蝮蛇毒抗凝蛋白组分对人结肠癌细胞株SW480凋亡的实验研究

Apoptosis of colon cancer SW480 cell line inducing by the anti- dotting protein component from venom of agkistrodon

目的探讨蝮蛇毒抗凝蛋白组分诱导人结肠癌细胞株SW480凋亡及其可能的作用机制。方法CCK-8比色法测定蝮蛇毒抗凝蛋白组分对体外培养的SW480细胞的细胞毒作用，流式细胞术（flow cytometry，FCM）检测细胞线粒体膜电位（mhochonal membrane potential，MMP）变化和细胞周期分析，Western blot检测凋亡相关蛋白半胱-天冬氨酸蛋白酶3（cysteine containing aspartate，capase-3）、bcl-2的表达。结果蝮蛇毒抗凝蛋白组分对SW480细胞具有明显的生长抑制作用，显微镜下观察到细胞圆缩，悬浮细胞增多，细胞分裂相减少，培养液内细胞碎片明显增多，流式细胞仪直方图上亦出现特征性的亚二倍体峰，随着蝮蛇毒抗凝蛋白组分浓度的增加，线粒体膜电位逐渐下降，凋亡率逐渐增加，capase-3蛋白表达增加，bcl-2蛋白表达无变化。结论皖南尖吻蝮蛇毒抗凝蛋白组分可诱导SW480细胞发生凋亡，通过线位体／半胱天冬氨酸蛋白酶3途径可能是SW480细胞发生凋亡的机制之一。

Objective To investigate the effects of the anti - dotting protein component from venom of Agkistrodon inducing SW480 apoptosis and its mechanism. Methods CCK - 8 method was used to detect the antitumor effect of CTX -d in vitro;flow cytometry were used to observe the apoptotic and cell cycle inducing effect of the anti - clotting protein component from venom of Agkistrodon in SW480 cells. Mitochondrial transmembrane potential change（Arpm） was analyzed by flow cytometry;The levels of caspase- 3 and bel- 2 were analyzed by Westem blotting. Results The generation depressant effects of SW480 ceils cultured in vitro were detected by CCK- 8 method（P 〈 0.05）, and there were dosage- time dependent relationships. Typical changes of apoptosis such as the suspending cells and the cellular debris got more,the process of cell division got less were observed, and subdiploid peak can be detected. Caspase -3 expression enhanced but bcl -2 expression did not changed obviously with the anti - clotting protein component from venom of Agkistrodon＇s coneentration increase. Conclusions The anti -dotting protein component from venom of Agkistrodon can induce apoptosis in SW480,the mitochondria/caspase- 3 -specific pathway may be involved in the mechanism.