人肝癌HepG2/5-Fu耐药细胞株多药耐药与凋亡关系的研究

Relation between multi-drug resistance and apoptosis of HepG2/5-Fu

目的探讨耐药株人肝癌细胞HepG2/5-Fu的耐药机制及耐药与细胞凋亡的关系。方法检测HepG2/5-Fu细胞株对不同抗肿瘤药物的敏感性,流式细胞仪分析HepG2/5-Fu细胞在5-Fu作用的凋亡情况以及对5-Fu的耐受分析,RT-PCR分析HepG2/5-Fu细胞株中凋亡相关基因p53、bcl-2、bcl-xl、bax等基因的表达情况。结果HepG2/5-Fu细胞与HepG2细胞相比除对5-Fu耐药外,对其它几种常用化疗药均有不同程度的耐药性。HepG2细胞中未经5-Fu处理的对照组、30μmol/L5-Fu组及150μmol/L5-Fu组的凋亡指数与对照组相比,凋亡分别增加了1.2倍和2.1倍,差异具有显著性(P<0.05)。而经同样浓度5-Fu处理的HepG2/5-Fu细胞与其对照组相比,细胞凋亡变化不明显。经30μmol/L和150μmol/L5-Fu处理后,HepG2细胞与其对照组相比,呈现明显的G0/G1期增加,S期、G2/M减少,而HepG2/5-Fu细胞与其对照组相比细胞周期未见改变。PCR分析发现:与HepG2细胞相比,HepG2/5-Fu细胞耐药株bcl-xl、bcl-xs和bax的表达增高,而p53和cpp32的表达减少,特别是p53基因,表达明显减少。在人肝癌细胞HepG2和HepG2/5-Fu细胞mRNA水平均没有检测到bcl-2的表达。结论wtp53基因的突变与肝癌细胞的细胞周期的改变、耐药性的产生相关,是肝癌细胞HepG2/5-Fu产生多药耐药性的机制之一。

[Objective] To discuss the mechanism of HepG2/5-Fu drug-resistant cell and the relation between apoptosis and drug resistance. [Methods] We analyzed multi-medicine drug resistance the of human liver cancer cell line HepG2/5-Fu, and analyzed the apoptosiss and the tolerance of HepG2/5-Fu cell induced by 5-Fu through AS. And we also analyzed apoptosiss correlation genes expression on the HepG2/5-Fu cell line such as p53, bcl-2, bcl-xl, bax et al. by RT-PCR. [Results] The HepG2/5-Fu cell had the different degree of drug resistance to 5-Fu and other several kinds of commonly used chemotherapy medicine compared with HepG2. Among them, the drug resistance to 5-Fu reached 107.0 times, and paclitaxel reached 49.0 times compared with the drug resistanct of HepG2. ②When treated the HepG2 with the concent ration of 5-Fu （0μmo/L, 30μmo/L or 150μmol/L）, the AI was （6.5±1.0）%, （14.0±4.0）% and （20.0±5.0）%, respectively. The apoptosis rate increased 1.2 time and 2.1 time compared with the one not treated with 5-Fu, which were significant （P 〈0.05）. But treated the HepG2/5-Fu with the same concent ration of 5-Fu （30 txmo/L or 150 μmol/L）, the Al was （6.7±0.7）%, （7.1±2.2）% and （6.5±2.0）%. When treated with the same concent ration of 5-Fu （30μmo/L or 150μmol/L）, the proportion of apoptosis significantly increased and G0/G1 phase increased and S and G2/M phases were reduced in HepG2 cells , but the proportion of apoptosis and cell cycle did not change in HepG2/5-Fu cells. The expression of bcl-xl, bcl-xs, and bax mRNA significantly increased and the expression of p53 and cpp32 mRNA significantly decreased in resistant HepG2/5-Fu cells, as compared with HepG2 cells by RT-PCR.[Conclusion] wtp53 gene is related to the apoptosis and drug resistance of liver cancer cell HePG2/5-Fu.