FasL、抗DR5单克隆抗体对结肠癌细胞株HT29的杀伤作用及其机制

Fas ligand and anti-human DR5 monoclonal antibody induce colon cancer cell line HT29 apoptosis

目的探讨FasL、抗DR5单克隆抗体（Anti—DR5mAb）对结肠癌细胞株HT29的杀伤作用及机制。方法采用逆转录-聚合酶链反应（RT—PCR）、噻唑蓝（MTF）比色法、DNA倍体分析，Western blot等。结果HT29细胞表面FasmRNA的表达低于DR5mRNA的表达。50mg／LFasL和Anti—DR5mAb对HT29细胞的杀伤率分别为（25．49±0．90）％和（48．90±3．15）％，这种作用呈剂量依赖性。流式细胞术分析细胞周期和凋亡实验表明FasL和Anti—DR5mAb能够抑制HT29细胞的生长，并且诱导它的凋亡。25mg／LFasL和Anti—DR5mAb对HT29细胞的凋亡指数分别为（13．8±1．5）％和（22．6±1．1）％。FasL和Anti—DR5mAb作用HT29细胞后，Caspase-3蛋白表达上升，bcl-2蛋白表达水平下降。结论FasL、Anti—DR5mAb能不同程度的诱导结肠癌细胞株HT29凋亡，其机制与其受体和Caspase-3、bcl-2的表达有关。

Objective To study the cytotoxic effects of Fas ligand and anti-human DR5 monoclonal antibodies （Anti-DR5 mAb） on colon cell line HT29 and the underlying mechanism. Methods Fas/DR5 mRNA was detected by RT-PCR. Cytotoxicity exerted by FasL/Anti-DR5 mAb on tumor cell lines was measured by MTT colorimetry and the induced apoptosis was determined by agarose gel electro- phoresis. The cell cycle and apoptosis was assessed by flow cytometry with PI staining. Caspase-3 and bcl- 2 expression was assayed by Western blot. Results The expression of Fas mRNA in HT29 cells was lower than DR5 mRNA. HT29 cells were sensitive to anti-DR5 mAb but partially sensitive to FasL in a dose dependent manner. The killing rate of 50 mg/L FasL and anti-DR5 mAb to HT29 cells was （25.49 ± 0.90）% and （48.90 ± 3.15 ）%, respectively. The cell cycle analysis suggested that FasL/ Anti-DR5 mAb could inhibit HT29 cells in G0/G1 phase, and then further inhibit cell growth. The apoptosis rate of 25 mg/L FasL and anti-DR5 mAb to HT29 cells was （ 13.8 ±1.5）% and （22.6± 1.1）% ,respectively. The expression of Caspase-3 was increased, but the expression of bcl-2 was decreased obviously in HT29 cells after treated with FasL/Anti-DR5 mAb. Conclusion FasL/ Anti-DR5 mAb could induce apoptosis of HT29 cells. The underlying mechanism may be relevant to Fas/DR5 mRNA expression, and the release of Caspase-3 and bcl-2.