本文選題：結(jié)直腸癌 + 雷公藤甲素　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：結(jié)直腸癌(Colorectal cancer,CRC)的發(fā)病率和致死率很高,分別位居腫瘤譜的第三位和第四位。好發(fā)于直腸及直腸與乙狀結(jié)腸交界處,是常見的消化道惡性腫瘤之一。臨床研究領(lǐng)域?qū)Y(jié)直腸癌預(yù)防與治療的方法也在不斷的改進(jìn)。雷公藤甲素(Triptolide,TPL)又稱為雷公藤內(nèi)酯或是雷公藤內(nèi)酯醇,來源于中藥雷公藤的根,是有著多種生物活性的天然產(chǎn)物�，F(xiàn)代醫(yī)藥研究表明,TPL不僅抗氧化,抗類風(fēng)濕,還有抗癌的作用。但TPL在結(jié)直腸癌中的治療作用的研究還很少見。細(xì)胞自噬和凋亡均為抗癌治療機(jī)制中的研究熱點,傳統(tǒng)的化療藥物在化療的過程中會引起或誘導(dǎo)細(xì)胞保護(hù)性自噬,從而降低了化療藥物抑制癌細(xì)胞增殖的作用。本研究旨在細(xì)胞自噬在TPL抗結(jié)直腸癌細(xì)胞增殖效應(yīng)中的作用進(jìn)行研究分析。(1)雷公藤甲素(TPL)誘導(dǎo)調(diào)亡作用抑制結(jié)直腸癌細(xì)胞增殖通過用預(yù)定濃度梯度的TPL分別處理結(jié)直腸癌細(xì)胞系SW480和HCT11648h。測定細(xì)胞生長曲線表明,TPL能夠顯著抑制兩種人結(jié)直腸癌細(xì)胞系的增殖。接著用Western Blot和Annexin-V/PI雙染法流式凋亡檢測法分析凋亡相關(guān)蛋白。數(shù)據(jù)結(jié)果顯示,與對照組相比較,TPL處理組細(xì)胞凋亡率隨濃度增加明顯升高;促凋亡因子Cleaved-Caspase3被激活,Cleaved-PARP1蛋白表達(dá)量增加,有一定的濃度依賴性。表明TPL可以誘導(dǎo)結(jié)直腸癌細(xì)胞凋亡,抑制其細(xì)胞增殖。(2)TPL可抑制結(jié)直腸癌細(xì)胞自噬發(fā)生和自噬流的形成TPL處理組與對照組比較,自噬相關(guān)因子LC3-Ι向LC3-Π轉(zhuǎn)化有明顯的減少,提示TPL可以抑制結(jié)直腸癌細(xì)胞本底水平自噬。利用m TOR抑制劑雷帕霉素或饑餓處理細(xì)胞誘導(dǎo)自噬,觀察TPL在應(yīng)激條件下抑制自噬的能力。吖啶橙染色后可見,隨著處理濃度的增加,HCT116細(xì)胞系中酸性自噬泡的數(shù)量明顯降低。另外,GFP-LC3斑點實驗顯示,TPL處理以后,由雷帕霉素(Rapamycin,Rapa)或血清饑餓(Serum starvation)誘導(dǎo)的GFP-LC3斑點減少。間接免疫熒光法顯示TPL處理組比對照組的內(nèi)源性LC3斑點數(shù)量顯著減少。Western Blot結(jié)果顯示,TPL可明顯抑制雷帕霉素或饑餓誘導(dǎo)的自噬相關(guān)蛋白LC3-II的表達(dá)。LC3-II和溶酶體的標(biāo)志物L(fēng)AMP1的免疫熒光共定位數(shù)據(jù)顯示,TPL抑制自噬溶酶體的產(chǎn)生;我們利用GFP-RFP-LC3串聯(lián)質(zhì)粒檢測TPL對結(jié)直腸癌細(xì)胞中自噬流的影響,結(jié)果顯示TPL處理以后,LC3紅色斑點顯著減少,表明TPL可以抑制結(jié)直腸癌細(xì)胞自噬流的形成。上述結(jié)果表明,在結(jié)直腸癌細(xì)胞中,TPL抑制自噬的發(fā)生。(3)TPL通過減少自噬相關(guān)基因的轉(zhuǎn)錄來抑制自噬發(fā)生為了解析TPL抑制結(jié)直腸癌細(xì)胞自噬的分子機(jī)制,我們應(yīng)用定量PCR、Western-blot法分別檢測了的自噬起始相關(guān)基因的表達(dá),發(fā)現(xiàn)TPL可以顯著抑制ATG5、ATG7和ATG12的轉(zhuǎn)錄,并具有一定濃度依賴性。結(jié)果表明TPL抑制自噬是通過減少自噬相關(guān)基因轉(zhuǎn)錄水平來實現(xiàn)的。(4)TPL自噬抑制作用可以增強一些化療藥物的抗癌效果傳統(tǒng)結(jié)直腸癌化療藥物5-氟尿嘧啶(5-Fluorouracil,5-FU)可以誘導(dǎo)保護(hù)性自噬,雷公藤甲素可以抑制自噬的發(fā)生,提示5-FU和雷公藤甲素聯(lián)用可能具有更好的抗癌效果。初步證實5-FU與TPL分別及共同處理細(xì)胞后,細(xì)胞增殖實驗結(jié)果顯示人結(jié)直腸癌細(xì)胞增殖被抑制,尤其在5-FU與TPL共同處理組;Western Blot結(jié)果也顯示共同處理后,細(xì)胞凋亡增加。為進(jìn)一步說明,進(jìn)行裸鼠異體移植瘤的種植,初步實驗結(jié)果顯示,5-FU與TPL聯(lián)合用藥化療的移植瘤與空白對照組腫瘤組織、TPL單獨用藥的移植瘤比較,移植瘤的直徑、質(zhì)量顯著減小。以上實驗結(jié)果均可提示雷公藤甲素和5-FU聯(lián)用具有更好的抗癌效果,雷公藤甲素自噬抑制作用可能是聯(lián)用的一個增敏機(jī)制。綜上所述,本課題研究表明,TPL誘導(dǎo)細(xì)胞凋亡抑制人結(jié)腸癌細(xì)胞增殖,并且通過減少自噬相關(guān)基因的轉(zhuǎn)錄來抑制自噬的發(fā)生,TPL可以和一些會誘導(dǎo)保護(hù)性自噬抑制作用的藥物聯(lián)用,從而增強這些藥物的化療效果。本研究對TPL抗結(jié)直腸癌的研究及其機(jī)制研究打下一定基礎(chǔ),旨在對發(fā)現(xiàn)結(jié)直腸癌中新的治療靶點提供方向。
