TMEM88真核表達(dá)質(zhì)粒的構(gòu)建及其功能研究
發(fā)布時間：2018-08-27 09:53
【摘要】：目的構(gòu)建TMEM88的真核表達(dá)質(zhì)粒,并研究其對肝癌細(xì)胞增殖和凋亡的影響。方法提取人肝星狀細(xì)胞的總RNA,逆轉(zhuǎn)錄成cDNA作為模板,利用PCR法擴增出TMEM88的CDS序列,雙酶切后連接到pEGFP-C2載體上,然后進(jìn)行轉(zhuǎn)化、質(zhì)粒抽提、酶切鑒定,最后挑取陽性克隆送生物公司測序。將pEGFP-C2-TMEM88真核表達(dá)質(zhì)粒分別轉(zhuǎn)染至人肝癌細(xì)胞株SMMC-7721中,通過MTT實驗和流式細(xì)胞術(shù)檢測其對細(xì)胞增殖和凋亡的影響。結(jié)果測序結(jié)果顯示pEGFP-C2-TMEM88真核表達(dá)質(zhì)粒構(gòu)建成功;MTT實驗結(jié)果顯示,過表達(dá)組細(xì)胞的增殖率為(0.625±0.07),顯著低于正常組的(0.880±0.09)(P0.05);流式細(xì)胞術(shù)結(jié)果顯示,過表達(dá)組細(xì)胞的凋亡率為22.1%,顯著高于正常組的9.1%。結(jié)論 TMEM88能夠顯著抑制人肝癌細(xì)胞株SMMC-7721的增殖,并促進(jìn)其凋亡,為進(jìn)一步了解TMEM88的功能、尋求肝癌治療的新方向奠定了基礎(chǔ)。
[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 鹵0.07), which was significantly lower than that of the normal group (0.880 鹵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.
【作者單位】： 安徽醫(yī)科大學(xué)附屬合肥醫(yī)院(合肥市第二人民醫(yī)院)腫瘤放療科;安徽醫(yī)科大學(xué)藥學(xué)院;
【基金】：合肥市科技攻關(guān)計劃項目(編號:合科[2015]163號-38) 安徽醫(yī)科大學(xué)博士科研資助基金(編號:XJ201536) 安徽高校自然科學(xué)研究重點項目(編號:KJ2016A348)
【分類號】：R735.7
，
本文編號：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 鹵0.07), which was significantly lower than that of the normal group (0.880 鹵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.
【作者單位】： 安徽醫(yī)科大學(xué)附屬合肥醫(yī)院(合肥市第二人民醫(yī)院)腫瘤放療科;安徽醫(yī)科大學(xué)藥學(xué)院;
【基金】：合肥市科技攻關(guān)計劃項目(編號:合科[2015]163號-38) 安徽醫(yī)科大學(xué)博士科研資助基金(編號:XJ201536) 安徽高校自然科學(xué)研究重點項目(編號:KJ2016A348)
【分類號】：R735.7
，
本文編號：2206921
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