雷帕霉素联合顺铂诱导肝癌细胞凋亡和自噬及对细胞侵袭力的影响

Effect of rapamycin and cisplatin on cell apoptosis, autophagy and invasion in hepatoma carcinoma cell

目的研究雷帕霉素联合顺铂诱导肝癌细胞凋亡、自噬及其对细胞侵袭转移能力的影响。方法将雷帕霉素以及顺铂分别或联合作用于HepG-2细胞,采用MTT法检测HepG-2细胞增殖抑制情况,采用透射电镜、流式细胞仪检测HepG-2细胞凋亡,采用MDC染色、转染pGFP-LC3检测细胞自噬,用Transwell小室测定HepG-2细胞对细胞外基质的侵袭力。结果与对照组相比,单独应用雷帕霉素组未出现明显的细胞凋亡情况;单独应用顺铂组细胞凋亡情况较明显,凋亡率达到(21.27±3.65)%;顺铂联合应用雷帕霉素组细胞凋亡率最高,达到(43.33±5.64)%。雷帕霉素组、顺铂组和顺铂联合应用雷帕霉素组HepG-2细胞均出现点状的自噬泡,顺铂联合应用雷帕霉素组自噬泡数量显著高于雷帕霉素组和顺铂组。雷帕霉素组、顺铂组和顺铂联合应用雷帕霉素组对HepG-2细胞侵袭抑制率分别为(19.2±5.1)%、(47.8±4.1)%和(78.0±2.4)%。结论顺铂可直接诱导HepG-2细胞凋亡,雷帕霉素本身不诱导HepG-2细胞凋亡,但其可显著促进顺铂诱导的HepG-2细胞凋亡;同时顺铂和雷帕霉素均可直接诱导HepG-2细胞自噬,但两者联合应用促进HepG-2细胞自噬情况非常明显。体外侵袭实验结果表明顺铂联合应用雷帕霉素可显著降低HepG-2细胞的体外侵袭能力。

Objective To observe the cell apoptosis, autophagy and invasion of rapamycin combined with cisplatin in hepatoma carcinoma cell. Methods Rapamycin and cisplatin were mixed with HepG-2 cells, the cell apoptosis was detected by transmission electron microscopy and flow cytometry,the cell autophagy was detected by MDC stain and transfected pGFP-LC3. Matrigel-transwell chamber method was used to evaluate the cell abilities of metastasis and chemotaxis. Results Compared with the control group, rapamycin group does not appear obvious apoptosis. The apoptosis rate of cisplatin alone group reaches to（21.27±3.65）%. The apoptosis rate of cisplatin combinated with rapamycin group is the highest, which reaches to（43.33±5.64）%. Punctate of autophagic vacuoles were detected in rapamycin group, cisplatin group and cisplatin combinated with rapamycin group. Autophagic vacuoles in cisplatin combinated with rapamycin group were significantly higher than rapamycin hormone group and cisplatin group. The HepG-2 cell invasion inhibition rate was（19.2±5.1）%,（47.8±4.1）% and（78.0±2.4）% in rapamycin group, cisplatin group and cisplatin combinated with rapamycin group. Conclusions Cisplatin can directly induce HepG-2 cell apoptosis, rapamycin itself does not induce HepG-2 cell apoptosis, but it can significantly promote apoptosis in HepG-2 cell induced by cisplatin. Meanwhile cisplatin and rapamycin can directly induce HepG-2 cell autophagy, but both combined to promote HepG-2 cell autophagy more obviously. In vitro invasion essay showed that cisplatin combinated with rapamycin can inhibit the invasion and metastasis of HepG-2 cell.