调控血红素加氧酶-1诱导K562A02细胞增殖、凋亡及耐药机制研究

发布时间：2017-12-27 15:04

本文关键词：调控血红素加氧酶-1诱导K562A02细胞增殖、凋亡及耐药机制研究　出处：《贵阳医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的:本文旨在通过HO-1诱导剂Hemin及抑制剂ZNPPIX调控HO-1,并联合阿霉素逆转K562A02细胞化疗耐药机制的研究,为慢性髓系白血病的逆转耐药提供新的策略。方法:培养K562及K562A02细胞,采用荧光原位杂交(FISH)法检测K562A02细胞中bcr-abl融合基因表达。MTT检测不同浓度阿霉素处理后,K562及K562A02细胞增殖抑制率,并根据IC50值计算药物逆转倍数。分别用HO-1诱导剂Hemin及抑制剂ZNPPIX调控HO-1基因表达联合阿霉素处理K562A02细胞后,MTT法检测不同浓度阿霉素处理24h、48h、72h后K562A02细胞增殖抑制率;流式细胞术检测药物诱导细胞凋亡情况。Realtime-PCR检测耐药相关基因MDR1、NF-κB、MRP1、Topo IIα、ABCD2m RNA的表达水平。western-blot检测耐药相关基因MDR1、NF-κB、MRP1、Topo IIα、ABCD2及凋亡基因蛋白表达水平。流式细胞术检测联合处理后的增殖凋亡率。结果:FISH法分析结果显示K562A02细胞中bcr-abl融合基因阳性细胞占92%。阿霉素对K562、K562A02细胞的IC50值分别为(12.320±1.720)ug/ml和(24.742±2.310)ug/ml,K562A02细胞相对于K562细胞的耐药倍数为2.01。MTT结果示阿霉素作用K562A02细胞24,48,72小时后,细胞增殖抑制率呈现浓度-时间依赖性。Real-time PCR及Western blot结果显示阿霉素处理细胞后,HO-1表达下降,耐药相关基因MDR1、NF-κB、MRP1、Topo IIα、ABCD2表达亦降低,用HO-1诱导剂Hemin、抑制剂ZNPP IX、阿霉素单药分别及联合处理K562A02细胞后,结果显示HO-1高表达后耐药相关基因表达升高,细胞凋亡率下降。而降低HO-1表达,耐药相关基因表达下降,细胞凋亡率增加。结论:阿霉素可抑制K562A02细胞增殖且诱导细胞凋亡,呈时间剂量梯度依赖关系,HO-1作为靶基因,当上调HO-1时候,可以增加药物对K562A02细胞耐药性,促进细胞增殖、减少细胞凋亡的效应。当下调HO-1时,可增加阿霉素对细胞的敏感性,抑制细胞增殖,促进细胞凋亡,从而达到逆转耐药的目的。HO-1可作为逆转耐药的靶基因,可以重新使K562A02对阿霉素重新敏感,起到增敏效应。
[Abstract]:Objective: the aim of this study is to regulate HO-1 through HO-1 inducer Hemin and inhibitor ZNPPIX, combined with doxorubicin reversing the mechanism of chemoresistance in K562A02 cells, so as to provide new strategies for reversing drug resistance in chronic myeloid leukemia. Methods: K562 and K562A02 cells were cultured and the expression of bcr-abl fusion gene in K562A02 cells was detected by fluorescence in situ hybridization (FISH). MTT was used to detect the proliferation inhibition rate of K562 and K562A02 cells after adriamycin treatment with different concentrations, and the drug reversal multiple was calculated according to the value of IC50. HO-1 inducer Hemin and inhibitor ZNPPIX were used to regulate HO-1 gene expression and adriamycin treatment of K562A02 cells. MTT assay was used to detect the proliferation inhibition rate of K562A02 cells treated with different concentrations of doxorubicin after 24h, 48h and 72h, and apoptosis was induced by flow cytometry. Realtime-PCR was used to detect the expression level of resistance related genes MDR1, NF- kappa B, MRP1, Topo II alpha and ABCD2m RNA. The expression level of resistance related genes MDR1, NF- kappa B, MRP1, Topo II alpha, ABCD2 and apoptotic gene protein were detected by Western-blot. Flow cytometry was used to detect the rate of proliferation and apoptosis after combined treatment. Results: the results of FISH analysis showed that the BCR-ABL fusion gene positive cells in K562A02 cells accounted for 92%. The IC50 values of adriamycin for K562 and K562A02 cells were (12.320 + 1.720) ug/ml and (24.742 + 2.310) ug/ml, respectively, and the resistance rate of K562A02 cells to K562 cells was 2.01. MTT results showed that the proliferation inhibition rate of adriamycin K562A02 cells showed a concentration time dependence after 24,48,72 hours. Real-time PCR and Western blot showed that cells treated with adriamycin, the expression of HO-1 decreased, the expression of multidrug resistance related genes MDR1, NF-, MRP1, Topo, II kappa B alpha, ABCD2 were also lower, respectively, and the combined treatment of K562A02 cells, ZNPP IX, agent Hemin inhibitor doxorubicin monotherapy after induction by HO-1, the results showed that high expression of HO-1 after drug resistance related gene expression increased, apoptosis rate decreased. The expression of HO-1 was decreased, the expression of resistance related genes decreased and the rate of apoptosis increased. Conclusion: doxorubicin can inhibit the proliferation and induce apoptosis of K562A02 cells in a time dose gradient dependent manner. When HO-1 is used as target gene, when HO-1 is up-regulated, it can increase drug resistance to K562A02 cells, promote cell proliferation and reduce cell apoptosis. When HO-1 is downregulated, it can increase the sensitivity of adriamycin to cells, inhibit cell proliferation and promote cell apoptosis, so as to reverse the drug resistance. HO-1 can be used as a target gene for reversing drug resistance, which can resensitize K562A02 to adriamycin and play an increasing sensitizing effect.
【学位授予单位】：贵阳医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.7

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