本文選題：腫瘤微環(huán)境 + 轉(zhuǎn)化生長(zhǎng)因子-β　； 參考：《廣州中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：背景和目的大腸癌(CRC)對(duì)人類(lèi)生活健康構(gòu)成了極大的危險(xiǎn),深入了解腸癌發(fā)生發(fā)展機(jī)制及開(kāi)發(fā)有效藥物的形勢(shì)已刻不容緩。研究發(fā)現(xiàn)腫瘤微環(huán)境(TME)中的腫瘤細(xì)胞與基質(zhì)細(xì)胞通過(guò)其分泌到微環(huán)境中的轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)發(fā)生串?dāng)_從而促進(jìn)基質(zhì)細(xì)胞之間的表型轉(zhuǎn)變,而且研究發(fā)現(xiàn)在表型轉(zhuǎn)變過(guò)程中伴隨有表觀遺傳學(xué)的改變,而組蛋白修飾作為表觀遺傳學(xué)中的重要成分因結(jié)構(gòu)特性因而可通過(guò)各種化學(xué)基團(tuán)修飾對(duì)周?chē)h(huán)境進(jìn)行即時(shí)快速響應(yīng),因此組蛋白修飾可能參與了細(xì)胞表型轉(zhuǎn)變相關(guān)機(jī)制。中藥單體吳茱萸堿和小檗堿對(duì)腫瘤細(xì)胞均具有較強(qiáng)的抗腫瘤活性,然而對(duì)其具體的抗癌機(jī)制尚不明確,因此,本研究旨在通過(guò)TGF-β1體外誘導(dǎo)結(jié)腸上皮細(xì)胞系NCM460構(gòu)建表型轉(zhuǎn)變模型探討組蛋白修飾變化機(jī)制以及吳茱萸堿和小檗堿的干預(yù)效應(yīng)。方法將含有10ng/mlTGF-β 1培養(yǎng)基培養(yǎng)永生化結(jié)腸上皮細(xì)胞系NCM460至15天(d)后采用細(xì)胞免疫熒光進(jìn)行細(xì)胞表型標(biāo)志物E-cadherin、ZO-1和vimentin的免疫熒光檢測(cè),同時(shí)采用免疫印跡(westernblotting)對(duì)此三種表型標(biāo)志物及表型相關(guān)轉(zhuǎn)錄因子Snail和Slug的蛋白表達(dá)進(jìn)行了測(cè)定。為了探索細(xì)胞表型轉(zhuǎn)變中染色體不穩(wěn)定及組蛋白修飾模式的變化,分別采用分選式流式細(xì)胞儀(FACS)和western blotting檢測(cè)TGF-β 1誘導(dǎo)表型轉(zhuǎn)變后的細(xì)胞中非整倍體細(xì)胞百分比情況和五種組蛋白修飾標(biāo)記物(H3K4me2、H3K4me3、H3K9me2、H3K9ac、H3K27me3)蛋白表達(dá)水平。為了探討左金丸主要活性成分吳茱萸堿和小檗堿對(duì)TGF-β 1的生物學(xué)效應(yīng)的干預(yù)作用,將TGF-β 1誘導(dǎo)表型轉(zhuǎn)變后的細(xì)胞進(jìn)行不同濃度吳茱萸堿(5μg/ml、10μg/ml和20μg/ml)和小檗堿(50μg/ml、75μg/ml和100μg/ml)處理后72h后,分別按照上述方法檢測(cè)表型標(biāo)志物及Snail、Slug表達(dá),非整倍體細(xì)胞百分比變化以及異常組蛋白修飾模式的影響。結(jié)果(1)TGF-β 1對(duì)NCM460細(xì)胞表型轉(zhuǎn)變及中藥單體干預(yù)的影響10ng/mlTGF-β 1誘導(dǎo)NCM460細(xì)胞15d后細(xì)胞發(fā)生了 EMT樣表型轉(zhuǎn)變,細(xì)胞呈現(xiàn)出紡錘樣的形態(tài)學(xué)改變,同時(shí)上皮細(xì)胞標(biāo)志物E-cadherin和緊密連接復(fù)合物ZO-1的表達(dá)出現(xiàn)顯著下調(diào),而間質(zhì)性標(biāo)志物vimentin的表達(dá)出現(xiàn)了明顯升高,其熒光強(qiáng)度與光密度值與未加入TGF-β 1處理的細(xì)胞相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。此外與TGF-β 1誘導(dǎo)表型轉(zhuǎn)變相關(guān)的轉(zhuǎn)錄因子Snail和Slug也出現(xiàn)了顯著上調(diào)(P0.05)。而加入不同濃度吳茱萸堿和小檗堿處理72h后,均能不同程度上調(diào)E-cadherin和ZO-1和下調(diào)vimentin表達(dá),表明吳茱萸堿和小檗堿可對(duì)抗TGF-β1引起的細(xì)胞表型轉(zhuǎn)變效應(yīng)。(2)表型轉(zhuǎn)變中TGF-β1對(duì)細(xì)胞中非整倍體性的影響及中藥單體的干預(yù)效應(yīng)NCM460細(xì)胞受到10ng/mlTGF-β 1誘導(dǎo)15d發(fā)生表型轉(zhuǎn)變后,非整倍體細(xì)胞數(shù)百分比明顯高于陰性對(duì)照組(P0.05),整倍體亞群分析顯示,轉(zhuǎn)化細(xì)胞中亞二倍體.細(xì)胞數(shù)明顯多于超二倍體和多倍體數(shù)目(P0.05),而誘導(dǎo)20d和25天后細(xì)胞中分別以超二倍體和多倍體細(xì)胞為主。但是經(jīng)高濃度和中濃度的吳茱萸堿和小檗堿處理72h后,非整倍體細(xì)胞數(shù)(亞二倍體和超二倍體)與對(duì)照組相比均出現(xiàn)了顯著的減少(P0.05)。(3)TGF-β1誘導(dǎo)表型轉(zhuǎn)變中組蛋白修飾變化及中藥單體干預(yù)的影響與對(duì)照組細(xì)胞相比,TGF-β 1誘導(dǎo)表型轉(zhuǎn)變細(xì)胞中H3K4me2、H3K4me3、H3K9me2、H3K9ac和H3K27me3均出現(xiàn)了顯著互不相同的變化,其中H3K4me2、H3K9me2和H3K9ac的表達(dá)顯著上調(diào)(P0.05),而H3K4me3和H3K27me3的表達(dá)出現(xiàn)了明顯下調(diào)反應(yīng)(P0.05)。然而H3K4me3和H3K27me3的表達(dá)經(jīng)吳茱萸堿和小檗堿處理出現(xiàn)了不同程度的上調(diào),而H3K9me2和H3K9ac的表達(dá)呈現(xiàn)出不同程度下調(diào),但是H3K4me2的表達(dá)經(jīng)10μg/ml吳茱萸堿和100μg/ml以及50μg/ml小檗堿處理后下調(diào)。結(jié)論(1)結(jié)腸上皮細(xì)胞在體外受TGF-β 1誘導(dǎo)表型轉(zhuǎn)變后出現(xiàn)了非整倍體性的染色體不穩(wěn)定以及異常組蛋白修飾,表明TGF-β1可能通過(guò)與組蛋白修飾相關(guān)機(jī)制從而導(dǎo)致基因組不穩(wěn)定性而發(fā)揮出促表型轉(zhuǎn)變效應(yīng)。(2)一定濃度吳茱萸堿和小檗堿可能通過(guò)多機(jī)制包括穩(wěn)定染色體數(shù)目或調(diào)控異常組蛋白修飾模式而發(fā)揮出一定的抗腫瘤活性作用。
