shRNA沉默肝癌细胞Kir6.2基因对K_(ATP)通道电流的影响  

Effect of shRNA silencing Kir6.2 gene in hepatoma cells on the K_(ATP) current.

作　　者：苏晓通[1] 梁平[1] 丁生才[1] 杨彤翰[1] 韩克强[1] 

机构地区：[1]第三军医大学新桥医院肝胆外科,重庆400037

出　　处：《四川医学》2008年第6期643-645,共3页Sichuan Medical Journal

摘　　要：目的研究shRNA干扰肝癌细胞Kir6.2基因后KATP通道电流的变化。方法脂质体法将构建的3种shRNA质粒表达载体HK(隐性对照质粒)、K1(质粒携带干扰序列1)、K2(质粒携带干扰序列2)分别转染肝癌细胞。实验分为4组:SK组(未转染)、HK组、K1组、K2组。采用膜片钳全细胞记录方法记录KATP通道电流。高阻封接后,用10mmol/L2-脱氧-D-葡萄糖灌流液灌流10min,测量各组的电流,最后换成100μM格列本脲灌流液灌流10min,再次测量电流,再次电流相减即可得格列本脲敏感的KATP电流。绘制Ⅰ-Ⅴ曲线。结果在SK、HK、K1、K2 4组中,KATP电流分别为(n=7)(nA)(1.80±0.673)、(1.87±0.541)、(5.16±1.011)、(3.89±1.287)。K1、K2组与SK组比较差异有统计学意义。K1、K2组KATP电流的幅度明显高于HK组。结论shRNA沉默肝癌细胞Kir6.2基因后KATP通道电流改变明显。Objective To study the change of the ATP-sensivive potassium（ KAy）current after shRNA silencing Kit6.2 gene in hepatoma cells. Methods Plassmid vectors containing negative control sequence HK and 2 kinds of different sequences of K1 and K2 were constructed and transfected into Sk-Hep1 cells by using Lipofectamine 2000 methods,respectively .The experiment was divided into 4 groups：SK（normal） ,HK,K1 and K2. The selected single clone was cultured after screening by G418. By using the whole-cell recording the current ATP-sensitive potassium channel was recorded with voltage-clamp mode. After the gigahom seal was formed, perfusion with 10mmol/12-deoxy-D-glucose was perfused 10 minutes to test the current in each group, then perfusion with 100uM gloibenclamide 10 minutest to test the current in each group. The glibenclamide-sensitive KATP current was obtained by substraction of the current of two times. Ⅰ-Ⅴ curves was drawed. Results The KATP current in SK, HK, K1,K2 groups（n = 7）（nA）was （1.80±0.673）,（1.87±0.541 ）, 15.16±1.011 ）, 13.89±1.287） ,respectively.Compared the curren in K1 and K2 gruops with that in SK group,there was significantly statistical.the current in HK group was much lower than that in K1 and K2 groups. Ⅰ -Ⅴ curves in K1 and K2 groups were close to the vertical axis.Conclusion The KATP current changed dramatically after the shRNA silencing the Kit6.2 gene in hepatoma cells.

关 键 词：SHRNA 肝癌细胞 全细胞记录 2-脱氧-D-葡萄糖 格列本脲 

分 类 号：R735.7[医药卫生—肿瘤]