[Abstract]:The incidence and fatality rate of Colorectal cancer (CRC) are high, ranking third and fourth in the tumor spectrum respectively. It is one of the common malignant tumors in the rectum and rectum and sigmoid colon. The methods of prevention and treatment for colorectal cancer are also constantly improved in clinical research. Tripterygium wilfordii (Tri Ptolide, TPL), also known as Tripterygium wilfordii or tripterylide, is derived from the root of Tripterygium wilfordii, a natural product with a variety of biological activities. Modern medicine studies have shown that TPL is not only antioxidation, anti rheumatoid, and anticancer, but the study of the therapeutic effect of TPL in colorectal cancer is rare. For the research hot spots in the mechanism of anticancer treatment, traditional chemotherapeutic drugs can induce or induce protective autophagy in the process of chemotherapy, which reduces the effect of chemotherapeutic drugs on cancer cell proliferation. The purpose of this study was to analyze the role of autophagy in the proliferation effect of TPL against colorectal cancer cells. (1) Tripterygium wilfordii (TPL Induction of apoptosis to inhibit the proliferation of colorectal cancer cells by using a predetermined concentration gradient of TPL to determine the cell growth curve of the colorectal cancer cell lines SW480 and HCT11648h. showed that TPL could significantly inhibit the proliferation of two human colorectal cancer cell lines. Then the Western Blot and Annexin-V/PI double staining flow apoptosis detection method was used to analyze the cell proliferation. The results of apoptosis related protein showed that the apoptosis rate of TPL treated group was significantly increased with the increase of concentration, the apoptosis factor Cleaved-Caspase3 was activated, the expression of Cleaved-PARP1 protein increased, and there was a certain concentration dependence. It showed that TPL could induce apoptosis of colorectal cancer cells and inhibit the proliferation of cells. (2) TPL can be inhibited. TPL treatment group with autophagy and autophagic flow formation in colorectal cancer cells compared with the control group, autophagy related factor LC3- decreased significantly to LC3-, suggesting that TPL could inhibit autophagy at the background level of colorectal cancer cells. Autophagy induced by rapamycin or starvation cells of M TOR inhibitor was used to observe the inhibition of TPL under stress conditions. The ability to make autophagy. After staining with acridine orange, the number of acidic autophagic bubbles in the HCT116 cell line decreased significantly with the increase of treatment concentration. In addition, the GFP-LC3 speckle experiment showed that after TPL treatment, the GFP-LC3 spots induced by rapamycin (Rapamycin, Rapa) or serum starvation (Serum starvation) were reduced. The indirect immunofluorescence method showed TPL. The number of endogenous LC3 spots in the treatment group was significantly reduced by.Western Blot results, and TPL significantly inhibited the immunofluorescence co localization data