【摘要】：目的:研究凋亡調(diào)控相關(guān)蛋白來了解順鉑耐藥成因,同時(shí)考察乙烷硒啉(Ethaselen)在K562耐藥細(xì)胞中逆轉(zhuǎn)順鉑耐藥的作用,并初步探討其作用機(jī)制。創(chuàng)新點(diǎn):首次研究乙烷硒啉在逆轉(zhuǎn)順鉑耐藥中的作用,且此作用與乙烷硒啉誘導(dǎo)細(xì)胞凋亡相關(guān)。方法:通過長(zhǎng)時(shí)間脈沖誘導(dǎo)得到順鉑耐藥K562細(xì)胞,并觀察耐藥細(xì)胞形態(tài)及倍增時(shí)間。采用MTT法考察乙烷硒啉、順鉑及其聯(lián)用組在不同細(xì)胞株間的生長(zhǎng)抑制作用。流式細(xì)胞術(shù)分析細(xì)胞凋亡情況以及細(xì)胞內(nèi)活性氧(ROS)水平。最后,通過蛋白質(zhì)免疫印跡(Western blot)考察凋亡調(diào)控相關(guān)蛋白水平的變化。結(jié)論:脈沖誘導(dǎo)得到的K562耐藥細(xì)胞對(duì)順鉑的耐受性是原K562細(xì)胞的5.34倍。形態(tài)學(xué)觀察發(fā)現(xiàn),耐藥細(xì)胞體積增大,粘附性進(jìn)一步降低。乙烷硒啉與順鉑聯(lián)用表現(xiàn)出協(xié)同效應(yīng)。當(dāng)加入少量的乙烷硒啉(順鉑與乙烷硒啉的摩爾比率為10:1),順鉑作用K562耐藥細(xì)胞的半抑制濃度(IC50)值可以減少21倍。流式細(xì)胞術(shù)及Western blot表明,乙烷硒啉能夠誘導(dǎo)耐藥細(xì)胞凋亡。其逆轉(zhuǎn)順鉑耐藥主要是通過調(diào)控Bcl-2及Bax蛋白比例以及通過提高細(xì)胞內(nèi)活性氧水平引起線粒體通透轉(zhuǎn)運(yùn)孔道(PTP)蛋白孔道的形成來促使釋放細(xì)胞色素c,進(jìn)而引起Caspase凋亡途徑。
[Abstract]:Aim: to study the apoptosis regulation related proteins to understand the cause of platinum resistance, and to investigate the reversal effect of ethane selenite (Ethaselen) on platinum resistance in K562 resistant cells, and to explore its mechanism. Innovation: the role of ethane selenite in reversal of platinum resistance was studied for the first time, and this effect was related to apoptosis induced by ethane selenite. Methods: DDP resistant K562 cells were induced by long pulse, and the morphology and doubling time of drug resistant cells were observed. MTT assay was used to investigate the growth inhibition of ethane selenite, DDP and their combination groups among different cell lines. Apoptosis and intracellular reactive oxygen species (Ros) (ROS) levels were analyzed by flow cytometry. Finally, the changes of apoptosis-regulated protein levels were investigated by Western imprinting (Western blot). Conclusion: the drug resistance of K562 cells induced by pulse is 5.34 times as much as that of original K562 cells. Morphological observation showed that the volume of drug-resistant cells increased and the adhesion decreased further. The combination of ethane selenite and DDP showed synergistic effect. When a small amount of ethane selenite was added (the molar ratio of DDP to ethane selenite was 10:1), the semi-inhibitory concentration (IC50) of K562 cells treated with DDP could be reduced by 21 times. Flow cytometry and Western blot showed that ethane selenite could induce apoptosis of drug-resistant cells. The reversal of DDP resistance is mainly due to the regulation of the proportion of Bcl-2 and Bax proteins and the formation of (PTP) protein pores in mitochondrial permeable transport channels by increasing the level of reactive oxygen species (Ros) in the cells to promote the release of cytochrome C. And then the apoptosis pathway of Caspase was induced.
【作者單位】： State
【基金】：supported in part by the National Natural Science Foundation of China(No.81372266) the National Science and Technology Major Project of the Ministry of Science and Technology of China(No.2011zx09101-001-03)
【分類號(hào)】：R96

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