自噬在雷公藤甲素抗结直肠癌细胞增殖效应中作用的研究

发布时间：2018-05-27 23:22

本文选题：结直肠癌 + 雷公藤甲素　；参考：《重庆医科大学》2017年硕士论文

【摘要】：结直肠癌(Colorectal cancer,CRC)的发病率和致死率很高,分别位居肿瘤谱的第三位和第四位。好发于直肠及直肠与乙状结肠交界处,是常见的消化道恶性肿瘤之一。临床研究领域对结直肠癌预防与治疗的方法也在不断的改进。雷公藤甲素(Triptolide,TPL)又称为雷公藤内酯或是雷公藤内酯醇,来源于中药雷公藤的根,是有着多种生物活性的天然产物。现代医药研究表明,TPL不仅抗氧化,抗类风湿,还有抗癌的作用。但TPL在结直肠癌中的治疗作用的研究还很少见。细胞自噬和凋亡均为抗癌治疗机制中的研究热点,传统的化疗药物在化疗的过程中会引起或诱导细胞保护性自噬,从而降低了化疗药物抑制癌细胞增殖的作用。本研究旨在细胞自噬在TPL抗结直肠癌细胞增殖效应中的作用进行研究分析。(1)雷公藤甲素(TPL)诱导调亡作用抑制结直肠癌细胞增殖通过用预定浓度梯度的TPL分别处理结直肠癌细胞系SW480和HCT11648h。测定细胞生长曲线表明,TPL能够显著抑制两种人结直肠癌细胞系的增殖。接着用Western Blot和Annexin-V/PI双染法流式凋亡检测法分析凋亡相关蛋白。数据结果显示,与对照组相比较,TPL处理组细胞凋亡率随浓度增加明显升高;促凋亡因子Cleaved-Caspase3被激活,Cleaved-PARP1蛋白表达量增加,有一定的浓度依赖性。表明TPL可以诱导结直肠癌细胞凋亡,抑制其细胞增殖。(2)TPL可抑制结直肠癌细胞自噬发生和自噬流的形成TPL处理组与对照组比较,自噬相关因子LC3-Ι向LC3-Π转化有明显的减少,提示TPL可以抑制结直肠癌细胞本底水平自噬。利用m TOR抑制剂雷帕霉素或饥饿处理细胞诱导自噬,观察TPL在应激条件下抑制自噬的能力。吖啶橙染色后可见,随着处理浓度的增加,HCT116细胞系中酸性自噬泡的数量明显降低。另外,GFP-LC3斑点实验显示,TPL处理以后,由雷帕霉素(Rapamycin,Rapa)或血清饥饿(Serum starvation)诱导的GFP-LC3斑点减少。间接免疫荧光法显示TPL处理组比对照组的内源性LC3斑点数量显著减少。Western Blot结果显示,TPL可明显抑制雷帕霉素或饥饿诱导的自噬相关蛋白LC3-II的表达。LC3-II和溶酶体的标志物LAMP1的免疫荧光共定位数据显示,TPL抑制自噬溶酶体的产生;我们利用GFP-RFP-LC3串联质粒检测TPL对结直肠癌细胞中自噬流的影响,结果显示TPL处理以后,LC3红色斑点显著减少,表明TPL可以抑制结直肠癌细胞自噬流的形成。上述结果表明,在结直肠癌细胞中,TPL抑制自噬的发生。(3)TPL通过减少自噬相关基因的转录来抑制自噬发生为了解析TPL抑制结直肠癌细胞自噬的分子机制,我们应用定量PCR、Western-blot法分别检测了的自噬起始相关基因的表达,发现TPL可以显著抑制ATG5、ATG7和ATG12的转录,并具有一定浓度依赖性。结果表明TPL抑制自噬是通过减少自噬相关基因转录水平来实现的。(4)TPL自噬抑制作用可以增强一些化疗药物的抗癌效果传统结直肠癌化疗药物5-氟尿嘧啶(5-Fluorouracil,5-FU)可以诱导保护性自噬,雷公藤甲素可以抑制自噬的发生,提示5-FU和雷公藤甲素联用可能具有更好的抗癌效果。初步证实5-FU与TPL分别及共同处理细胞后,细胞增殖实验结果显示人结直肠癌细胞增殖被抑制,尤其在5-FU与TPL共同处理组;Western Blot结果也显示共同处理后,细胞凋亡增加。为进一步说明,进行裸鼠异体移植瘤的种植,初步实验结果显示,5-FU与TPL联合用药化疗的移植瘤与空白对照组肿瘤组织、TPL单独用药的移植瘤比较,移植瘤的直径、质量显著减小。以上实验结果均可提示雷公藤甲素和5-FU联用具有更好的抗癌效果,雷公藤甲素自噬抑制作用可能是联用的一个增敏机制。综上所述,本课题研究表明,TPL诱导细胞凋亡抑制人结肠癌细胞增殖,并且通过减少自噬相关基因的转录来抑制自噬的发生,TPL可以和一些会诱导保护性自噬抑制作用的药物联用,从而增强这些药物的化疗效果。本研究对TPL抗结直肠癌的研究及其机制研究打下一定基础,旨在对发现结直肠癌中新的治疗靶点提供方向。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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