原癌基因pim-3的克隆及其对肝癌细胞凋亡的影响  

Cloning of pro-oncogene pim-3 cDNA and its effect on apoptosis of hepatocellular carcinoma cells

作　　者：邓欢[1] 刘亮明[1] 张吉翔[1] 罗杰[1] 尹东[2] 熊瑛[1] 汤蕾[1] 谢正元[1] 

机构地区：[1]南昌大学第二附属医院消化内科 [2]江西省分子医学重点实验室,江西省南昌市330006

出　　处：《世界华人消化杂志》2006年第23期2288-2293,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金项目;No.30160032;No.30360037;江西省科委重点项目;No.2001 10300101;No.2004180300300~~

摘　　要：目的:克隆大鼠原癌基因pim-3并构建真核表达重组质粒pEGFP-N2／pim-3,观察他在真核细胞SMMC-7721中的表达情况以及对细胞凋亡的影响．方法:利用TRIzol从液氮保存的大鼠骨骼肌组织中提取总RNA,采用RT-PCR方法获取pim-3 cDNA,并构建重组质粒pEGFP-N2／pim-3,转化用CaCl2法制备的大肠杆菌JM-109菌株,酶切以及测序鉴定．将重组质粒通过脂质体介导转染肝癌SMMC-7721细胞后利用倒置荧光显微镜观察基因表达情况,并利用流式细胞术及MTT比色实验对转染细胞的生物学行为进行检测．结果:将RT-PCR产物全部用于低熔点琼脂糖凝胶电泳、回收,在DL 2000 Marker 1000 bp附近可见清晰条带,与实验设计符合;阳性克隆质粒的酶切电泳和测序结果表明,大鼠pim-3 cDNA的克隆和重组质粒pEGFP-N2／pim-3的构建成功;重组质粒pEGFP-N2/pim-3转染组凋亡细胞占3．5％,质粒pEGFP-N2转染组凋亡细胞占10．7％,而仅加入转染液的空白组凋亡细胞占11．0％,重组质粒转染组与两对照组之间差异有统计学意义(P<0．05);倒置荧光显微镜可以观察到pim-3在SMMC-772 1细胞中的正常表达;MTT比色实验显示原癌基因pim-3能够明显抑制细胞凋亡．结论:真核表达重组质粒pEGFP-N2/pim-3构建成功;原癌基因pim-3能明显抑制肝癌细胞凋亡．AIM： To clone rat pim-3 cDNA and construct its eukaryotic expressing plasmid, and to investigate the expression of pim-3 and its effect on the apoptosis in SMMC-7721 cells. METHODS： Total RNA skeletal muscle tissues was isolated from rat with TRIzol method. pim-3 cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR） and constructed into eukaryotic expression vector pEGFP-N2, which expressed green fluorescent protein. The recombinant pEGFP-N2/pim-3 was then transformed into ria. After identified by E.coli JM109 host bacterestriction endonuclease digestion and sequencing, plasmid pEGFP-NJ pim-3 was transfected into SMMC-7721 cells by liposome mediation. Fluorescent microscopy was used to examine the expression of pim-3 gene, and the biological behaviors of the transfected cells were tested by flow cytometry and MTT colorimetric assay. RESULTS： Restriction enzyme digestion analysis and sequencing showed that the recombinant plasmid of rat pim-3 was correctly constructed; Flow cytometry showed the apoptosis rate was 3.48% in the cells transfected with the recombinant plasmid pEGFP-N2/pim-3, 10.74% in those transfected with the plasmid pEGFP-N2, and 11.01% in those without transfection. There were signicant differences between the recombinant plasmid transfected group and the other two control groups （both P 〈 0.05）. Fluorescent microscopy and MTT showed that pim-3 was expressed in SMMC-7721 cells and the apoptosis of the transfected cells was significantly inhibited. CONCLUSION： pim-3 gene is successfully cloned, and it can induce apoptosis of hepatocel- lular carcinoma cells.

关 键 词：肝癌 原癌基因pim-3 克隆 凋亡 荧光显微镜 MTT比色试验 流式细胞术 

分 类 号：R735.7[医药卫生—肿瘤]