[Abstract]:Background and objective colorectal cancer (CRC) poses a great danger to human life and health. It is urgent to understand the development mechanism of colon cancer and the development of effective drugs. It is found that tumor cells and stromal cells in the tumor microenvironment (TME) have crosstalk from the transforming growth factor beta (TGF- beta) secreted into the microenvironment. It promotes phenotypic transformation between stromal cells, and studies have found that there are epigenetic changes in the process of phenotypic transformation, and histone modification is an important component in epigenetics as a result of structural properties so that the surrounding environment can be quickly responsive to the surrounding environment by various chemical groups, so histone modification may be modified. It is involved in the mechanism of cell phenotypic transformation. The mono evodiine and berberine have strong anti-tumor activity to tumor cells. However, the specific anticancer mechanism is not clear. Therefore, the aim of this study is to explore the change machine of histone modification by the construction of the phenotype transformation model of the colonic epithelial cell line NCM460 by TGF- beta 1 in vitro. The intervention effect of evodiine and berberine. Methods the 10ng/mlTGF- beta 1 medium was cultured for the immortalized colonic epithelial cell line from NCM460 to 15 days (d). Cell immunofluorescence was used to detect the cell phenotypic markers E-cadherin, ZO-1 and vimentin, and the immunoblotting (westernblotting) was used at the same time for three forms. The protein expression of type markers and phenotypic related transcription factors Snail and Slug was measured. In order to explore the chromosomal instability and the changes in the histone modification patterns during cell phenotypic transition, the percentage of aneuploidy cells in the cells induced by TGF- beta 1 was detected by selective flow cytometry (FACS) and Western blotting, respectively. The protein expression level of the five histone modifier markers (H3K4me2, H3K4me3, H3K9me2, H3K9ac, H3K27me3). In order to explore the biological effects of evodiine and berberine on the biological effects of TGF- beta 1, the main active components of Zuojin Pill were investigated. The cells after the induction of the phenotypic transformation of TGF- beta 1 were different concentrations of evodipine (5 mu, 10 micron g/ml). After 20 mu g/ml) and berberine (50 g/ml, 75 mu g/ml and 100 g/ml) after 72h, the phenotypic markers and the expression of Snail, Slug, aneuploidy and abnormal histone modification were detected respectively. Results (1) the effect of TGF- beta 1 on the phenotype transformation of NCM460 cells and the effect of 10ng/mlTGF- beta on the intervention of traditional Chinese medicine. 