重组分泌型Fas配体对肝癌细胞株HepG2凋亡的影响  

Study on the apoptosis mediated by recombinant secretory Fas ligand in hepatoma cell line HepG2

作　　者：尚小玲[1,3] 汪宏良 冯琴琴[4] 胡芳 杨浩 谌章舟[1,2,3] SHANG Xiaoling;WANG Hongliang;FENG Qinqin;HU Fang;YANG Hao;SHEN Zhangzhou(Department of Clinical Laboratory,Huangshi Central Hospital/Affiliated Hospital of Hubei Polytechnic University,Huangshi,Hubei 435000,China;Medical School,Hubei Polytechnic University,Huangshi,Hubei 435003,China;Hubei Key Laboratory of Kidney Diseases Pathogenesis and Intervention,Huangshi,Hubei 435003,China;Department of Obstetrics,Huangshi Maternal and Children′s Health Hospital,Huangshi,Hubei 435003,China)

机构地区：[1]黄石市中心医院/湖北理工学院附属医院医学检验科,湖北黄石435000 [2]湖北理工学院医学院,湖北黄石435003 [3]肾脏疾病发生与干预湖北省重点实验室,湖北黄石435003 [4]黄石市妇幼保健院产科,湖北黄石435003

出　　处：《国际检验医学杂志》2023年第4期450-454,460,共6页International Journal of Laboratory Medicine

基　　金：湖北省卫生健康委员会科研基金项目(WJ2019M044);黄石市科技局项目(2019A02);肾脏疾病发生与干预湖北省重点实验室开放基金项目(SB202105)。

摘　　要：目的构建重组表达Fas配体(FasL)基因(N端含有IL2信号肽)的慢病毒,探讨其介导肝癌细胞凋亡的可行性及其可能的分子机制。方法将含有IL2信号肽的FasL基因克隆至慢病毒载体pCDH-CMV-MCS-EF1-GFP+Puro(简称pCDH)中,然后将重组pCDH-IL2sig-FasL与慢病毒辅助质粒共同转染至HEK293细胞中,获得重组慢病毒LV-IL2sig-FasL,阴性对照为LV,分别感染HepG2细胞。最后将成功构建的稳转细胞HepG2-LV-IL2sig-FasL及HepG2-LV同时培养至96 h,采用实时荧光定量聚合酶链反应(qPCR)检测细胞中相关凋亡信号因子的转录水平,Western blot检测蛋白表达水平,MTT检测细胞增殖抑制水平。结果HepG2-LV-IL2sig-FasL细胞相对于阴性对照株HepG2-LV,其凋亡因子转录及表达水平均上调;MTT检测结果显示HepG2-LV-IL2sig-FasL细胞生长明显受到抑制。结论外源表达IL2sig-Fas基因可诱导肿瘤细胞HepG2凋亡,有可能作为肿瘤基因治疗的新途径。Objective To construct a recombinant lentivirus expressing Fas ligand(FasL)gene containing IL2 signal peptide at N-terminal,and to investigate the feasibility and possible molecular mechanism of FasL gene mediated apoptosis of liver cancer cells.Methods FasL gene containing IL2 signal peptide was cloned into lentiviral vector pCDH-CMV-MCS-EF1-GFP+Puro(pCDH),and then the recombinant pCDH-IL2sig-FasL and lentiviral helper plasmid were transfected into HEK293 cell line.Recombinant lentivirus LV-IL2sig-FasL was obtained,and the negative control was LV,which was infected with HepG2 cells respectively.Finally,the successfully constructed stable cell lines HepG2-LV-IL2sig-FasL and HepG2-LV were simultaneously cultured for 96 h,and the transcription levels of apoptosis signal factors in the cell lines were detected by using real-time fluorescent quantitative polymerase chain reaction(qPCR),the protein expression levels were detected by using western blot,and the cell proliferation inhibition levels were detected by using MTT.Results Compared with hepG2-LV,the transcription and expression levels of apoptosis factors in HepG2-LV-IL2sig-FasL cell line were up-regulated.MTT showed that the growth of HepG2-LV-IL2sig-FasL cell line was significantly inhibited.Conclusion The expression of IL2sig-FasL gene can induce the apoptosis of HepG2,which may be used as a new approach for gene therapy.

关 键 词：FAS配体 肝癌细胞 慢病毒 凋亡 

分 类 号：Q291[生物学—细胞生物学]