發(fā)布時間：2018-06-13 05:25

  本文選題：雷公藤紅素 + 結(jié)直腸癌�。� 參考：《北京協(xié)和醫(yī)學院》2017年博士論文

【摘要】：近年來,中藥防治腫瘤受到了人們廣泛的關(guān)注。雷公藤是我國傳統(tǒng)中藥材衛(wèi)矛科雷公藤屬植物,它的有效成分之一——雷公藤紅素,被證明具有廣泛的藥理學作用。臨床上,雷公藤紅素作為傳統(tǒng)藥物,用于治療多種炎癥疾病和自身免疫性疾病,如過敏性哮喘、肌萎縮側(cè)索硬化癥以及類風濕性關(guān)節(jié)炎等。隨著研究的深入,人們逐漸發(fā)現(xiàn),雷公藤紅素還可以抑制多種腫瘤的發(fā)生發(fā)展,如乳腺癌、肝細胞肝癌、膀胱癌、胃癌和前列腺癌等,但在結(jié)直腸癌中的報道卻非常少見。本研究的目的是系統(tǒng)地探究雷公藤紅素對結(jié)直腸癌的作用以及其具體的分子機制。MTT和細胞克隆的實驗結(jié)果表明,雷公藤紅素可以顯著地抑制結(jié)直腸癌細胞系SW480和HCT116細胞的生長。我們用雷公藤紅素腹腔注射兩種經(jīng)典的結(jié)直腸癌動物模型——-C57BL/6J-ApcMin/+(APCMin/+)小鼠和AOM/DSS小鼠,結(jié)果發(fā)現(xiàn)雷公藤紅素在小鼠體內(nèi)也有明顯的抑瘤作用。同時,Western blot和TUNEL的實驗結(jié)果證明,雷公藤紅素在體內(nèi)、外可以促進細胞凋亡。此外,我們還發(fā)現(xiàn)雷公藤紅素可以通過泛素化-蛋白酶體途徑降解β-catenin,從而抑制Wnt/β-catenin信號通路的活性。面臨床上,大多數(shù)患者的結(jié)直腸癌組織都存在Wnt/β-catenin信號通路的異常活化。因此,這提示我們,抑制Wnt/β-catenin信號通路可能是雷公藤紅素抑制結(jié)直腸癌的機制之一。Hippo信號通路與Wnt/β-catenin信號通路有著千絲萬縷的聯(lián)系。Hippo信號通路是一條高度保守的信號通路,參與調(diào)控器官大小等。有研究指出,Hippo信號通路的下游主要效應(yīng)分子——Yes相關(guān)蛋白(yes-associated protein,YAP)與β-catenin可以相互作用,從而調(diào)節(jié)腸道再生以及結(jié)直腸癌的發(fā)生發(fā)展。我們的研究表明,在雷公藤紅素降解β-catenin的過程中,YAP存在于β-catenin降解的復合體上,并且YAP的磷酸化對β-catenin的降解至關(guān)重要。最近的文獻報道,肝臟激酶1(liver kinase B1,LKB1),也被稱作絲氨酸-蘇氨酸激酶 11(serine-threonine kinase 11,STK11)作為抑癌基因,可以促進YAP的磷酸化,使得YAP從細胞核移至細胞漿中,最后被蛋白酶體降解。我們在SW480和HCT116細胞中利用CRISPR-Cas9體系構(gòu)建了LKB1缺失的穩(wěn)定細胞系。在體內(nèi),我們利用Loxp-Cre體系構(gòu)建了在腸道特異性敲除LKB1的小鼠,并且用AOM/DSS誘導其發(fā)展為結(jié)直腸癌。我們用雷公藤紅素處理后,結(jié)果發(fā)現(xiàn)LKB1的缺失不僅可以在體內(nèi)外促進結(jié)直腸癌細胞的生長,還能拮抗雷公藤紅素降解β-catenin,從而削弱雷公藤紅素的抑瘤作用。我們進一步還發(fā)現(xiàn)了雷公藤紅素可以上調(diào)熱休克因子1(heat shock factor 1,HSF1)的表達,從而增強LKB1的轉(zhuǎn)錄活性。LKB1激活其下游分子——AMP活化的蛋白激酶α(AMP-activated protein kinase α,AMPKα),進一步磷酸化 YAP,從而促進了β-catenin泛素化-蛋白酶體途徑的降解。綜上所述,我們的研究表明,雷公藤紅素通過泛素化-蛋白酶體途徑降解β-catenin,從而在體內(nèi)、外明顯地抑制結(jié)直腸癌細胞的生長,而找到雷公藤紅素降解β-catenin的具體途徑是關(guān)鍵。我們的研究發(fā)現(xiàn)了其潛在的一條通路——雷公藤紅素可以通過提高HSF1的蛋白水平,上調(diào)LKB1的轉(zhuǎn)錄活性,從而激活AMPKα,進一步磷酸化YAP。磷酸化的YAP存在于β-catenin降解的復合體上,促進β-catenin泛素化-蛋白酶體途徑的降解。在此過程中,YAP和LKB1在結(jié)直腸癌發(fā)生發(fā)展和雷公藤紅素抑瘤過程中起著不可或缺的作用。以上結(jié)果為雷公藤紅素在臨床上用于結(jié)直腸癌的治療提供了很好的理論基礎(chǔ)。Hippo信號通路是一條高度保守的信號通路,它參與機體組織和器官的發(fā)育過程和維持體內(nèi)細胞增殖與凋亡的動態(tài)平衡,也被證明與腫瘤的發(fā)生發(fā)展有著密切的聯(lián)系。Hippo信號通路的主要下游效應(yīng)分子YAP,是一個轉(zhuǎn)錄激活因子,參與調(diào)控下游很多靶基因,而這些靶基因多與細胞的增殖與存活相關(guān)。作為癌基因,YAP可以促進細胞生長和抑制細胞凋亡。而Hippo信號通路下游的另一個主要效應(yīng)分子TAZ,與YAP具有將近60%的同源性。因此,我們推測TAZ可能具有與YAP相似的生物學功能。為了驗證我們的猜想,我們分別在T-Rex-293細胞和HEK293細胞中構(gòu)建了 TAZ誘導表達和穩(wěn)定表達的細胞系。細胞生長曲線、細胞克隆形成以及裸鼠皮下成瘤實驗結(jié)果證明了,高表達TAZ可以在體內(nèi)外促進細胞的生長。同時,我們還發(fā)現(xiàn)上調(diào)TAZ的表達可以部分逆轉(zhuǎn)雷公藤紅素誘導的細胞凋亡。接著,我們又找到了 TAZ抵抗細胞凋亡的可能的分子機制——誘導的TAZ高表達可以上調(diào)其下游靶基因包括ANKRD,CYR61以及CTGF,并且還可以增加Bcl-2的表達,降低Bax的表達,活化PI3K/Akt信號通路。以上發(fā)現(xiàn)揭示了 TAZ作為癌基因可以參與調(diào)控細胞增殖和凋亡。
