凋亡抵抗与人肝癌细胞对紫杉醇耐药关系的研究  

The Relationship between Apoptosis Resistance and Paclitaxel-resistant in Human Hepatoma Cell Line Hep3B

作　　者：周明杰[1] 韩子午 范春雷[1] 钱颖[1] 田男[1] 

机构地区：[1]浙江中医药大学生命科学学院,杭州310053

出　　处：《浙江中医药大学学报》2015年第2期128-132,138,共6页Journal of Zhejiang Chinese Medical University

基　　金：高等学校博士学科点专项科研基金(20133322120002);浙江中医药大学校级重点项目(20132208)~~

摘　　要：[目的]体外建立紫杉醇(Taxol)耐药的人肝癌细胞株Hep3B/TAX,并以此为模型对凋亡抵抗现象与紫杉醇耐药的相关性进行研究。[方法]采用浓度梯度间歇刺激法建立Hep3B/TAX耐药细胞株,MTT法检测Hep3B/TAX对紫杉醇的耐药性,流式细胞术检测Hep3B/TAX的细胞凋亡水平,荧光定量PCR检测Hep3B和Hep3B/TAX细胞株中Bcl-2、Bax、caspase3、caspase8和caspase9基因的表达水平,蛋白质免疫印迹(Western-blot)检测Hep3B、Hep3B/TAX细胞株中Bcl-2、Bax、caspase3、caspase8和caspase9的蛋白表达。[结果]Hep3B细胞对紫杉醇的IC50为0.2mΜ,而耐药细胞株Hep3B/TAX对紫杉醇的IC50升为4.33μM。耐药指数(RI)为21.65。流式细胞术结果显示,0.1μM、0.2μM紫杉醇处理24h后,亲本细胞Hep3B的凋亡率显著高于耐药细胞株Hep3B/TAX。RT-PCR结果显示,与Hep3B细胞株相比,Hep3B/TAX细胞株中Bcl-2 m RNA的表达量显著升高(P<0.01),而caspase3、caspase8、caspase9和Bax m RNA的表达量显著降低(P<0.01)。Western-blot实验结果显示,与Hep3B细胞株相比,Hep3B/TAX耐药细胞株的抗凋亡蛋白Bcl-2的表达量显著升高,相关促凋亡蛋白caspase3、caspase8、caspase9和Bax的表达量降低。[结论]成功建立紫杉醇耐药的人肝癌细胞株Hep3B/TAX,人肝癌细胞株Hep3B对紫杉醇耐药的产生与改变凋亡相关因子的表达而导致的凋亡抵抗现象有关。[Objective] To establish a taxol-resistant cell line Hep3B/TAX and investigate the relationship between the apoptosis resistance and taxol resistance. [Methods] The cell line Hep3 B was cultured by gradually increasing dose of taxol to generate its resistance cell line Hep3B/TAX. The resistant index of Hep3B/TAX to taxol was determined by MTT assay,and its apoptosis rate was determined by Flow Cytometers,real time PCR analysis showed the m RNA expression level of Bcl-2, caspase3, caspase8, caspase9 and Bax in Hep3B/TAX and Hep3 B cells,and Western-blotting assay was used to detect the protein level of Bcl-2, caspase3, caspase8, caspase9 and Bax in Hep3B/TAX and Hep3 B cells. [Results] The IC50 of the cell line Hep3 B to taxol is 0.2m M,but the cell line Hep3B/TAX rose to 4.33μM,the resistant index of taxol（RI）was 21.65. The result of Flow Cytometers showed that the apoptosis rate of Hep3 B was higher than Hep3B/TAX,after treated with taxol（0.1μM,0.2μM）for 24 h. Compared with the cell of Hep3 B,the expression of Bcl-2 had, a significant increase in Hep3B/TAX（P 0.01）, while the expression of caspase3, caspase8, caspase9 and Bax in Hep3B/TAX was reduced（P0.01）.The result of the Western-blotting assay showed that the expression of anti-apoptosis related protein Bcl-2 was significantly higher in Hep3B/TAX,the expression of the mitochondrial pro-apoptotic related proteins such as caspase3,caspase8,caspase9 and Bax was significantly reduced in Hep3B/TAX. [Conclusions] Hep3B/TAX cell line was successfully established. The phenomenon of Hep3 B cell line resistant to taxol was related to the phenomenon of anti-apoptosis which caused by the changes of the apoptosis related proteins.

关 键 词：凋亡抵抗 原发性肝癌 紫杉醇耐药 线粒体 

分 类 号：R331[医药卫生—人体生理学]