of the LAMP1 of the expression of.LC3-II and lysosomes of the autophagy associated protein LC3-II, which was induced by rapamycin or starvation. TPL inhibited the production of autophagic lysosomes; we used GFP-RFP-LC3. The effect of TPL on autophagic flow in colorectal cancer cells was detected by tandem plasmids. The results showed that after TPL treatment, the red spots of LC3 decreased significantly, indicating that TPL could inhibit the formation of autophagic flow in colorectal cancer cells. The results showed that TPL inhibited autophagy in colorectal cancer cells. (3) TPL was suppressed by the reduction of autophagy related genes. Autophagy was made to analyze the molecular mechanism of TPL inhibition of autophagy in colorectal cancer cells. We used quantitative PCR and Western-blot to detect the expression of autophagy initiation related genes. It was found that TPL could significantly inhibit the transcription of ATG5, ATG7 and ATG12, with a certain concentration dependent dependence. The results showed that TPL inhibited autophagy by reducing autophagy by reducing autophagy. (4) the inhibition of autophagy (4) inhibition of autophagy can enhance the anticancer effect of some chemotherapeutic drugs, the traditional colon cancer chemotherapy drug 5- fluorouracil (5-Fluorouracil, 5-FU) can induce protective autophagy, and triptolide can inhibit the occurrence of autophagy, suggesting that the combination of 5-FU and triptolide may be better. Anticancer effect. It was preliminarily confirmed that 5-FU and TPL were respectively treated with cells, and the cell proliferation experiment showed that the proliferation of human colorectal cancer cells was inhibited, especially in the group of 5-FU and TPL, and the result of Western Blot showed that the cell apoptosis increased after CO treatment. The results showed that the tumor tissue of 5-FU combined with TPL was compared with the blank control group, and the diameter of the transplanted tumor was significantly lower than that of the TPL alone. The above results could suggest that Triptolide and 5-FU have better anti-cancer effect. The autophagy inhibition of triptolide may be one of the combined use. In summary, this study shows that TPL induced apoptosis inhibits the proliferation of human colon cancer cells and inhibits autophagy by reducing autophagy related genes, and TPL can be combined with some drugs that can induce protective autophagy. This study enhances the efficacy of these drugs. This study is a study on the resistance of these drugs to TPL. The research and mechanism research of colorectal cancer has laid a foundation for the discovery of new therapeutic targets in colorectal cancer.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34

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