[Abstract]:In recent years, traditional Chinese medicine for the prevention and control of tumor has attracted wide attention. Tripterygium wilfordii is a traditional Chinese medicinal herb of the genus Corey, one of its effective components, tripterin, has been proved to have extensive pharmacological effects. Diseases, such as allergic asthma, amyotrophic lateral sclerosis and rheumatoid arthritis, have gradually been found, with the further research, that tripterin can also inhibit the development of various tumors, such as breast cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, and prostate cancer, but it is rarely reported in colorectal cancer. The purpose of this study was to systematically explore the effect of tripterin on colorectal cancer and its specific molecular mechanism.MTT and cell clones. The results showed that tripterin could significantly inhibit the growth of SW480 and HCT116 cells in the colorectal cancer cell lines. We injected two classic colorectal cancer models with tripterin abdominal cavity. -C57BL/6J-ApcMin/+ (APCMin/+) mice and AOM/DSS mice found that tripterine was also significantly antitumor in mice. Meanwhile, the results of Western blot and TUNEL showed that tripterine could promote apoptosis in the body. In addition, we also found that tripterine could be used as a ubiquitin protease. The body pathway degrades beta -catenin and inhibits the activity of the Wnt/ beta -catenin signaling pathway. In the face of the bed, the colorectal cancer tissues in most patients have abnormal activation of the Wnt/ beta -catenin signaling pathway. Therefore, this suggests that the inhibition of Wnt/ beta -catenin signaling pathway may be one of the.Hippo signals of Lei Gongteng erythropoietin inhibition of colorectal cancer. The pathway is inextricably linked to the Wnt/ beta -catenin signaling pathway. The.Hippo signaling pathway is a highly conservative signal pathway and participates in the regulation of the size of the organ. There is a study that the main effector molecules of the Hippo signaling pathway, the Yes related protein (Yes-associated protein, YAP), can interact with the beta -catenin, thereby regulating the interaction between the Hippo signaling pathway and the beta -catenin. Intestinal regeneration and the development of colorectal cancer. Our study showed that in the course of tripterine degradation of beta -catenin, YAP exists on the complex of beta -catenin degradation, and the phosphorylation of YAP is essential for the degradation of beta -catenin. Recent literature reports that liver irritable enzyme 1 (liver kinase B1, LKB1), also known as serine - Su. As a tumor suppressor gene, ammonia kinase 11 (serine-threonine kinase 11, STK11) can promote the phosphorylation of YAP, causing YAP to move from the nucleus to the cytoplasm and eventually degrade the proteasome. We construct a LKB1 deletion stable cell line in SW480 and HCT116 cells. In vivo, we use the Loxp-Cre system to construct the cell. The mice in the intestinal specific knockout of LKB1 were induced to develop colorectal cancer with AOM/DSS. After treatment with tripterine, we found that the absence of LKB1 could not only promote the growth of colorectal cancer cells in vivo and in vivo, but also antagonize the degradation of beta -catenin by tripterine and weaken the tumor suppressor effect of tripterin. It is also found that tripterin