【摘要】：本論文對(duì)氯代亞甲基硫色滿酮抗腫瘤機(jī)制進(jìn)行了初步研究。以人肝癌HepG-2細(xì)胞，人宮頸癌Hela細(xì)胞為模型，采用臺(tái)盼藍(lán)染色法檢測(cè)CMCT、CMMT對(duì)細(xì)胞增殖的影響；Hoechst33258染色，流式細(xì)胞術(shù)(FCM)檢測(cè)CMCT、CMMT對(duì)細(xì)胞凋亡的影響；ELISA法檢測(cè)CMMT對(duì)凋亡相關(guān)蛋白表達(dá)的影響；RT-PCR方法檢測(cè)凋亡相關(guān)基因轉(zhuǎn)錄水平的變化；ELISA法檢測(cè)CMMT對(duì)小鼠巨噬細(xì)胞分泌因子激活作用。實(shí)驗(yàn)結(jié)果表明CMCT、CMMT能夠明顯抑制HepG-2和Hela細(xì)胞的增殖，表現(xiàn)為低濃度抑制，高濃度致死；Hoechst33258熒光染色和流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示CMCT、CMMT可誘導(dǎo)腫瘤細(xì)胞凋亡；CMMT作用24h后，caspase-3，-8和-9的蛋白含量增高，并呈濃度依賴性；ELISA法檢測(cè)顯示CMMT可使HepG-2細(xì)胞中死亡受體TNFR1、Hela細(xì)胞中DR3表達(dá)量顯著增高；CMMT可以上調(diào)促調(diào)往基因Bax、p53的表達(dá)，下調(diào)抑凋亡基因Bcl-2的表達(dá)。 基于硫色滿酮類(lèi)化合物可能具有免疫促進(jìn)的猜測(cè)，我們對(duì)CMMT可否激活巨噬細(xì)胞進(jìn)行了實(shí)驗(yàn)。實(shí)驗(yàn)結(jié)果表明，CMMT可激活小鼠的原代腹腔巨噬細(xì)胞，提高TNF-α、IL-6、IL-12的分泌量，具有類(lèi)似LPS的作用，提示其有可能是TLRs的激動(dòng)劑。 以上結(jié)果表明CMMT的抗癌機(jī)制可能是通過(guò)死亡受體和線粒體兩個(gè)途徑誘導(dǎo)腫瘤細(xì)胞凋亡；并能通過(guò)免疫激活作用激活體內(nèi)先天免疫系統(tǒng)，，間接發(fā)揮抗腫瘤作用。
[Abstract]:In this paper, the anti-tumor mechanism of chloromethylene thiolone was studied. The effect of HepG-2 on cell proliferation was detected by trypan blue staining, and the apoptosis was detected by flow cytometry (FCM) (FCM). Effects of CMMT on the expression of apoptosis-related proteins by ELISA; RT-PCR method was used to detect the transcription level of apoptosis-related genes. Elisa was used to detect the activation of macrophage secreting factors by CMMT. The results showed that the proliferation of HepG-2 and Hela cells was significantly inhibited by CMCTT. The results of high concentration lethal Hoechst33258 fluorescence staining and flow cytometry showed that CMCTMT-CMMT could induce apoptosis of tumor cells. The protein levels of caspase-3, caspase-8 and -9 were increased 24 hours after treatment with CMMT. The results of concentration dependent Elisa showed that CMMT could significantly increase the expression of DR3 in HepG-2 cell line TNFR1 and Hela cells. CMMT could up-regulate the expression of Bax-p53 gene. The expression of Bcl-2 was down-regulated. Based on the hypothesis that thiolemone compounds may be immune-promoting, we have studied whether CMMT can activate macrophages. The results showed that CMMT could activate the primary peritoneal macrophages of mice and increase the secretion of TNF- 偽 and IL-6 IL-12, which was similar to that of LPS, suggesting that it might be an agonist of TLRs. These results suggest that the anticancer mechanism of CMMT may be induced apoptosis of tumor cells by death receptor and mitochondria, and can activate innate immune system through immune activation, and indirectly play an anti-tumor role.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R965

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