本文關(guān)鍵詞： ApoG2 放射增敏 鼻咽癌 Bcl-2 自噬 裸鼠　出處：《暨南大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的：程序性細胞死亡包括凋亡和自噬性死亡,越來越多的研究表明自噬在腫瘤中起重要作用。Beclin 1與Ⅲ型PI3K結(jié)合啟動自噬的發(fā)生,Bcl-2/Bcl-xL結(jié)合Beclin 1阻斷其自噬誘導功能。Bcl-2/Bcl-xL等在鼻咽癌中高表達且與其預(yù)后呈負相關(guān)。鼻咽癌以放療為主,放療除了誘導凋亡還能誘導自噬,在抗腫瘤中起重要作用。Apogossypolone (ApoG2)是經(jīng)過改造的棉酚衍生物,它是一種新型的抗凋亡Bcl-2家族蛋白的小分子抑制劑。我們研究發(fā)現(xiàn)ApoG2能與Bcl-2/Bcl-xl結(jié)合,在鼻咽癌細胞中阻斷Bcl-2的功能,并在體內(nèi)外增強化療敏感性；還發(fā)現(xiàn)抗癌藥物、射線等調(diào)控Beclin 1的表達誘導鼻咽癌細胞自噬性死亡。本課題擬進一步研究ApoG2抑制Bcl-2/Bcl-xL與Beclin 1的結(jié)合,阻斷Beclin 1誘導自噬的功能,體內(nèi)外增強放射線誘導鼻咽癌細胞自噬性死亡以及放療敏感性,為Bcl-2小分子抑制劑類型的抗腫瘤藥物聯(lián)合放射治療鼻咽癌提供實驗依據(jù),同時為將ApoG2研發(fā)成新型的抗腫瘤藥物奠定基礎(chǔ)。 方法：體外培養(yǎng)人鼻咽癌CNE1、CNE2和SUNE1細胞及鼻咽上皮細胞NP69,MTT法檢測不同濃度Bcl-2抑制劑ApoG2對人鼻咽癌CNE1、CNE2和SUNE1細胞的抑制作用及對鼻咽上皮細胞NP69的細胞毒作用,計算各自的IC50值,選擇低于IC50的藥物濃度作為后續(xù)實驗濃度。克隆形成實驗檢測不同劑量放射線加或不加ApoG2對人鼻咽癌CNE1、CNE2和SUNE1細胞生存曲線的影響,計算其存活率,繪制細胞存活曲線。免疫共沉淀法檢測ApoG2對Beclin 1和Bcl-2結(jié)合的影響。慢病毒感染CNE2細胞,Knock down Atg5,建立穩(wěn)定細胞株,觀察自噬的缺失對ApoG2引起的鼻咽癌細胞放療增敏的影響。GFP-LC3質(zhì)粒轉(zhuǎn)染腫瘤細胞,熒光顯微鏡觀察自噬標志物LC3的聚集。透射電鏡檢測細胞自噬形態(tài)。免疫印跡法(Western blot)檢測自噬相關(guān)蛋白LC3、P62的表達。建立裸鼠移植瘤模型,觀察ApoG2口服、單獨放療及兩者聯(lián)合對鼻咽癌細胞移植腫瘤的生長抑制作用；免疫組化檢測移植瘤組織中LC3表達的變化。 結(jié)果： 1. ApoG2可增強CNE1、CNE2、SUNE1細胞的放射敏感性 MTT結(jié)果顯示Bcl-2抑制劑ApoG2對人鼻咽癌CNE1、CNE2、SUNE1細胞均具有增殖抑制作用,呈劑量依賴性,IC50值分別為2.84、2.18、5.64μM。對正常鼻咽上皮細胞無細胞毒作用。體外克隆形成實驗分析顯示,CNE2細胞中放療+ApoG2不同濃度組的劑量效應(yīng)因子(DEF)值均大于1,分別為1.92、1.52和1.13。ApoG2可以提高放射增敏作用,并且放射增敏效果呈藥物劑量依賴性。 2. ApoG2可促使Bcl-2與Beclin 1解離 Beclin 1是一種BH3-only蛋白家族成員,起初被鑒定為Bcl-2相互作用蛋白,也是第一個被發(fā)現(xiàn)的哺乳動物自噬相關(guān)基因,在腫瘤的發(fā)生發(fā)展中起關(guān)鍵作用。免疫共沉淀檢測結(jié)果顯示ApoG2可與Beclin 1競爭性抑制Bcl-2,從而促使Bcl-2與Beclin 1解離。 3. ApoG2可誘導鼻咽癌細胞發(fā)生自噬 激光共聚焦顯微鏡觀察可見ApoG2及放射處理后的轉(zhuǎn)染GFP-LC3質(zhì)粒的CNE2細胞內(nèi),LC3呈現(xiàn)明顯的點狀聚集,且ApoG2聯(lián)合放射可以增加LC3的點狀聚集,對照組胞漿內(nèi)LC3呈均勻彌散分布；透射電鏡觀察2Gy放射聯(lián)合20μMApoG2組照射48 h后可觀察到自噬小體的形成。Western blot檢測可見放療組和ApoG2組均出現(xiàn)LC3的表達,而ApoG2聯(lián)合放療可以顯著增加LC3的表達；并呈時間劑量依賴性。提示ApoG2聯(lián)合放療可進一步誘導鼻咽癌細胞發(fā)生自噬。流式細胞術(shù)檢測細胞壞死,結(jié)果顯示ApoG2聯(lián)合放療處理可以誘導CNE2細胞發(fā)生凋亡和壞死。 4. knock down Atg5可抑制CNE2細胞自噬的發(fā)生,并提高ApoG2+放療組的克隆形成率 建立Atg5 knock down CNE2細胞株,Western blot檢測到與VEC組(轉(zhuǎn)染空載體)相比shAtg5組(轉(zhuǎn)染質(zhì)粒shAtg5)中Atg5的表達被抑制。Western blot檢測ApoG2不能誘導shAtg5細胞發(fā)生自噬�？寺⌒纬蓪嶒灆z測放療、ApoG2處理及兩者聯(lián)合處理VEC組和shAtg5組細胞的細胞存活率,shAtg5組細胞存活率明顯高于VEC組,細胞死亡率平均從67%升至82%。 5. ApoG2可抑制裸鼠移植瘤的生長 建立荷人鼻咽癌細胞的裸鼠放療增敏模型,與對照組對比,ApoG2聯(lián)合放射線可減小裸鼠的瘤體積及瘤重。ApoG2組、放療組、ApoG2+放療組對CNE 2裸鼠移植瘤生長的抑制率分別是46.89%、19.34%和61.64%。免疫組化結(jié)果顯示ApoG2聯(lián)合放射可增加瘤組織中LC3的表達。 結(jié)論： 1. ApoG2可誘導鼻咽癌細胞發(fā)生自噬。 2. ApoG2可增強鼻咽癌細胞對放療的敏感。 3. ApoG2可促使Bcl-2與Beclin 1解離,游離出的Beclin 1活化下游自噬信號通路,這可能是ApoG2提高放射增敏作用的機制。 4. ApoG2聯(lián)合放療可顯著抑制裸鼠(CNE2細胞)移植瘤的生長。
