mHCN4基因修饰小鼠骨髓间充质干细胞重建起搏离子流通道  

The reconstruction of I_f channels in mouse mesenchymal stem cells with mHCN4

作　　者：仝识非[1] 宋治远[1] 姚青[1] 万瑛[2] 邹丽云[2] 钟理[1] 

机构地区：[1]第三军医大学西南医院心内科,重庆400038 [2]第三军医大学免疫学研究所

出　　处：《中国心脏起搏与心电生理杂志》2007年第1期51-54,共4页Chinese Journal of Cardiac Pacing and Electrophysiology

基　　金：国家自然科学基金资助项目(项目编号:30370585)

摘　　要：目的观察超极化激活的环核苷酸门控通道亚型4(mHCN4)外源基因对小鼠骨髓间充质干细胞(mMSCs)的转染及其功能表达。方法2个月大昆明小鼠拉颈处死,无菌条件下分离股、胫骨,收集骨髓细胞种植于塑料培养瓶,24h后更换培养液,保留贴壁细胞,待细胞达90%融合时胰酶消化传代培养。收集第3代贴壁细胞,用免疫磁珠法分选CD11b-细胞继续扩增培养。以pUC18、pcDNA3-mHCN4、pIRES2-EGFP、pMSCV-puro等质粒载体为架构,用不同酶类将目的片段连接构建pMSCV-mHCN4-EGFP及pMSCV-EGFP逆转录病毒载体。载体转染PT67包装细胞,收集逆转录病毒上清转染第3代扩增的mMSCs细胞,观察mMSCs的转染效果及阳性转染细胞的mHCN4通道动力学特性。结果mMSCs的转染效率约10%～20%。mHCN4基因转染组可记录到明显的时间依赖性的超极化激活内向电流(If),If的半数最大激活电压为-99.0±5.8mV,在-140mV电压时的激活时间常数为451±61ms。该电流对CsCl高度敏感;对照组细胞在超激化状态下无明显的电流出现。结论mHCN4外源基因可在mMSCs中成功表达起搏离子流通道,基因修饰后的mMSCs有可能成为一种有效的生物起搏种子细胞。Objective To study the transfection and functional expression of mHCN4 gene in mouse mesenchymal stem cells （mMSCs）. Methods Bone marrow was collected from 2-month-old Kunming mice by flushing femurs and tibias with complete medium constituted of DMEM-LG. Cells were plated in a Petri dish at a density of 1 × 10^6 cells/cm^2, After 24 hours, non-adherent cells were removed by two to three washes with PBS, adherent cells were further euhured in com- plete medium and retrieved by trypsinisation and immuno-depleted of granulo-monoeytic cells using a biotinylated antibody against CDllb. CDllb-negative cells were subsequently seeded on human fibronectin-coated Petri dishes, pMSCV-mHCN4-EGFP and pMSCV-EGFP retrovirus vectors were constructed by enzymatic reconnection of target segments from vectors pUC18, pcDNA3-mHCN4, pIRES2-EGFP and pMSCV-puro. The stably expressed packaging cell clones were collected by transfecting PT67 packaging cell with different constructed vectors, transfected 3rd generation of mMSCs,purified by puromycin, and finally examined the positively transfected cells with whole-cell patch clamp experiments. Results Transfection ratio for mMSCs was 10% - 20%. The recorded hyperpolarization-activated inward current （ If ） from mHCN4 target gene after 3rd day transfection had obvious time dependency. The half-maximum activated voltage of If was - 99 ± 5.8 mV, activation time constant was 451 ±61 ms at -140 mV. The current was very sensitive to CsCl. The control cells didn＇t show the current at the same clamp voltage. Conclusion The functional Is channels can be reconstructed in mMSCs transfected with mHCN4 gene. The mMSCs modified with mHCN4 gene maybe a source of effective biological pacemaker cells.

关 键 词：电生理学 间充质干细胞 超极化激活的环核苷酸门控通道 小鼠 起搏离子流 

分 类 号：R318.11[医药卫生—生物医学工程]