1 induced EMT like phenotype change after 15d induced NCM460 cells, the cells showed a spindle like morphological change, while the expression of the epithelial marker E-cadherin and the tightly connected complex ZO-1 decreased significantly, while the expression of the interstitial marker vimentin increased obviously. The fluorescence intensity and light density value were not added to the cells. The cells treated by TGF- beta 1 had statistical significance (P0.05). In addition, the transcription factors related to the TGF- beta 1 induced phenotypic transformation, Snail and Slug were also significantly up-regulated (P0.05). After adding evodiine and berberine to 72h, the expression of E-cadherin and ZO-1 and down regulated vimentin were up to a different extent, indicating Wu Zhu Oodipine and berberine can antagonize the cell phenotypic transformation effect caused by TGF- beta 1. (2) the effect of TGF- beta 1 on the aneuploidy of cells during the phenotypic transformation and the intervention effect of Chinese medicine monomers, NCM460 cells with 10ng/mlTGF- beta 1 induced 15d phenotype transformation, the ratio of aneuploidy cells is significantly higher than that of the negative control group (P0.05), aneuploidy Subgroup analysis showed that the number of cells in the transformed cells was more than the hyper diploid and polyploid number (P0.05), and the cells were induced by hyper diploid and polyploid cells in 20d and 25 days, but the number of aneuploid cells (diploid and super two) after the treatment of 72h with evodipine and berberine at high concentration and concentration. Compared with the control group, there was a significant decrease (P0.05). (3) the changes of histone modification in the TGF- beta 1 induced phenotypic transformation and the effect of the intervention of traditional Chinese medicine were compared with those of the control group. The changes of H3K4me2, H3K4me3, H3K9me2, H3K9ac and H3K27me3 in the TGF- beta 1 induced phenotypic transition cells were significantly different, H3K4me2, H3, and H3. The expression of K9me2 and H3K9ac was significantly up-regulated (P0.05), while the expression of H3K4me3 and H3K27me3 was obviously down regulated (P0.05). However, the expression of H3K4me3 and H3K27me3 was up up to varying degrees by evodiine and berberine, and the expression of H3K9me2 and H3K9ac decreased in varying degrees, but the H3K4me2 expression was 10 mu g/ml Wu. After the treatment of cornel and 100 g/ml and 50 g/ml berberine. Conclusion (1) the aneuploidy chromosomal instability and abnormal histone modification of the colonic epithelial cells after the induced phenotypic transformation of TGF- beta 1 in vitro show that TGF- beta 1 may play a role in genomic instability by the mechanism associated with histone modification. (2) a certain concentration of evodiine and berberine may play a role in antitumor activity through multiple mechanisms including the number of stable chromosomes or the regulation of abnormal histone modification.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 Jinlu Dai;Yi Lu;Hernan Roca;Jill M.Keller;Jian Zhang;Laurie K.Mc Cauley;Evan T.Keller;;Immune mediators in the tumor microenvironment of prostate cancer[J];Chinese Journal of Cancer;2017年03期