[Abstract]:Objective: programmed cell death, including apoptosis and autophagic cell death, a growing number of studies suggest that autophagy plays an important role in.Beclin 1 and type PI3K in tumors with start autophagy, Bcl-2/Bcl-xL combined with Beclin 1 to block the function of.Bcl-2/Bcl-xL in autophagy induced high expression in nasopharyngeal carcinoma and its prognosis was negatively related to nasopharyngeal carcinoma radiotherapy. In addition, radiotherapy induced apoptosis can induce autophagy,.Apogossypolone plays an important role in anti-tumor (ApoG2) after gossypol derivative transformation, it is a kind of novel small molecule inhibitors of anti apoptotic Bcl-2 family proteins. We found that ApoG2 can bind to Bcl-2/Bcl-xl, blocking the function of Bcl-2 in nasopharyngeal carcinoma cells, and enhance the chemosensitivity in vitro and in vivo; also found anticancer drugs, radiation and other expression regulation of Beclin 1 induced autophagic cell death in nasopharyngeal carcinoma. This article intends to With the further study of ApoG2 inhibition of Bcl-2/Bcl-xL and Beclin 1, Beclin 1 blockade induced autophagy function induced by radiotherapy in nasopharyngeal carcinoma cells and enhance the radiosensitivity of autophagic cell death in vitro and in vivo antitumor drugs combined with radiotherapy for nasopharyngeal carcinoma and provide experimental evidence of small molecule inhibitors of Bcl-2 type, and ApoG2 will be developed into new anticancer drugs laid the foundation.
Methods: human nasopharyngeal carcinoma CNE1 in vitro, CNE2 and SUNE1 cells and nasopharyngeal epithelial cells NP69, detection of different concentrations of Bcl-2 inhibitor ApoG2 MTT on human nasopharyngeal carcinoma CNE1, inhibition of CNE2 and SUNE1 cells of nasopharyngeal epithelial cells and the cytotoxicity of NP69, to calculate their IC50 value, choose a lower drug concentration of IC50 as a follow-up experiment concentration. Colony formation assay of different doses of radiation with or without ApoG2 CNE1 on human nasopharyngeal carcinoma, effect of CNE2 and SUNE1 cell survival curve, calculate the survival rate, cell survival curve. Co immunoprecipitation method was used to detect the effect of Beclin 1 and ApoG2 Bcl-2 binding. Lentivirus infected CNE2 cells, Knock down Atg5. To establish a stable cell line, NPC cells lack of observation of radiotherapy autophagy induced by ApoG2 sensitized.GFP-LC3 plasmid was transfected into tumor cells, fluorescence microscope was used to observe autophagy marker LC3 Aggregation. Transmission electron microscope was used to detect autophagy morphology. Immunoblotting (Western blot) detection of autophagy related protein LC3, P62 expression. Nude mice, observe ApoG2 orally, radiotherapy alone or in combination on nasopharyngeal carcinoma cell transplantation tumor growth inhibition; expression of LC3 immunohistochemical detection of tumor tissues.
Result錛，

本文編號：1506100