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目的:探讨TFPI-2基因对人肝癌细胞Hep3B生长增殖、凋亡及甲胎蛋白AFP表达的影响。方法:将重组质粒PCDNA3.1-TFPI-2转染Hep3B细胞并经G418稳定筛选后,RT-PCR和Westernblot检测转染前后TFPI-2 mRNA和蛋白表达水平,采用CCK-8法、生长曲线观察TFPI-2对人肝癌细胞Hep3B生长增殖的影响,通过平板克隆形成实验观察单个细胞的增殖能力,RT-PCR检测AFP mRNA的表达,并用电化学发光法测定培养上清液中甲胎蛋白AFP含量,流式细胞仪检测细胞早晚期凋亡情况。结果:转染成功的Hep3B细胞检测到TFPI-2 mRNA和蛋白的表达;与转染空载体及未转染的细胞相比,转染TFPI-2的细胞生长增殖能力明显减弱;AFP mRNA表达抑制率为16.51%,AFP蛋白分泌明显低于对照组(P<0.01);流式细胞术检测转染TFPI-2的Hep3B细胞早期凋亡率明显增加(24.03%±7.28%vs 8.77%±3.66%)。结论:TFPI-2表达可显著抑制肝癌细胞生长和AFP的表达,同时还能诱导细胞早期凋亡。为进一步探讨靶向TFPI-2的肝癌基因治疗提供了实验依据。
Objective: To investigate the effect of TFPI-2 on the proliferation, apoptosis and the expression of AFP in Hep3B human hepatoma cells. Methods: The recombinant plasmid pcDNA3.1-TFPI-2 was transfected into Hep3B cells and stably screened by G418. The expression of TFPI-2 mRNA and protein was detected by RT-PCR and Western blot. The growth curve was observed by CCK-8 TFPI-2 on the growth and proliferation of human hepatocellular carcinoma Hep3B cells. The proliferation of single cells was observed by plate clone formation assay. The expression of AFP mRNA was detected by RT-PCR. The expression of AFP in the culture supernatant was determined by electrochemiluminescence Content, flow cytometry detection of early and late cell apoptosis. Results: The expression of TFPI-2 mRNA and protein was detected in Hep3B cells transfected with TFPI-2. The proliferation of TFPI-2 transfected cells was significantly lower than that of untransfected and untransfected cells. AFP mRNA expression was inhibited The rate of AFP protein secretion was significantly lower than that of the control group (P <0.01). Flow cytometry showed that the apoptotic rate of Hep3B cells transfected with TFPI-2 was significantly increased (24.03% ± 7.28% vs 8.77% ± 3.66% ). Conclusion: The expression of TFPI-2 can significantly inhibit the growth of hepatocellular carcinoma cells and the expression of AFP, but also induce the early apoptosis of cells. This study provides the experimental basis for further exploring the gene therapy of liver cancer targeting TFPI-2.