2 焦延娜;韓淑燕;;抗癌中藥單體對(duì)腫瘤細(xì)胞自噬的調(diào)控[J];中國(guó)實(shí)驗(yàn)方劑學(xué)雜志;2017年07期

3 劉擁軍;沈衛(wèi)星;程海波;尤建良;;尤建良運(yùn)用中醫(yī)藥治療惡性腫瘤四大特點(diǎn)[J];遼寧中醫(yī)雜志;2016年11期

4 袁龍;陳益;劉澤洪;王芬;李科瓊;李靜;陳地龍;;吳茱萸堿抑制HDAC6促進(jìn)人白血病K562細(xì)胞周期阻滯和凋亡的機(jī)制研究[J];中草藥;2016年17期

5 張曉亮;邵磊;;扶正祛毒方聯(lián)合鹽酸小檗堿對(duì)乳腺癌細(xì)胞生長(zhǎng)抑制作用的臨床觀察[J];中華中醫(yī)藥學(xué)刊;2016年09期

6 錢(qián)東;丁小鳳;程菁菁;袁智勇;;靶向端粒 端粒酶的抗腫瘤治療研究進(jìn)展[J];中國(guó)腫瘤臨床;2016年15期

7 Norio Kubo;Kenichiro Araki;Hiroyuki Kuwano;Ken Shirabe;;Cancer-associated fibroblasts in hepatocellular carcinoma[J];World Journal of Gastroenterology;2016年30期

8 葉青;王瑞平;鄒璽;;中藥逆轉(zhuǎn)胃癌多藥耐藥作用的研究進(jìn)展[J];中華中醫(yī)藥雜志;2016年08期

9 姚璐;張秀麗;趙越;張萍;王雪梅;池志宏;;小檗堿對(duì)TGF-β1誘導(dǎo)的腎小管上皮細(xì)胞轉(zhuǎn)分化及TGF-β/Smad通路的影響[J];解剖科學(xué)進(jìn)展;2016年04期

10 曾智豪;陳小伍;朱達(dá)堅(jiān);羅仲燃;楊敏;;鹽酸小檗堿對(duì)MCF-7乳腺癌小鼠體內(nèi)抑制腫瘤生長(zhǎng)的實(shí)驗(yàn)研究[J];河北醫(yī)學(xué);2016年06期

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