄SD大鼠，采用改良全骨髓贴壁培养法分离培养BMSCs。以LVs作为转染载体，增强绿色荧光蛋白（enhancedgreenfluorescentprotein，EGFP）为标记，构建LVs．HCN4．EGFP病毒液。取第3代BMSCs分别转染LVs—HCN4．EGFP病毒液（实验组）和LVs．EGFP空白病毒液（对照组），荧光显微镜观察转染24、48、72h后两组绿色荧光表达情况，72h时Westernblot检测HCN4蛋白表达；电生理检测实验组转染72h后BMSCs的起搏电流。结果实验组BMSCs形态正常、生长良好；48h后荧光显微镜下可见散在的绿色荧光，转染效率约10％；72h荧光表达稍增多，转染效率为20％～25％。对照组未见绿色荧光表达。Westernblot检测示转染72h后，实验组可见与HCN4蛋白相对分子质量相同的条带表达，而对照组仅有微弱表达；实验组蛋白条带灰度值为33．75±0．41，显著高于对照组的23．39±0．33（t=17．524，P=-0．013）。实验组转染后的BMSCs检测到起搏电流，并能被CsCl完全阻断，符合起搏电流特点。结论重组LVs介导HCN4基因成功转染大鼠BMSCs，并检测到HCN4蛋白表达及起搏电流。Objective To investigate the feasibility of recombinant lentivirus （LVs） mediated hyperpolarization- activated cyclic nucleotide-gated cation channel 4 （HCN4） gene transfecting rat bone mesenchymal stem cells （BMSCs） so as to construct the biological pacemaker cells. Methods Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein （EGFP） as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid （experimental group） and LVs-EGFP null virus liquid （control group）. Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group. Results After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group （33.75±0.41） was significantly higher than that of the control group （23.39 ± 0.33） （t=-17.524, P=0.013）. In the experimental group, the pacemaker current was recorded, and it could be blocked by CsC1, in accordance with the characteristics of pacemaker current. Conclusion The recombinant LVs mediated HCN4 gene is successfully transfected into ra

关 键 词：超极化激活的环核苷酸门控阳离子通道4 BMSCS 慢病毒转染 起搏电流 

分 类 号：R541.7[医药卫生—心血管疾病]