石蒜碱增强顺铂对肝癌HepG2.2.15细胞的细胞毒作用并抑制HBV复制的研究

Study on lycorine enhancing cytotoxic effect of cisplatin on hepatocellular carcinoma HepG2.2.15 cells and inhibiting HBV replication

目的探讨石蒜碱对顺铂的肝癌HepG2.2.15细胞毒作用及乙肝病毒(HBV)复制的影响。方法将HepG2.2.15细胞随机分为对照组、顺铂组、石蒜碱组和顺铂+石蒜碱组。采用细胞计数试剂盒-8(CCK-8)实验检测HepG2.2.15细胞存活率,流式细胞术检测HepG2.2.15细胞凋亡率,Hoechst 33258染色检测HepG2.2.15细胞凋亡形态,蛋白免疫印迹(Western blot)检测HepG2.2.15细胞B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、裂解的天冬氨酸特异性半胱氨酸蛋白酶3(cleaved Caspase-3)、乙肝免疫球蛋白(HBsAg)和乙肝核心抗原(HBcAg)蛋白表达水平,Transwell检测HepG2.2.15细胞迁移、侵袭水平,qRT-PCR检测HBV复制水平。结果与对照组比较,顺铂组HepG2.2.15细胞存活率、迁移数、侵袭数和Bcl-2表达水平明显降低,差异有统计学意义(t值分别为4.508、5.943、4.165、3.799,P<0.05),凋亡率、Bax、cleaved Caspase-3、HBsAg、HBcAg表达水平、HBV复制水平明显增加,差异有统计学意义(t值分别为-11.857、-4.685、-4.609、-10.180、-12.408、-13.295,P<0.05)。与顺铂组比较,顺铂+石蒜碱组HepG2.2.15细胞存活率、迁移数、侵袭数、Bcl-2、HBsAg、HBcAg表达水平和HBV复制水平明显降低(t值分别为3.135、5.260、6.534、4.999、4.852、5.580、8.192,P<0.05),凋亡率、Bax、cleaved Caspase-3表达水平明显增加,差异有统计学意义(t值分别为-8.895、-6.410、-6.431,P<0.05)。Hoechst 33258染色显示,对照组和石蒜碱组细胞核为淡蓝色,形态均匀,顺铂组细胞核为亮蓝色,且出现浓缩、碎裂等凋亡特征,顺铂+石蒜碱组细胞核更亮,凋亡特征更明显。结论石蒜碱可增强顺铂对HepG2.2.15细胞的细胞毒作用并抑制顺铂引起的HBV复制。

Objective To investigate the effect of lycorine on cytotoxic effect of cisplatin-induced hepatocellular carcinoma HepG 2.2.15 and hepatitis B virus(HBV)replication.Methods HepG2.2.15 cells were randomly divided into control group,cisplatin group,lycorine group and cisplatin+lycorine group.Cell counting Kit 8(CCK-8)assay was used to detect the survival rate of HepG2.2.15 cells.Flow cytometry was used to detect the apoptosis rate of HepG2.2.15 cells.Hoechst 33258 staining was used to detect the apoptotic morphology of HepG2.2.15 cells.Western blot was used to detect the protein expression levels of B-lymphocytoma-2-associated X protein(Bax),B-lymphocytoma-2(Bcl-2),cleaved cysteine-containing aspartate-specific proteases-3(cleaved Caspase-3),Hepatitis B immunoglobulin(HBsAg),hepatitis B core antigen(HBcAg)of HepG2.2.15 cells.Transwell was used to detect the migration and invasion level of HepG2.2.15 cells.QRT-PCR was used to detect the HBV replication level of HepG2.2.15 cells.Results Compared with the control group,the survival rate,migration number,invasion number and bcl-2 expression level of HepG2.2.15 cells in cisplatin group were significantly decreased(t values were 4.508,5.943,4.165 and 3.799,respectively,P<0.05),while the apoptosis rate,Bax,cleaved Caspase-3,HBsAg,HBcAg expression level,HBV replication level were significantly increased(t values were-11.857,-4.685,-4.609,-10.180,-12.408 and-13.295,respectively,P<0.05),the difference was statistically significant.Compared with the cisplatin group,the survival rate,migration number,invasion number,bcl-2,HBsAg and HBcAg expression level,HBV replication level of HepG 2.2.15 cells in cisplatin+lycorine group were significantly decreased(t values were 3.135,5.260,6.534,4.999,4.852,5.580 and 8.192,respectively,P<0.05),while the apoptosis rate,Bax and cleaved Caspase-3 expression levels were significantly increased(t values were-8.895,-6.410 and-6.431,respectively,P<0.05),the difference was statistically significant.Hoechst 33258 staining showed that the nuclei of the control group and lycorine group were light blue with uniform morphology,while the nuclei of the cisplatin group were bright blue with apoptotic characteristics such as concentration and fragmentation,while the nuclei of the cisplatin+lycorine group were brighter with more obvious apoptotic characteristics.Conclusion Lycorine can enhance the cytotoxic effect of cisplatin on HepG2.2.15 cells and inhibit HBV replication induced by cisplatin.