重组人胰岛素原基因在培养的大鼠肝癌细胞中基因转移和表达  

Gene transfer and expression of recombined human proinsulin in hepatoma cells of rats

作　　者：王玉霞[1] 袁凤山[1] 吴志香[1] 杨立新[1] 杜宁[1] 刘兆喆[1] 

机构地区：[1]中国医科大学附属第一医院内分泌科,辽宁沈阳110001

出　　处：《中国病理生理杂志》2007年第2期242-245,共4页Chinese Journal of Pathophysiology

基　　金：辽宁省自然科学基金资助项目(No.962293)

摘　　要：目的:探讨含有葡萄糖反应元件的重组人胰岛素原基因在培养的大鼠肝癌细胞中基因转移及表达情况。方法:将含有3点突变及2点突变的人胰岛素原基因转染至大鼠肝癌CBRH7919细胞,测定不同葡萄糖浓度下瞬时及用G418筛选稳定表达后胰岛素的分泌量,并检测基因整合情况。结果:①转染后38 h及1个月,不同葡萄糖浓度下,含有3点突变胰岛素原基因的肝癌细胞胰岛素分泌量不同;含有2点突变胰岛素原基因的肝癌细胞,只有在25 mmol/L的葡萄糖浓度培养液中,才能测出胰岛素分泌量;②阳性克隆细胞基因组扩增出特异目的电泳条带,未转染细胞无此条带。结论:①用逆转录病毒为载体能将重组人胰岛素原基因转染到肝癌细胞中,并使之稳定表达。②所构建的重组人胰岛素原基因能指导靶细胞随葡萄糖浓度的变化调节胰岛素分泌。AIM： To explore the recombined human proinsulin gene containing glucose reaction element （GL- RE） expression in transfected CBRH7919 cells. METHODS： The packaged retrovirus encoding genetically modified human proinsulin PLXSN - （GLRE）3 - BP - 1MpINS3 and PLXSN - （GLRE）3 - BP - 1MpINS2 were transfected into CBRH7919 cells. Insulin values in cells after transient and steady expression screened by G418 at different glucose levels were detected. Chromosome DNA was isolated from transfected and untransfected cells and polymerase chain reaction （PCR） was performed. PCR products were analyzed by electrophoresis. RESULTS： 38 h after transfeetion, at the glucose levels of 0 - 25 mmol/L, the levels of insulin produced by ceils including PLXSN - （ GLRE ） 3 - BP - 1MpINS3 were （ 3.57 ±0.21）U/L, （5.30 ±0.20）U/L, （16.27 ±0.87）U/L, （23.23 ±1.12）U/L, respectively （P〈0.05）. One month after transfection, under above glucose levels, insulin values were （ 3.57 ± 0. 21 ） U/L, （ 5. 30 ± 0. 20 ） U/L, （ 16. 27 ± 0. 87）U/L, （23.23 ± 1.12）U/L （P 〈0. 05）. CBRH7919 cells including PLXSN - （ GLRE）3 - BP - 1MpINS2 secreted detectable insulin value at the level of 25 mmol/L, they were （2. 10 ± 0.23 ）U/L and （2.05 ± 0. 17 ）U/L, respectively. PCR products of transfected ceils showed target band, but control cells did not. CONCLUSIONS ： Recombined proinsulin gene was transfected successfully in CBRH7919 ceils. The ceils combined human proinsulin gene has the ability of producing insulin with increase in glucose concentration in vitro.

关 键 词：葡萄糖 胰岛素 肝肿瘤 基因转移 基因表达 

分 类 号：R363[医药卫生—病理学]