缺氧状态下丝裂原活化蛋白激酶磷酸酶-1在胃癌细胞系SGC7901中的过表达及其意义  

The overexpression and significance of MKP-1 in oxygen-deprived gastric cancer cell line SGC7901

作　　者：刘长江[1] 时永全[1] 韩者艺[1] 薛妍[1] 潘阳林[1] 申慧琴[1] 杜静平[1] 韩霜[1] 乔泰东[1] 樊代明[1] 

机构地区：[1]第四军医大学西京医院消化病研究所,西安710032

出　　处：《中华医学杂志》2004年第4期306-311,共6页National Medical Journal of China

基　　金：国家自然科学基金资助项目 (3 0 10 0 0 79)

摘　　要：目的　探讨丝裂原活化蛋白激酶磷酸酶 1　 (MKP 1)在缺氧胃癌细胞系SGC790 1中的表达及对缺氧诱导因子 1(HIF 1)的影响。方法　采用半定量逆转录 聚合酶链反应 (RT PCR)和Western印迹检测MKP 1在常氧及缺氧状态下胃癌细胞系SGC790 1中的表达 ;借助DNA重组技术构建MKP 1基因的正义真核表达载体 ;利用脂质体将正义真核表达载体和空载体转入SGC790 1细胞 ;双荧光素酶报告基因 (DualLuciferaseReporter,DLR)检测系统检测荧光素酶活性并采用ELISA法检测SGC790 1细胞培养上清血管内皮生长因子 (VEGF)的变化。结果　 (1)半定量RT PCR和Western印迹结果提示 ,MKP 1在胃癌细胞系SGC790 1中表达 ,缺氧时其表达上调 ;(2 )分别将MKP 1正义真核表达载体和pcDNA3.1空载体瞬时转入SGC790 1细胞 ;转染 4 8h后 ,缺氧 12h磷酸化HIF 1α在正义真核表达载体转染细胞 (SGC790 1 MKP 1)中的表达低于空载体转染细胞 (SGC790 1 空载体 )和未转染细胞(SGC790 1)。 (3)报告基因试验显示常氧情况下 3、6和 12h荧光素酶活性在SGC790 1细胞、SGC790 1 空载体细胞和SGC790 1 MKP 1细胞中差异无显著意义 (P >0 .0 5 ) ;缺氧情况下 ,SGC790 1 MKP 1细胞中荧光素酶活性在 3、6和 12h均明显低于SGC790 1细胞和SGC790 1 空载体 (P <0 .0 1)Objective To investigate the expression status of Mitogen-activated Protein Kinase Phosphatase-1 in oxygen-deprived gastric cancer cell line SGC7901 and its role in HIF-1 regulation. Methods The expression of MKP-1 in gastric cell line SGC7901 was detected with Western blot and semiquantity RT-PCR; Eukaryotic sense expression vector was constructed based on DNA recombination technology. Transfections of SGC7901 were performed using liposome; The luciferase activity was determined using Dual Luciferase Reporter System and the levels of VEGF in SGC7901cells under normoxia and hypoxia were measured by ELISA. Results (1) Semiquantity RT-PCR and Western blot suggested that the expression of MKP-1 was elevated in oxygen-deprived gastric cancer cell line SGC7901; (2) 48 hours after transfection, the phosphorylated form of HIF-1α in cell line transfected with recombinant plasmids was lower compared with that in cell line transfected with empty vectors after 12 hours of exposure to hypoxia; (3) There was very low luciferase activity under nomoxia while under hypoxia luciferase activity increased in a time-dependent manner and at all time points there was significant lower luciferase activity in SGC7901 cells than in cells transfected with empty plasmids. (4) At different points of time course, the expression of VEGF in SGC7901 was significantly higher under hypoxia than that in SGC7901 under nomorxia, while under hypoxia, the expression of VEGF in SGC7901 transfected with recombinant plasmids was significantly lower than that in SGC7901 transfected with empty vectors at all time points as indicated. Conclusion The expression of MKP-1 in SGC7901 was elevated under hypoxia, which could downregulate the HIF-1 trans-activition activity thereby repressing the expression of downstream target gene VEGF.

关 键 词：缺氧状态 丝裂原活化蛋白激酶磷酸酶-1 胃癌 SGC7901细胞 DNA重组技术 基因表达调控 

分 类 号：R735.2[医药卫生—肿瘤]