Huh7耐药肝癌细胞系的建立及其与SP细胞的关系

The establishment of chemoresistance cell line Huh7 and its relation with SP cell

目的建立多柔比星(doxorubicin,D ox)耐药人肝癌细胞系H uh7/D ox,初步探讨H uh7/D ox与侧群细胞(side population cell,SP)的关系。方法以D ox为诱导剂,采用低浓度反复间歇诱导法建立人肝癌耐药细胞系H uh7/D ox,用M TT法测定药物敏感性;最后将耐药细胞和亲本细胞分别行H oechest33342染色,用流式细胞仪检测SP细胞含量;采用逆转录-聚合酶链反应和定量PC R检测干细胞标记分子(C D 133,C D 90,O C T4,N anog)的表达变化。结果成功建立D ox耐药细胞系H uh7/D ox,对D ox的耐药指数为15;H oech-est33342染色分析显示H uh7和H uh7/D ox中SP细胞含量分别为0.2%和1.3%;定量PC R结果显示H uh7/D ox干细胞标记C D 133、C D 90、O C T4和N anog的m R N A水平显著增加。结论建立了稳定的人肝癌耐药细胞系H uh7/D ox,H uh7/D ox耐药细胞中SP细胞的含量明显增加,提示与化疗耐药相关。

【Objective】 To establish doxorubicin（Dox） resistant human hepatoma cell line（Huh7/Dox） and to study the relation between drug-resistant cell and side population cell（SP cell）.【Methods】 The Establishment of Dox resistant human hepatoma cell line Huh7/Dox was used by low-does and interval-inactivation methods.The characteristic of drug-resistant cell was detected by MTT assay.The percentage of SP cell was determined by Hoechst33342 and Flow Cytometry（FCM） assay.The mRNA expression level of CD133,CD90,OCT4 and Nanog were analyzed by real-time PCR.【Results】 Huh7/Dox cell line was established after 3-month treatment,which could resist to Dox with resistance index of 15.The percentage of SP cells was about 0.2% in parental Huh7 cell line and 1.3% in Huh7/Dox cell line.The mNRA expression levels of CD133,CD90,OCT4 and Nanog in Huh7/Dox cells were higher compared with parental Huh7 cells.【Conclusion】 A stable Dox-resistant Huh7/Dox cell line was established,and the percentage of SP cells in Huh7/Dox cells was elevated in compared with parental Huh7 cells,which might have a function in chemotherapy resistance.