【摘要】：背景及目的:很多實(shí)體腫瘤包括黑色素瘤對(duì)治療的耐受或者在反復(fù)治療的情況下引起的獲得性耐受成為臨床治療的重大障礙,這很大程度上是由于腫瘤細(xì)胞對(duì)化療藥物誘導(dǎo)凋亡不敏感。目前研究表明除了死亡受體和線粒體途徑外,內(nèi)質(zhì)網(wǎng)也在調(diào)節(jié)化療藥物誘發(fā)的細(xì)胞凋亡中起著重要的作用。一些化療藥物能夠引發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激,盡管內(nèi)質(zhì)網(wǎng)應(yīng)激時(shí)的未折疊蛋白反應(yīng)可通過減緩內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)而保護(hù)細(xì)胞,但過強(qiáng)或持續(xù)時(shí)間過長(zhǎng)的應(yīng)激反應(yīng)可引起細(xì)胞凋亡。近期研究結(jié)果表明:黑色素瘤對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的凋亡并不敏感,這種抗凋亡機(jī)制目前尚未完全明確。但已經(jīng)清楚和線粒體途徑密切相關(guān)的BH3-only家族蛋白對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡起到重要調(diào)節(jié)作用,其中Bim在很多對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡敏感的細(xì)胞中發(fā)生上調(diào)并發(fā)揮重要的促凋亡作用,但目前對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)下的黑色素瘤細(xì)胞中Bim的表達(dá)情況了解甚少,本課題旨在探討B(tài)im在黑色素瘤細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)中的調(diào)控以及其在黑色素瘤細(xì)胞對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡耐受中的作用,從而更加完善黑色素瘤對(duì)化療誘導(dǎo)凋亡不敏感的分子機(jī)制。 方法:采用天然核苷抗生素衣霉素(TM)處理黑色素瘤Mel-RM細(xì)胞株、MM200細(xì)胞株和對(duì)照組HEK293細(xì)胞株,建立內(nèi)質(zhì)網(wǎng)應(yīng)激模型。通過流式細(xì)胞術(shù)(FCM)AnnexinⅤ/ PI雙染法及Hoechst染色法檢測(cè)細(xì)胞凋亡;Western blot檢測(cè)caspase-3,-9及PARP的活化和Bim,GRP78,CHOP及FOXO1等蛋白水平;實(shí)時(shí)熒光定量PCR檢測(cè)Bim,CHOP及FOXO1的mRNA水平;小干擾RNA(siRNA)技術(shù)特異性“沉默”Bim基因,Western blot檢測(cè)Bim的“沉默”效率和“沉默”前后caspase-3的活化情況;采用流式細(xì)胞術(shù)檢測(cè)“沉默”Bim前后TM誘導(dǎo)HEK293細(xì)胞凋亡率的變化。 結(jié)果:TM作用黑色素瘤細(xì)胞和HEK293細(xì)胞后,兩株細(xì)胞對(duì)藥物誘導(dǎo)的凋亡均呈劑量和時(shí)間依賴性,但黑色素瘤細(xì)胞較HEK293細(xì)胞相比,對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡表現(xiàn)為耐受。對(duì)照組HEK293細(xì)胞中caspase-3,-9明顯活化,但在黑色素瘤細(xì)胞中卻不明顯。在內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)下的黑色素瘤細(xì)胞中Bim的蛋白水平?jīng)]有上調(diào),mRNA水平下調(diào)。檢測(cè)黑色素瘤細(xì)胞中內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)下調(diào)控Bim的轉(zhuǎn)錄因子CHOP的表達(dá)水平上調(diào),另一轉(zhuǎn)錄因子FOXO1的表達(dá)水平下調(diào)。在TM誘導(dǎo)的HEK293細(xì)胞中特異性“沉默”Bim基因后caspase-3的活化程度下降,細(xì)胞凋亡率也明顯下降。 結(jié)論:Bim在內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)下的黑色素瘤細(xì)胞中受到異常調(diào)控,這可能和轉(zhuǎn)錄因子CHOP及FOXO1有關(guān),這種異常調(diào)控可能是導(dǎo)致黑色素瘤細(xì)胞對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡耐受的重要原因,因此Bim是提高黑色素瘤對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡敏感性的一個(gè)重要靶點(diǎn)。
[Abstract]:Background and objective: the tolerance of many solid tumors, including melanoma, to treatment or acquired tolerance in the case of repeated treatment has become a major obstacle to clinical treatment. This is largely due to the insensitivity of tumor cells to chemotherapeutic drugs. In addition to death receptors and mitochondrial pathways, endoplasmic reticulum plays an important role in regulating apoptosis induced by chemotherapeutic drugs. Some chemotherapeutic drugs can induce endoplasmic reticulum stress. Although the unfolded protein response to endoplasmic reticulum stress can protect cells by slowing down the endoplasmic reticulum stress state, excessive or prolonged stress response can induce apoptosis. Recent studies have shown that melanoma is not sensitive to endoplasmic reticulum stress-induced apoptosis, and the mechanism of anti-apoptosis is not completely clear. However, it is well known that BH3-only family proteins, which are closely related to mitochondrial pathway, play an important role in regulating endoplasmic reticulum stress-induced apoptosis, in which Bim up-regulates and plays an important role in promoting apoptosis in many cells sensitive to endoplasmic reticulum stress apoptosis. However, little is known about the expression of Bim in melanoma cells under endoplasmic reticulum stress. The aim of this study was to investigate the role of Bim in the regulation of endoplasmic reticulum stress in melanoma cells and its role in the tolerance of melanoma cells to endoplasmic reticulum stress-induced apoptosis. Thus, the molecular mechanism of melanoma insensitivity to chemotherapy-induced apoptosis was improved. Methods: the endoplasmic reticulum (ER) stress model was established by treating melanoma Mel-RM cell line, MM200 cell line and control HEK293 cell line with natural nucleoside antibiotic itamycin (TM). The activation of caspase-3,-9 and PARP and the levels of Bim,GRP78,CHOP and FOXO1 were detected by (FCM) Annexin 鈪，

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