本文關鍵詞： 食管鱗癌 JQ1 治療 小分子抑制劑 BET　出處：《北京協(xié)和醫(yī)學院》2015年博士論文　論文類型：學位論文

【摘要】：我國食管癌發(fā)病率居世界首位,又是食管癌死亡率最高的國家；其中95%為鱗癌,與歐美國家70%左右為腺癌有明顯差異。我國食管鱗癌在城市位于惡性腫瘤發(fā)病第4位,死亡第5位；在農(nóng)村發(fā)病和死亡率均為第4位,是危害我國人民健康的主要惡性腫瘤之一。目前,食管鱗癌的治療以手術和放療為主,尚缺少有效的化療藥物,急需研究開發(fā)新的對食管鱗癌有效的治療藥物。染色質(zhì)的重構是細胞表型調(diào)節(jié)的重要機制,在包括食管癌在內(nèi)的惡性腫瘤的發(fā)生發(fā)展中發(fā)揮重要的作用。組蛋白乙�；揎検侨旧|(zhì)重構的重要方式之一,組蛋白賴氨酸的乙酰化使染色質(zhì)結構打開,呈轉錄激活狀態(tài)。BET (Bromodomain and extra terminal domain)蛋白家族,是一類含有Bromodomain結構域的分子,是與乙�；嚢彼峤Y合的活性區(qū)域。BET蛋白通過與乙�；嚢彼峤Y合,招募其他轉錄因子,并組裝成轉錄復合物,激活下游靶基因的轉錄。JQ1是一種新的BET小分子抑制劑,能夠競爭性結合其活性結合區(qū),使轉錄復合物從染色質(zhì)區(qū)解離,從而抑制其下游分子的轉錄。研究已發(fā)現(xiàn)JQ1對血液系統(tǒng)腫瘤及胰腺癌、卵巢癌有明顯的抑制活性。本論文研究了JQ1對食管鱗癌細胞的抑制活性,并初步探討了其分子機制。我們用不同濃度的JQ1處理了不同的食管鱗癌細胞,在不同的時間點用CCK8檢測了細胞的活性,繪制細胞增殖曲線。利用流式細胞分析了JQ1處理細胞的細胞周期分布。利用real time PCR和Western Blot檢測了JQ1處理細胞后c-Myc和p21的表達水平。我們首先用20μM的JQ1處理了7株食管鱗癌細胞120h進行了敏感性初篩,發(fā)現(xiàn)JQ1能夠顯著抑制食管鱗癌細胞的增殖,對不同的細胞系表現(xiàn)出不同的抑制活性。隨后分別用0.1μM、0.5μM、1μM、2μM、5μM、10μM口20μM的JQ1處理了食管鱗癌細胞系KYSE-30、KYSE-70、KYSE-140、KYSE-150、KYSE-410、KYSE-450和KYSE-510,分別在處理0h、24h、48h、72h、96h和120h檢測了細胞活性,增殖曲線表明不同濃度的JQ1對不同的食管鱗癌細胞系具有不同的抑制活性,除了KYSE-140細胞對JQ1敏感性較差以外,其他6株食管鱗癌細胞系對JQ1都具有較高的敏感性。我們進一步分析了JQ1對食管鱗癌細胞增殖抑制活性的劑量效應關系,結果表明,JQ1對KYSE-30、KYSE-70、KYSE-150和KYSE-510的增殖抑制活性具有明顯的劑量效應關系；JQ1對KYSE-410和KYSE-450的抑制活性的劑量效應關系并不明顯,但JQ1在較低的0.1μM濃度時就有較高的抑制活性,72h抑制率就達到28.67%,對KYSE-450的抑制率達到29.07%。我們進一步分析了JQ1對不同食管鱗癌細胞系增殖抑制活性的IC50值,結果表明JQ1對食管鱗癌細胞增殖抑制的IC50值范圍為0.19μM-9.95μM,其中KYSE-410和KYSE-450的IC50值最低,分別為0.92μM和0.19μM。進一步機制研究發(fā)現(xiàn),JQ1處理食管鱗癌細胞后G1期細胞比例明顯增高,說明JQ1通過引起食管鱗癌細胞G0/G1期阻滯來發(fā)揮其增殖抑制活性。下游蛋白表達分析表明,JQ1抑制了食管鱗癌細胞c-Myc的轉錄和表達,進而使p21的轉錄和表達增加,發(fā)揮其細胞周期阻滯和增殖抑制活性。綜述所述,本論文的研究結果發(fā)現(xiàn)新的BET蛋白小分子抑制劑JQ1通過抑制c-Myc和p21的表達誘導了細胞周期G0/G1期阻滯,發(fā)揮了其對食管鱗癌細胞的增殖的抑制活性,并具有明顯的劑量效應關系。BET蛋白小分子抑制劑JQ1是潛在的食管鱗癌新的治療藥物。
[Abstract]:The incidence of esophageal cancer in China ranks first in the world, and is the country with the highest mortality rate of esophageal cancer; 95% were squamous cell carcinoma, and 70% adenocarcinoma around Europe and the United States have obvious difference. Our city is located in the esophageal squamous cell carcinoma in the incidence of malignant tumor fourth, Fifth deaths in rural areas; the morbidity and mortality rate was fourth. China is one of the major malignant tumors harm people's health. At present, the treatment of esophageal cancer with surgery and radiotherapy, there is a lack of effective chemotherapy drugs, in urgent need of research and development of esophageal squamous cell carcinoma with new treatment. The reconstruction of chromatin is an important mechanism in the regulation of cell types, including esophageal cancer, malignant the occurrence and development of tumor play an important role. Histone acetylation is one of the important ways of chromatin remodeling and histone lysine acetylation to open chromatin structure, a transcription activation state of the.BET (Bro Modomain and extra terminal domain) is a protein family, proteins containing Bromodomain domain, is combined with acetylated lysine with acetyl lysine binding activity of.BET protein, recruit other transcription factors and assembled transcription complexes,.JQ1 activates transcription of downstream target genes is a kind of new small molecule inhibitors of BET, can compete with the activity of the transcription complex binding region from chromatin region dissociation, thereby inhibiting the transcription of the downstream molecules. Studies