PTEN-PI3K/AKT信号传导途径对胃癌细胞株MKN28凋亡的影响  被引量:3

Effects of PTEN-PI3K/AKT pathway on the apoptosis of gastric cancer MKN28 cells

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作  者:许小涛[1] 陶泽璋[1] 宋启斌[1] 姚颐[1] 阮鹏[1] 

机构地区:[1]武汉大学人民医院肿瘤科,430060

出  处:《中华消化外科杂志》2012年第4期377-381,共5页Chinese Journal of Digestive Surgery

基  金:国家重点基础研究发展计划(973计划)项目(2006CB705706)

摘  要:目的探讨PTEN—P13K/AKT信号传导途径对胃癌细胞株MKN28凋亡的调控作用及其机制。方法构建PTEN真核表达载体,转染至胃癌细胞株MKN28中(转染组);同样方法转染空载体和等量的PBS分别作为阴性对照组和空白对照组,检测对胃癌细胞株MKN28凋亡的影响及对P13K、AKT、Caspase-3、Caspase-9的影响。MTT检测细胞生长曲线,TUNEL染色法检测细胞凋亡,Westernblot检测蛋白的表达。P13K抑制剂LY294002处理未转染PTEN的胃癌细胞株MKN28(处理组);对照组加入等量的PBS,抑制P13K活性后检测细胞凋亡蛋白及凋亡相关蛋白的表达。组间比较采用t检验,时间依赖性检验采用相关分析方法。结果成功构建PTEN真核表达质粒并转染至胃癌细胞株MKN28中,获得稳定过表达PTEN的细胞模型。MTT检测发现转染组胃癌细胞株MKN28生长明显受到抑制,且呈时间依赖性(r=0.938,P〈0.05)。转染组平均凋亡率达到27.86%±4.78%,明显高于阴性对照组的0.01%±0.01%(t=9.527,P〈0.05)。转染PTEN后,转染组P13K蛋白表达量为0.25±0.03,明显低于空白对照组的0.93±0.16及阴性对照组的0.96±0.15(t=7.235,8.883,尸〈0.05)。转染组活化的AKT(P.AKT)蛋白表达量为0.214-0.03,明显低于空白对照组的0.93±0.13及阴性对照组的0.91±0.12(t=9.347,9.802,P〈0.05)。而转染组凋亡相关蛋白Caspase-3及Caspase-9表达量分别为0.86±0.11和0.87±0.12,明显高于阴性对照组的0.16±0.03和0.18±0.04及空白对照组的0.15±0.02和0.16±0.03(t=10.634,10.999,9.448,9.942,P〈0.05)。抑制P13K活性后,处理组细胞凋亡率为28.60%±4.50%,明显高于对照组的0.12%±0.06%(t=10.961,P〈0.05)。Westernblot检测发现处理组P13K和P—AKT的蛋白表达量分别为0.18±0.02和0.11±0.叭,明显低于对�Objective To investigate the regulatory effects of PTEN-PI3K/AKT pathway on the apoptosis of gastric cancer MKN28 cells and the possible mechanisms. Methods The specific sequence of PTEN was transfected to MKN28 cells by eukaryotic expression vector (transfection group), and then vacant vector (negative control group) and PBS (blank control group) were transfected to the MKN28 cells, respectively. The effects of FFEN-PI3K/AKT pathway on the apoptosis of MKN28 cells and the expressions of PI3K, AKT, Caspase-3 and Caspase-9 were investigated. The growth curve and apoptosis of the MKN28 cells were detected by MTF assay and TUNEL staining, respectively, and the protein expression was detected by the Western blot. MKN28 cells which did not transfeeted by the PTEN were processed by inhibitor of PI3K (LY294002) (treated group), and MKN28 cells in the control group were processed by PBS. The expressions of apoptosis protein and apoptosis-related protein were detected after inhibition of PI3K. All data were analyzed using the t test. Results The model of over-expression of PTEN was obtained and transfected into MKN28 cells. The survival of MKN28 cells in the transfection group was significantly inhibited in a time-dependant manner ( r = 0. 938, P 〈 0.05 ). The mean apoptotic rate of the transfection rate was 27.86% ± 4.78% , which was significantly higher than 0.01% ± 0.01% of the negative control group ( t = 9. 527, P 〈 0.05). The protein expression of PI3K in the transfection group was 0.25 ±0.03, which was significantly lower than 0.93 ± 0.16 of the blank control group and 0.96 ± 0. 15 of the negative control group ( t = 7. 235, 8. 883, P 〈 0.05 ). The protein expression of P-AKT in the transfection group was 0.21 ± 0.03, which was significantly lower than 0.93 _± 0.13 of the blank control group and 0.91 ± 0.12 of the negative control group (t = 9. 347, 9. 802, P 〈 0.05). The expressions of Caspase-3 and Caspase-9 of the transfection group were 0.86 ±0.11 and 0.87-±

关 键 词:胃肿瘤 PTEN PI3K AKT 凋亡 

分 类 号:R735.2[医药卫生—肿瘤]

 

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