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目的探讨转化生长因子β1(TGF-β1)诱导人肝癌细胞系凋亡及其与p53基因及Smad4的关系。方法选用3种含有不同p53基因状态的人肝癌细胞系,均分为TGF-β1诱导组(实验组)和对照组;应用脱氧核糖核苷酸末端转移酶介导的dUTP缺口末端标记技术(TUNEL)对TGF-β1诱导的肝癌细胞的凋亡进行定量检测;另外,应用Lipofectamine2000把含有Smad4结合元件和荧光素酶基因的TGF-β1可诱导的荧光素酶报告质粒对细胞进行转染,再经TGF-β1作用,分别检测其相对荧光素酶活性。结果在应用TUNEL检测的3个细胞系中,TGF-β1能诱导HepG2细胞(野生型p53)凋亡,其细胞凋亡率实验组明显高于对照组(P<0.05);而Huh-7(突变型p53)和Hep3B细胞(缺失型p53)凋亡细胞较少,其细胞凋亡率实验组与对照组差异无统计学意义(P>0.05)。荧光素酶检测提示,HepG2细胞的Smad4表达活性实验组明显高于对照组(P<0.05);而Huh-7和Hep3B细胞的Smad4表达较低,实验组与对照组差异无统计学意义(P>0.05)。结论HepG2细胞系比Huh-7和Hep3B细胞系更易发生TGF-β1诱导的凋亡,Smad4是TGF-β1信号转导途径的主要调控因子之一。
Objective To investigate the apoptosis of hepatocellular carcinoma cell line induced by transforming growth factor-β1 (TGF-β1) and its relationship with p53 gene and Smad4. Methods Three human hepatocellular carcinoma cell lines with different p53 gene status were selected and divided into TGF-β1-induced group (experimental group) and control group. TUNEL-mediated dUTP nick end labeling ) On TGF-β1-induced apoptosis in hepatocellular carcinoma cells. In addition, TGF-β1-induced luciferase reporter plasmids containing Smad4-binding element and luciferase gene were transfected into cells by Lipofectamine2000. TGF-β1 role, were detected relative luciferase activity. Results The apoptosis of HepG2 cells (wild-type p53) was induced by TGF-β1 in three cell lines detected by TUNEL, and the apoptosis rate of HepG2 cells was significantly higher than that of the control group (P <0.05) Mutant p53) and Hep3B cells (deletion type p53) had fewer apoptotic cells. There was no significant difference between the experimental group and the control group (P> 0.05). The luciferase assay indicated that Smad4 expression in HepG2 cells was significantly higher than that in control cells (P <0.05), while Smad4 expression was lower in Huh-7 and Hep3B cells (P <0.05), but there was no significant difference between HepG2 cells and control cells > 0.05). Conclusion HepG2 cells are more prone to TGF-β1-induced apoptosis than Huh-7 and Hep3B cells, one of the major regulators of TGF-β1 signal transduction pathway.