重组pcDNA 3.0-WWOX构建对胆囊癌细胞表达及△Ψm的影响  

The Influence of pcDNA 3.0-WWOX Recombinant Vector on the Expression of WWOX and △ψm in Gallbladder Carcinoma Cells

作　　者：魏东[1] 杨珮 吴雪松[3] 戈佳云[1] 施智甜 王琳[1] 邹浩[1] 张小文[1] 

机构地区：[1]昆明医科大学第二附属医院肝胆胰外科,云南昆明650101 [2]曲靖市第二人民医院胃肠外科,云南曲靖655000 [3]昆明医科大学第二附属医院胃肠外科,云南昆明650101

出　　处：《昆明医科大学学报》2016年第4期15-21,共7页Journal of Kunming Medical University

基　　金：云南省应用基础研究计划项目(2011FZ124);昆明医科大学博士研究生创新基金资助项目(2012D05)

摘　　要：目的体外构建及鉴定Pc DNA3.0-WWOX真核表达载体,探讨过表达WWOX对人胆囊癌细胞WWOXm RNA表达、蛋白表达及跨膜电位(△Ψm)的影响.方法提取人胆囊癌细胞总RNA,RT-PCR反应合成WWOX的c DNA,PCR产物回收纯化.目的基因WWOX和pc DNA3.0载体经限制性内切酶ECORⅠ和XhoⅠ酶切,将双酶切WWOX片段与pc DNA 3.0载体在T4 DNA连接酶的作用下进行连接重组.pc DNA 3.0-WWOX重组质粒转化后,提取质粒经双酶切鉴定及测序鉴定.将pc DNA 3.0-WWOX重组质粒转染人胆囊癌GBC-SD细胞,应用RT-PCR、Western Blot检测WWOX基因的表达水平、JC-1染色检测胆囊癌细胞线粒体跨膜电位(△Ψm)变化.结果成功构建了pc DNA3.0-WWOX重组载体,经测序DNA序列与Gene Bank中WWOX序列一致.成功转染pc DNA3.0-WWOX重组质粒至胆囊癌GBC-SD细胞中.RT-PCR及Western blot结果显示:胆囊癌GBC-SD细胞转染pc DNA3.0-WWOX重组质粒48 h后pc DNA-3.0-WWOX组灰度比值明显高于各对照组(P<0.05),对照组间m RNA表达量差异无统计学意义(P>0.05).pc DNA3.0-WWOX组蛋白表达灰度比值均明显高于各对照组(P<0.05),对照组间蛋白表达量差异无统计学意义(P>0.05).pc DNA3.0-WWOX组细胞线粒体内绿色荧光信号较对照组增高(P<0.05),而对照组间细胞线粒体内绿色荧光信号差异无统计学意义(P>0.05).结论体外成功构建了Pc DNA3.0-WWOX重组载体,顺利进行了重组载体的转化和转染实验.靶向Pc DNA3.0-WWOX重组载体可有效抑制胆囊癌细胞WWOXm RNA降解,促进其蛋白的表达,同时有效促进细胞的早期凋亡.WWOX可能参与线粒体依赖的凋亡途径调控胆囊癌细胞的生长.Objective To explore the influence of Pc DNA3.0- WWOX eukaryotic expression vector on the expression of WWOXm RNA, protein and transmembrane potential of mitochondrion（ △Ψm） in human gallbladder carcinoma cells. Me thods Total RNA in the human gallbladder carcinoma cells was extracted. Then the c DNA of WWOX gene was synthesized by RT- PCR and PCR products were reclaimed and purified. Both of WWOX gene and pc DNA3.0 vector were cut by restriction enzymes ECOR Ⅰ and Xho Ⅰ. The double enzyme WWOX fragments and pc DNA 3.0 vector were restructured under the action of T4 DNA ligase. After Pc DNA 3.0- WWOX recombinant plasmid transformation,the extraction of the plasmid by double enzyme cutting was sent to identification and sequencing identification. Then the pc DNA 3.0- WWOX recombinant plasmid was used to transfect gallbladder cancer GBC- SD cells. RT- PCR and Western Blot were used to detect the expression level of WWOX gene and JC- 1dyeing was used to detect the transmembrane potential（△Ψm） of gallbladder cancer cells＇ mitochondria.Re s ults We successfully construct pc DNA3.0- WWOX recombinant plasmid, and the sequencing analysis showed that the DNA sequence was the same to WWOX sequence in Gene Bank. pc DNA3.0- WWOX recombinant plasmid was successfully transfected to gallbladder cancer GBC- SD cells. RT- PCR and Western blot results showed that 48 hours after plasmid transfection in gallbladder cancer GBC- SD cells, the m RNA gray ratio of pc DNA- 3.0- WWOX group was obviously higher than that of the control groups（P〈0.05）,the amount of m RNA expression between the control groups had no significant difference（P〉0.05）. The protein gray ratio of pc DNA- 3.0- WWOX group was obviously higher than that of the control group（P〈0.05）, and the amount of protein expression between the control groups had no significant difference（P〉0.05）. The green fluorescent signals in mitochondria in cells in the pc DNA- 3.0- WWOX group was higher than the control groups（P〈0.05）,while ha

关 键 词：抑癌基因WWOX 胆囊癌细胞 转染 过表达 跨膜电位 

分 类 号：R735.8[医药卫生—肿瘤]