can increase the expression of heat shock factor 1 (heat shock factor 1, HSF1), thereby enhancing the transcriptional activity of LKB1 to activate its downstream molecules - AMP activated protein kinase alpha (AMP-activated protein kinase A, AMPK alpha), and further phosphorylation YAP, thus promoting beta ubiquitination - protease. In summary, our study shows that tripterin degrades beta -catenin through the ubiquitin proteasome pathway and thus significantly inhibits the growth of colorectal cancer cells in the body, and the specific pathway for the discovery of tripterin to degrade beta -catenin is the key. Our study found a potential pathway - Lei Gongteng erythropoietin can activate the transcriptional activity of LKB1 by raising the protein level of HSF1 and activating the AMPK alpha, and further phosphorylated YAP. phosphorylation YAP exists on the complex of beta -catenin degradation, promoting the degradation of beta -catenin ubiquitination proteasome pathway. In this process, YAP and LKB1 are developed in colorectal cancer and in the development of colorectal cancer and Lei Gongteng Erythropoietin plays an indispensable role in tumor suppression. The above results provide a good theoretical basis for the clinical application of tripterin in the treatment of colorectal cancer. The.Hippo signal pathway is a highly conservative signal pathway. It participates in the development of tissues and organs and maintains the dynamic balance of cell proliferation and apoptosis in the body. It has also been shown that the main downstream effector, YAP, which is closely related to the development of the.Hippo signaling pathway, is a transcriptional activator that participates in the regulation of many downstream target genes, which are related to cell proliferation and survival. As a oncogene, YAP can promote cell growth and inhibit apoptosis. Hippo TAZ, another major effector downstream of the signal pathway, has nearly 60% homology with YAP. Therefore, we speculate that TAZ may have a biological function similar to that of YAP. In order to verify our conjecture, we constructed the cell lines of TAZ induced and stable expression in T-Rex-293 and HEK293 cells. The cell growth curve, The results of cell cloning and subcutaneous tumor formation in nude mice showed that high expression of TAZ could promote cell growth in vitro and in vivo. Meanwhile, we also found that up regulation of TAZ could partly reverse the apoptosis induced by tripterin. Then, we found the possible molecular mechanism of TAZ resistance to apoptosis - induced TA The high expression of Z can increase its downstream target genes including ANKRD, CYR61 and CTGF, and also increase the expression of Bcl-2, reduce the expression of Bax and activate the PI3K/Akt signaling pathway. These findings reveal that TAZ as a oncogene can participate in the regulation of cell proliferation and apoptosis.
【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】：2017
【分類號】：R285
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本文編號：2012885