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目的:探讨重组水蛭素对人肝癌细胞增殖、凋亡能力的影响及可能的分子机制。方法:体外培养人肝癌SMMC-7721细胞,分4组:空白对照组,以PBS溶液进行假干预;重组水蛭素组,以1.0 U/m L重组水蛭素干预;凝血酶组,以1.0 U/m L凝血酶干预;混合组,以1.0 U/m L凝血酶+1.0 U/m L重组水蛭素干预。采用MTT、流式细胞技术分别观察细胞增殖能力及细胞凋亡的变化。采用qRT-PCR技术检测Bcl-2及Bax的mRNA表达变化,免疫蛋白印记技术检测细胞内Bcl-2及Bax蛋白的表达变化。结果:与空白对照组相比,重组水蛭素组细胞增殖能力较空白对照组与凝血酶组均显著降低、凋亡比例显著升高(P<0.05),而凝血酶组细胞增殖能力显著升高、凋亡比例显著降低(P<0.05);混合组细胞增殖能力较凝血酶组均显著降低、凋亡比例显著升高(P<0.05),而与空白对照组相比无明显统计学差异(P>0.05)。与空白对照组相比,凝血酶组细胞内Bcl-2 mRNA及蛋白的表达水平显著升高而Bax mRNA及蛋白的表达水平明显降低(P<0.05);重组水蛭素组细胞内Bcl-2mRNA及蛋白的表达水平明显降低而Bax mRNA及蛋白的表达水平明显升高(P<0.05);混合组Bcl-2及Bax表达未发生显著变化(P>0.05)。结论:重组水蛭素不仅可以显著抑制人肝癌SMMC-7721细胞的增殖,并促进其凋亡发生,而且能够有效逆转凝血酶导致的生长促进及凋亡抵抗作用。
Objective: To investigate the effect of recombinant hirudin on the proliferation and apoptosis of human hepatoma cells and its possible molecular mechanism. Methods: Human hepatoma SMMC-7721 cells were cultured in vitro and divided into 4 groups: blank control group, sham intervention with PBS solution; recombinant hirudin group with 1.0 U / mL recombinant hirudin intervention; thrombin group, 1.0 U / m L thrombin intervention; mixed group, with 1.0 U / m L thrombin + 1.0 U / m L recombinant hirudin intervention. MTT and flow cytometry were used to observe the changes of cell proliferation and apoptosis respectively. QRT-PCR was used to detect the mRNA expression of Bcl-2 and Bax, and the protein expression of Bcl-2 and Bax was detected by Western blotting. RESULTS: Compared with the blank control group, the proliferation of recombinant hirudin group was significantly lower than that of the blank control group and thrombin group (P <0.05), while the proliferation of thrombin group was significantly increased (P <0.05). The proliferation of the mixed group was significantly lower than that of the thrombin group (P <0.05), but not significantly different from the control group (P <0.05) P> 0.05). Compared with the blank control group, the expression of Bcl-2 mRNA and protein in the thrombin group was significantly increased, while the expression of Bax mRNA and protein was significantly decreased (P <0.05); Bcl-2 mRNA and protein expression in the recombinant hirudin group (P <0.05). The expression of Bcl-2 and Bax in the mixed group did not change significantly (P> 0.05). CONCLUSION: Recombinant hirudin can not only significantly inhibit the proliferation and promote the apoptosis of human hepatocellular carcinoma SMMC-7721 cells, but also effectively reverse thrombin-induced growth-promoting and apoptosis-resistant effects.