TMEM88真核表达质粒的构建及其功能研究
发布时间：2018-08-27 09:53
【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)(P0.05);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。
[Abstract]:Objective to construct eukaryotic expression plasmid of TMEM88 and to study its effect on proliferation and apoptosis of hepatoma cells. Methods the total RNA, of human hepatic stellate cells was extracted by reverse transcription into cDNA as template, and the CDS sequence of TMEM88 was amplified by PCR method, then ligated to pEGFP-C2 vector by double enzyme digestion, then transformed, plasmid extracted and identified by enzyme digestion. Finally, the positive clones were selected and sent to the biological company for sequencing. The eukaryotic expression plasmid of pEGFP-C2-TMEM88 was transfected into human hepatoma cell line SMMC-7721, and the effects of MTT and flow cytometry on cell proliferation and apoptosis were detected. Results the results of sequencing showed that the pEGFP-C2-TMEM88 eukaryotic expression plasmid was successfully constructed. The proliferative rate of the overexpression group was (0.625 卤0.07), which was significantly lower than that of the normal group (0.880 卤0.09) (P0.05). The rate of apoptosis in overexpression group was 22.1g, which was significantly higher than that in normal group (9.1%). Conclusion TMEM88 can significantly inhibit the proliferation of human hepatoma cell line SMMC-7721 and promote its apoptosis, which lays a foundation for further understanding the function of TMEM88 and seeking a new direction for the treatment of HCC.
【作者单位】： 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;安徽医科大学药学院;
【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)
【分类号】：R735.7
，
本文编号：2206921
[Abstract]:Objective to construct eukaryotic expression plasmid of TMEM88 and to study its effect on proliferation and apoptosis of hepatoma cells. Methods the total RNA, of human hepatic stellate cells was extracted by reverse transcription into cDNA as template, and the CDS sequence of TMEM88 was amplified by PCR method, then ligated to pEGFP-C2 vector by double enzyme digestion, then transformed, plasmid extracted and identified by enzyme digestion. Finally, the positive clones were selected and sent to the biological company for sequencing. The eukaryotic expression plasmid of pEGFP-C2-TMEM88 was transfected into human hepatoma cell line SMMC-7721, and the effects of MTT and flow cytometry on cell proliferation and apoptosis were detected. Results the results of sequencing showed that the pEGFP-C2-TMEM88 eukaryotic expression plasmid was successfully constructed. The proliferative rate of the overexpression group was (0.625 卤0.07), which was significantly lower than that of the normal group (0.880 卤0.09) (P0.05). The rate of apoptosis in overexpression group was 22.1g, which was significantly higher than that in normal group (9.1%). Conclusion TMEM88 can significantly inhibit the proliferation of human hepatoma cell line SMMC-7721 and promote its apoptosis, which lays a foundation for further understanding the function of TMEM88 and seeking a new direction for the treatment of HCC.
【作者单位】： 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;安徽医科大学药学院;
【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)
【分类号】：R735.7
，
本文编号：2206921
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