本文關(guān)鍵詞：調(diào)控血紅素加氧酶-1誘導(dǎo)K562A02細(xì)胞增殖、凋亡及耐藥機(jī)制研究　出處：《貴陽(yáng)醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 慢性髓系白血病 阿霉素 K562 K562A02 血紅素加氧酶-1

【摘要】：目的:本文旨在通過(guò)HO-1誘導(dǎo)劑Hemin及抑制劑ZNPPIX調(diào)控HO-1,并聯(lián)合阿霉素逆轉(zhuǎn)K562A02細(xì)胞化療耐藥機(jī)制的研究,為慢性髓系白血病的逆轉(zhuǎn)耐藥提供新的策略。方法:培養(yǎng)K562及K562A02細(xì)胞,采用熒光原位雜交(FISH)法檢測(cè)K562A02細(xì)胞中bcr-abl融合基因表達(dá)。MTT檢測(cè)不同濃度阿霉素處理后,K562及K562A02細(xì)胞增殖抑制率,并根據(jù)IC50值計(jì)算藥物逆轉(zhuǎn)倍數(shù)。分別用HO-1誘導(dǎo)劑Hemin及抑制劑ZNPPIX調(diào)控HO-1基因表達(dá)聯(lián)合阿霉素處理K562A02細(xì)胞后,MTT法檢測(cè)不同濃度阿霉素處理24h、48h、72h后K562A02細(xì)胞增殖抑制率;流式細(xì)胞術(shù)檢測(cè)藥物誘導(dǎo)細(xì)胞凋亡情況。Realtime-PCR檢測(cè)耐藥相關(guān)基因MDR1、NF-κB、MRP1、Topo IIα、ABCD2m RNA的表達(dá)水平。western-blot檢測(cè)耐藥相關(guān)基因MDR1、NF-κB、MRP1、Topo IIα、ABCD2及凋亡基因蛋白表達(dá)水平。流式細(xì)胞術(shù)檢測(cè)聯(lián)合處理后的增殖凋亡率。結(jié)果:FISH法分析結(jié)果顯示K562A02細(xì)胞中bcr-abl融合基因陽(yáng)性細(xì)胞占92%。阿霉素對(duì)K562、K562A02細(xì)胞的IC50值分別為(12.320±1.720)ug/ml和(24.742±2.310)ug/ml,K562A02細(xì)胞相對(duì)于K562細(xì)胞的耐藥倍數(shù)為2.01。MTT結(jié)果示阿霉素作用K562A02細(xì)胞24,48,72小時(shí)后,細(xì)胞增殖抑制率呈現(xiàn)濃度-時(shí)間依賴性。Real-time PCR及Western blot結(jié)果顯示阿霉素處理細(xì)胞后,HO-1表達(dá)下降,耐藥相關(guān)基因MDR1、NF-κB、MRP1、Topo IIα、ABCD2表達(dá)亦降低,用HO-1誘導(dǎo)劑Hemin、抑制劑ZNPP IX、阿霉素單藥分別及聯(lián)合處理K562A02細(xì)胞后,結(jié)果顯示HO-1高表達(dá)后耐藥相關(guān)基因表達(dá)升高,細(xì)胞凋亡率下降。而降低HO-1表達(dá),耐藥相關(guān)基因表達(dá)下降,細(xì)胞凋亡率增加。結(jié)論:阿霉素可抑制K562A02細(xì)胞增殖且誘導(dǎo)細(xì)胞凋亡,呈時(shí)間劑量梯度依賴關(guān)系,HO-1作為靶基因,當(dāng)上調(diào)HO-1時(shí)候,可以增加藥物對(duì)K562A02細(xì)胞耐藥性,促進(jìn)細(xì)胞增殖、減少細(xì)胞凋亡的效應(yīng)。當(dāng)下調(diào)HO-1時(shí),可增加阿霉素對(duì)細(xì)胞的敏感性,抑制細(xì)胞增殖,促進(jìn)細(xì)胞凋亡,從而達(dá)到逆轉(zhuǎn)耐藥的目的。HO-1可作為逆轉(zhuǎn)耐藥的靶基因,可以重新使K562A02對(duì)阿霉素重新敏感,起到增敏效應(yīng)。
[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.
【學(xué)位授予單位】：貴陽(yáng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.7

【參考文獻(xiàn)】

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1 柴柏勝;方琴;王季石;陳s，

本文編號(hào)：1342176