have found that JQ1 of blood cancer and pancreatic cancer, ovarian cancer has obvious inhibitory activity. This paper studied the inhibitory activity of JQ1 for esophageal squamous cell carcinoma, and to explore the molecular mechanism of esophageal squamous cell carcinoma. We treated with different concentrations of JQ1 were different, detection of cell viability by CCK8 at different time points, draw the cell growth curve using. Flow cytometric analysis of cell cycle distribution of the cells treated with JQ1. To detect the expression of JQ1 cells treated with c-Myc and p21 by real time PCR and Western Blot. We first used 20 M JQ1 with 7 strains of esophageal squamous cell carcinoma cell line 120h were sensitive screening, found that JQ1 could significantly inhibit the esophageal squamous cell carcinoma the proliferation of different cell lines showed different inhibition activity. Followed by 0.1 M, 0.5 M, 1 M, 2 M, 5 M, 10 M and 20 M JQ1 treatment KYSE-30, esophageal cancer cell line KYSE-70 KYSE-140, KYSE-150, KYSE-410, and KYSE-450 KYSE-510, 24h, 0h respectively in treatment, 48h, 72h, 96h and 120h to detect cell activity and proliferation curve showed that different concentrations of JQ1 could inhibit the activity of esophageal squamous cell carcinoma cell lines of different, in addition to KYSE-140 cells except for the poor sensitivity of JQ1, other 6 strains of esophageal squamous cell carcinoma cell lines have JQ1 High sensitivity. We further analyze the dose effect relationship, inhibitory activity of JQ1 on the proliferation of esophageal squamous cell carcinoma. The results show that the JQ1 of KYSE-30, KYSE-70, KYSE-150 and KYSE-510 inhibited proliferation activity had obvious dose effect relationship; dose effect relationship of KYSE-410 inhibited the activity of JQ1 and KYSE-450 is not obvious, but at lower JQ1 the 0.1 M concentration had higher inhibitory activity, the inhibition rate of 72h reached 28.67%, the inhibition rate of KYSE-450 reached 29.07%., we analyzed the JQ1 in different esophageal squamous cell carcinoma cell line proliferation inhibitory activity of IC50 values, the results show that inhibition of JQ1 on proliferation of esophageal squamous cell carcinoma of the IC50 range of 0.19 mu M-9.95 mu M among them, KYSE-410 and KYSE-450 of the lowest IC50 value were 0.92 M and 0.19 M. further study mechanism showed that JQ1 treatment of esophageal squamous cell carcinoma after G1 phase cells were increased, indicating JQ1 caused by esophageal squamous cell carcinoma G0/G1 cell cycle arrest to play its anti proliferative activity. Analysis shows that the downstream protein expression, inhibit JQ1 transcription and expression in esophageal squamous cell carcinoma cell line c-Myc, and the transcription and expression of p21 increased, exert its inhibitory activity on cell cycle arrest and proliferation. Review the research results of this paper found that BET the new protein small molecule inhibitors of JQ1 induced G0/G1 cell cycle arrest by inhibiting expression of c-Myc and p21, play the inhibitory activity of esophageal squamous cell carcinoma cell proliferation, and has obvious dose effect relationship between.BET protein small molecule inhibitors of JQ1 therapeutics is a new potential of esophageal squamous cell carcinoma.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.1

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