慢病毒介导KCNMA1体外转染大鼠间充质干细胞及功能测定  

Transfection of the KCNMA1 Lentiviral into the Mesenchymal Stem Cells in vitro and Its Function Assay

作　　者：何跃[1] 肖明朝[1] 聂永华[1] 何卫阳[1] 秦国东[1] 罗家宇[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科,重庆400016

出　　处：《中国细胞生物学学报》2012年第11期1110-1116,共7页Chinese Journal of Cell Biology

基　　金：重庆市自然科学基金(No.2009BB5411)资助项目~~

摘　　要：间充质干细胞具有高度增殖、自我更新和多向分化的潜能。大电导钙离子激活的钾通道M亚族α亚基(potassium large conductance calcium-activated channel,subfamily M,alpha member 1,KCNMA1)介导细胞内K+的外流,使细胞膜超极化,降低细胞的兴奋性。该研究通过制备KCNMA1重组慢病毒载体和空白对照慢病毒载体,将其转染至间充质干细胞内,测定转染复数值(MOI),并通过RT-PCR和Western blot比较转染前后KCNMA1的表达变化情况,检测转染前后细胞微环境中电解质浓度变化。结果成功包装了KCNMA1慢病毒载体和空白病毒载体并转染入干细胞内;含有目的基因的慢病毒转染间充质干细胞后,RT-PCR和Western blot提示KCNMA1过表达,且细胞微环境中K+浓度升高。证实成功地将含KCNMA1的慢病毒载体转染进入大鼠间充质干细胞内,并在细胞内过表达且发挥功能,为体内研究KCNMA1结合干细胞治疗相关疾病奠定了基础。Mesenchymal stem cells have a high degree potential of proliferation, self-renewal and multi-directional differentiation. Large conductance calcium-dependent potassium channel, subfamily M, alpha member 1 （KCNMA1） mediated intracellular K^＋ outflow, so that it made the membrane hyperpolarization and reduced cell excitability. The study through the preparation KCNMA1 recombinant lentivirus and blank vectors lentivirus to transfected into the stem cells, tested the multiplicity of infection （MOI）, detected the expression changes of KC- NMA1 and the microenvironment electrolyte concentration before and after KCNMA1 transfected into cells. Results showed that we transfected KCNMA1 and blank vectors lentiviral successfully into the stem cells. The results of RT-PCR and Western blot showed that KCNMA1 over-expressed in the transfected KCNMA1 cells compared with the blank vector cells and untransfected cells, and K^＋ concentration in the microenvironmental of transfected KCNMA 1 cells was higher than blank vector cells and untransfected cells. It is confirmed that we had successfully transfected the lentiviral containing KCNMA1 into the BM-MSCs and the cells over-expressed KCNMA1 and performed its functions, laying the foundation for in vivo studies about KCNMA1 and stem cell therapy related diseases.

关 键 词：大电导钙离子依赖的钾通道M亚族α亚基 骨髓间充质干细胞 慢病毒 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]