TGF-β1诱导肠上皮表型转变相关表观遗传机制及中药单体干预

发布时间：2018-06-04 14:28

本文选题：肿瘤微环境 + 转化生长因子-β　；参考：《广州中医药大学》2017年博士论文

【摘要】：背景和目的大肠癌(CRC)对人类生活健康构成了极大的危险,深入了解肠癌发生发展机制及开发有效药物的形势已刻不容缓。研究发现肿瘤微环境(TME)中的肿瘤细胞与基质细胞通过其分泌到微环境中的转化生长因子-β(TGF-β)发生串扰从而促进基质细胞之间的表型转变,而且研究发现在表型转变过程中伴随有表观遗传学的改变,而组蛋白修饰作为表观遗传学中的重要成分因结构特性因而可通过各种化学基团修饰对周围环境进行即时快速响应,因此组蛋白修饰可能参与了细胞表型转变相关机制。中药单体吴茱萸碱和小檗碱对肿瘤细胞均具有较强的抗肿瘤活性,然而对其具体的抗癌机制尚不明确,因此,本研究旨在通过TGF-β1体外诱导结肠上皮细胞系NCM460构建表型转变模型探讨组蛋白修饰变化机制以及吴茱萸碱和小檗碱的干预效应。方法将含有10ng/mlTGF-β 1培养基培养永生化结肠上皮细胞系NCM460至15天(d)后采用细胞免疫荧光进行细胞表型标志物E-cadherin、ZO-1和vimentin的免疫荧光检测,同时采用免疫印迹(westernblotting)对此三种表型标志物及表型相关转录因子Snail和Slug的蛋白表达进行了测定。为了探索细胞表型转变中染色体不稳定及组蛋白修饰模式的变化,分别采用分选式流式细胞仪(FACS)和western blotting检测TGF-β 1诱导表型转变后的细胞中非整倍体细胞百分比情况和五种组蛋白修饰标记物(H3K4me2、H3K4me3、H3K9me2、H3K9ac、H3K27me3)蛋白表达水平。为了探讨左金丸主要活性成分吴茱萸碱和小檗碱对TGF-β 1的生物学效应的干预作用,将TGF-β 1诱导表型转变后的细胞进行不同浓度吴茱萸碱(5μg/ml、10μg/ml和20μg/ml)和小檗碱(50μg/ml、75μg/ml和100μg/ml)处理后72h后,分别按照上述方法检测表型标志物及Snail、Slug表达,非整倍体细胞百分比变化以及异常组蛋白修饰模式的影响。结果(1)TGF-β 1对NCM460细胞表型转变及中药单体干预的影响10ng/mlTGF-β 1诱导NCM460细胞15d后细胞发生了 EMT样表型转变,细胞呈现出纺锤样的形态学改变,同时上皮细胞标志物E-cadherin和紧密连接复合物ZO-1的表达出现显著下调,而间质性标志物vimentin的表达出现了明显升高,其荧光强度与光密度值与未加入TGF-β 1处理的细胞相比差异具有统计学意义(P0.05)。此外与TGF-β 1诱导表型转变相关的转录因子Snail和Slug也出现了显著上调(P0.05)。而加入不同浓度吴茱萸碱和小檗碱处理72h后,均能不同程度上调E-cadherin和ZO-1和下调vimentin表达,表明吴茱萸碱和小檗碱可对抗TGF-β1引起的细胞表型转变效应。(2)表型转变中TGF-β1对细胞中非整倍体性的影响及中药单体的干预效应NCM460细胞受到10ng/mlTGF-β 1诱导15d发生表型转变后,非整倍体细胞数百分比明显高于阴性对照组(P0.05),整倍体亚群分析显示,转化细胞中亚二倍体.细胞数明显多于超二倍体和多倍体数目(P0.05),而诱导20d和25天后细胞中分别以超二倍体和多倍体细胞为主。但是经高浓度和中浓度的吴茱萸碱和小檗碱处理72h后,非整倍体细胞数(亚二倍体和超二倍体)与对照组相比均出现了显著的减少(P0.05)。(3)TGF-β1诱导表型转变中组蛋白修饰变化及中药单体干预的影响与对照组细胞相比,TGF-β 1诱导表型转变细胞中H3K4me2、H3K4me3、H3K9me2、H3K9ac和H3K27me3均出现了显著互不相同的变化,其中H3K4me2、H3K9me2和H3K9ac的表达显著上调(P0.05),而H3K4me3和H3K27me3的表达出现了明显下调反应(P0.05)。然而H3K4me3和H3K27me3的表达经吴茱萸碱和小檗碱处理出现了不同程度的上调,而H3K9me2和H3K9ac的表达呈现出不同程度下调,但是H3K4me2的表达经10μg/ml吴茱萸碱和100μg/ml以及50μg/ml小檗碱处理后下调。结论(1)结肠上皮细胞在体外受TGF-β 1诱导表型转变后出现了非整倍体性的染色体不稳定以及异常组蛋白修饰,表明TGF-β1可能通过与组蛋白修饰相关机制从而导致基因组不稳定性而发挥出促表型转变效应。(2)一定浓度吴茱萸碱和小檗碱可能通过多机制包括稳定染色体数目或调控异常组蛋白修饰模式而发挥出一定的抗肿瘤活性作用。
[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.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.3

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