TMEM88真核表达质粒的构建及其功能研究  

Construction of TMEM88 eukaryotic expression plasmid and its function research

作　　者：倪升丽 胡富勇[1] 李增[2] 孟晓明[2] 王学富[2] 徐涛[2] 

机构地区：[1]安徽医科大学附属合肥医院(合肥市第二人民医院)肿瘤放疗科,合肥230011 [2]安徽医科大学药学院,合肥230032

出　　处：《安徽医科大学学报》2017年第6期839-842,共4页Acta Universitatis Medicinalis Anhui

基　　金：合肥市科技攻关计划项目(编号:合科[2015]163号-38);安徽医科大学博士科研资助基金(编号:XJ201536);安徽高校自然科学研究重点项目(编号:KJ2016A348)

摘　　要：目的构建TMEM88的真核表达质粒,并研究其对肝癌细胞增殖和凋亡的影响。方法提取人肝星状细胞的总RNA,逆转录成cDNA作为模板,利用PCR法扩增出TMEM88的CDS序列,双酶切后连接到pEGFP-C2载体上,然后进行转化、质粒抽提、酶切鉴定,最后挑取阳性克隆送生物公司测序。将pEGFP-C2-TMEM88真核表达质粒分别转染至人肝癌细胞株SMMC-7721中,通过MTT实验和流式细胞术检测其对细胞增殖和凋亡的影响。结果测序结果显示pEGFP-C2-TMEM88真核表达质粒构建成功;MTT实验结果显示,过表达组细胞的增殖率为(0.625±0.07),显著低于正常组的(0.880±0.09)(P<0.05);流式细胞术结果显示,过表达组细胞的凋亡率为22.1%,显著高于正常组的9.1%。结论 TMEM88能够显著抑制人肝癌细胞株SMMC-7721的增殖,并促进其凋亡,为进一步了解TMEM88的功能、寻求肝癌治疗的新方向奠定了基础。Objective To construct the eukmyotic expression plasmid TMEM88, and study its effects on cell prolif- eration and apoptosis of liver cancer cells. Methods Extract total RNA of human hepatic stellate cells, reverse transcription into cDNA as templates, and obtain amplification TMEM88 CDS sequence by PCR, after connect to pEGFP-C2 carrier by using the double enzyme, and then make the conversion, plasmid extraction, and enzyme i- dentification, finally take positive clones to be sequenced in biological company, pEGFP-C2-TMEM88 eukaryotic expression plasmid was transfected respectively to human liver cancer cell line SMMC-7721 , then, MTF and flow cytometry methods were used to detect their effects on cell proliferation and apoptosis. Results The sequencing re- suits showed that pEGFP-C2-TMEM88 eukaryotic expression plasmid was built successfully. MTF experiment re- suits showed that the cell proliferation rate of transfection group was （0. 625 ± 0.07 ）, was significantly lower than that of normal group （0. 880±0.09） （P 〈0.05） ; Flow cytometry results showed that cell apoptosis rate of trans- fection group was 22. 1% , significantly higher than that of normal group（9. 1% ）. Conclusion TMEM88 can sig- nificantly inhibit the proliferation of human liver cancer cell line SMMC-7721, and promote its apoptosis, further understand the function of TMEM88, and lay a foundation for searching a new direction in the therapy of liver canc- er.

关 键 词：TMEM88 转染 增殖 凋亡 

分 类 号：R349.65[医药卫生—基础医学]