去甲斑蝥素对人胆囊癌GBC-SD细胞系增殖及侵袭的影响

目的　探讨去甲斑蝥素 (NCTD)对人胆囊癌GBC SD细胞系增殖、侵袭的影响及其机制。方法　应用细胞培养技术培养GBC SD细胞 ;以MTT法检测NCTD对GBC SD细胞的杀伤抑制率 ;以Matrigel侵袭实验、过河实验和SABC法检测NCTD对GBC SD细胞的侵袭力和对PCNA、Ki 6 7、MMP2 、TIMP2 蛋白表达的影响。结果　NCTD可明显抑制GBC SD细胞的增殖和生长 ,且随浓度提高或时间延长作用增强 ,呈剂量 时间效应关系 ;IC50 为 5 6 .18μg/ml,最强抑制作用时间为第 4 8小时。Matrigel侵袭、过河实验显示 ,NCTD在 5 μg/ml时 ,即能抑制GBC SD细胞的体外侵袭和运动能力 ,随浓度提高 ,过膜细胞减少 ,过膜死亡细胞增多 ,过河时间延长 (P <0 .0 1)。免疫组化测定显示 ,NCTD(IC50 )作用 4 8h后 ,GBC SD细胞PCNA、Ki 6 7、MMP2 表达下降 ,TIMP2 表达上升 ,MMP2 /TIMP2 比值下降 (P <0 .0 5 )。结论　NCTD可抑制人胆囊癌GBC SD细胞的生长 ,低浓度下也能抑制其体外侵袭能力 ;其机制可能与直接抑制GBC SD细胞迁移运动 ,干扰GBC SD细胞增殖相关基因蛋白PCNA、Ki 6 7和细胞基质溶解相关基因蛋白MMP2 、TIMP2 的表达有关。

The in vitro effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells and its mechanism

OBJECTIVE: To study the effect and mechanism of action of norcantharidin on proliferation and invasion of GBC-SD cells. METHODS: GBC-SD cells of human gallbladder carcinoma were cultured by cell culture technique. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The Matrigel experiment and the crossing-river test were used to examine the invasiveness of GBC-SD cells. Expression of MMP(2), TIMP(2), PCNA and Ki-67 proteins of GBC-SD cells was determined by streptavidin-biotin complex method. RESULTS: Norcantharidin inhibited the growth and proliferation of GBC-SD cells in a dose and time dependent manner, with an IC(50) value of 56.18 micro g/ml at 48 h. The Matrigel experiment showed that norcantharidin began to inhibit the in vitro invasion of GBC-SD cells at the concentration of 5 micro g/ml. At 40 micro g/ml, the invasive action of GBC-SD cells was inhibited completely and their crossing-river time was prolonged significantly. After treatment with norcantharidin, the expression of PCNA, Ki-67, MMP(2) was significantly decreased. With the increase in TIMP(2) expression, the MMP(2) to TIMP(2) ratio was decreased significantly (P < 0.05). CONCLUSION: Norcantharidin inhibits the in vitro proliferation and growth of human gallbladder carcinoma cells at relatively low concentrations by inhibiting PCNA and Ki-67 expression. Its anti-invasive activity may be the results of decrease in MMP(2) to TIMP(2) ratio and reduced cell motility.