| 摘要: |
| ［摘要］目的 体外构建及鉴定PcDNA3.0-WWOX真核表达载体，探讨过表达WWOX对人胆囊癌细胞WWOXmRNA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cDNA，PCR产物回收纯化．目的基因 WWOX和 pcDNA3.0载体经限制性内切酶ECORⅠ和XhoⅠ酶切，将双酶切WWOX片段与 pcDNA 3.0载体在T4 DNA连接酶的作用下进行连接重组．pcDNA 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pcDNA 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pcDNA3.0-WWOX重组载体，经测序DNA 序列与Gene Bank中WWOX序列一致．成功转染pcDNA3.0-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pcDNA3.0-WWOX重组质粒48 h后pcDNA-3.0-WWOX组灰度比值明显高于各对照组（P＜0.05），对照组间mRNA表达量差异无统计学意义（P＞0.05）．pcDNA3.0-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）．pcDNA3.0-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PcDNA3.0-WWOX重组载体，顺利进行了重组载体的转化和转染实验．靶向PcDNA3.0-WWOX重组载体可有效抑制胆囊癌细胞WWOXmRNA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． |
| 关键词: ［关键词］抑癌基因WWOX 胆囊癌细胞 转染 过表达 跨膜电位 |
| DOI： |
| 分类号: |
| 基金项目:［基金项目］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项目（2012D05） |
|
| The Influence of pcDNA 3.0-WWOX Recombinant Vector on the Expression of WWOX and △Ψm in Gallbladder Carcinoma Cells |
|
WEI Dong1,2,3
|
|
1.（1）Dept. of Hepatopancreatobiliary Surgery;2）Dept. of Gastrointestinal Surgery ， The Second People’s Hospital of Qujing，Qujing Yunnan 655000;3）Dept. of Gastrointestinal Surgery，The Second Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650101，China）
|
| Abstract: |
| ［Abstract］Objective To explore the influence of PcDNA3.0-WWOX eukaryotic expression vector on the expression of WWOXmRNA，protein and transmembrane potential of mitochondrion（△Ψm） in human gallbladder carcinoma cells. Methods Total RNA in the human gallbladder carcinoma cells was extracted. Then the cDNA of WWOX gene was synthesized by RT-PCR and PCR products were reclaimed and purified. Both of WWOX gene and pcDNA3.0 vector were cut by restriction enzymes ECOR Ⅰ and XhoⅠ. The double enzyme WWOX fragments and pcDNA 3.0 vector were restructured under the action of T4 DNA ligase. After PcDNA 3.0-WWOX recombinant plasmid transformation，the extraction of the plasmid by double enzyme cutting was sent to identification and sequencing identification. Then the pcDNA 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-1 dyeing was used to detect the transmembrane potential （△Ψm） of gallbladder cancer cells' mitochondria．Results We successfully construct pcDNA3.0 - WWOX recombinant plasmid，and the sequencing analysis showed that the DNA sequence was the same to WWOX sequence in Gene Bank. pcDNA3.0 - 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 mRNA gray ratio of pcDNA-3.0-WWOX group was obviously higher than that of the control groups（P＜0.05），the amount of mRNA expression between the control groups had no significant difference（P＞0.05）. The protein gray ratio of pcDNA-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 pcDNA-3.0-WWOX group was higher than the control groups（P＜0.05），while had no significant difference between the control groups（P＞0.05）. Conclusions We have successfully constructed PcDNA3.0-WWOX recombinant vector and performed transformation and transfection of this vector in vitro. Targeted PcDNA3.0 - WWOX recombinant vector can effectively inhibit WWOX mRNA degradation in the gallbladder cancer cells， effectively promote the expression of the protein and early apoptosis of the cells. WWOX gene may participate in mitochondria dependent apoptotic pathways which regulates gallbladder cancer cells growth. |
| Key words: ［Key words］Tumor suppressor gene WWOX Gallbladder cancer cell Transfection Overexpression Transmembrane potential |