CYP24A1参与炎症因子调控Wnt信号通路的机制研究

发布时间：2018-05-29 10:40

本文选题：CYP24A1 + IL-6　；参考：《北京协和医学院》2017年博士论文

【摘要】：第一部分:CYP24A1参与炎症因子调控Wnt信号通路的机制研究目的:CYP24A1编码1,25-双羟维生素D3特异性代谢酶,对1,25-双羟维生素D3局部组织浓度有决定性影响,后者能够下调Wnt/β-catenin通路活化程度。CYP24A1在结肠息肉与结肠癌中有异常高表达,其SNP分布也与溃疡性结肠炎发病相关。细胞因子IL-6和TNF-α的活化和NF-κB通路的激活在炎癌转化过程中起到关键作用,本课题组前期研究发现IL-6和TNF-α可通过NF-κB通路调控Wnt/β-catenin通路活化。有研究报道,IL-6和TNF-α能够诱导CYP24A1转录增多,但对蛋白含量影响尚不明确,且CYP24A1基因的表达调控是否参与IL-6或TNF-α通过NF-κB通路调控Wnt/β-catenin通路活化尚无相关研究。本研究旨在探讨CYP24A1在IL-6和TNF-α通过NF-κB通路调控Wnt通路过程中的作用,进一步明确炎症与肿瘤发生之间的关系与具体机制,为特异性抑制CYP24A1酶活性、增加结肠组织对1,25-双羟维生素D3抑癌作用敏感性在预防UC癌变中的应用提供了研究基础。方法:本研究将细胞因子IL-6和TNF-α作用于结肠癌细胞株HCT-116、Caco-2,使用Western Blot检测对CYP24A1蛋白表达的影响,使用凝胶迁移实验(EMSA)检测对NF-κB通路活化程度的影响,使用Western Blot检测NF-κB通路特异性抑制剂PDTC对IL-6和TNF-α诱导CYP24A1表达的影响;利用siRNA敲减CYP24A1表达后,双荧光素酶报告基因实验检测IL-6和TNF-α对Wnt信号通路调控作用的变化,使用Western Blot检测细胞核β-catenin蛋白水平。结果:IL-6与TNF-α作用于Caco-2和HCT-116细胞可使CYP24A1表达量增加,且表现为时间、剂量依赖。在Caco-2细胞系选取IL-6 100ng/ml、TNF-α50ng/ml 分别作用 6h,HCT-116 细胞系选取 IL-6 50ng/ml、TNF-α100ng/ml 分别作用6h,NF-κB通路活化程度增加,使用NF-κB通路特异性抑制剂PDTC可明显抑制IL-6与TNF-α对CYP24A1表达的诱导作用。利用siRNA敲减CYP24A1表达可部分拮抗IL-6和TNF-α诱导的β-catenin核内分布增多以及Wnt通路活化(相对荧光素酶活性Caco-2细胞系IL-6 3.297±0.1610 vs 2.527±0.03323,p=0.0221;TNF-α 4.130±0.4289 vs 2.780±0.3092,p=0.0249;HCT116 细胞系 IL-6 1.357±0.03422 vs1.086±0.07545,p=0.0048;TNF-α2.374±0.1955 vs 1.802±0.06163,p=0.0313)。结论:在Caco-2和HCT-116细胞系中,IL-6和TNF-α可能通过诱导NF-κB通路活化进一步促进CYP24A1表达量增加,促进核内β-catenin增多,上调Wnt通路活化程度。第二部分:溃疡性结肠炎癌变模型小鼠中CYP24A1基因表达与炎症因子的关系目的:本实验通过建立AOM联合DSS小鼠UC癌变模型,给予阻断炎症因子TNF-α、NF-κB处理,探究炎症相关途径在UC癌变中对CYP24A1表达的影响。方法:将C57BL/6小鼠随机分为4组,第1组为空白对照组,第2-4组建立UC癌变小鼠模型(腹腔注射12.5mg/kg AOM,1周后给予2.5%DSS的饮用水,连续5天,之后换成普通饮用水),其中,第2组为模型对照组,第3组给予NF-κB反义寡聚核苷酸每两周一次灌肠,第4组给予TNF-α单克隆抗体每两周一次腹腔注射。12周后处死小鼠,统计瘤负荷,进行病理学评估,使用免疫组化方法检测CYP24A1表达情况。结果:CYP24A1表达在模型对照组(n=8,2.500±0.3273)中较空白组(n=6,1.167±0.1667)升高(p=0.0067)。NF-κB反义寡聚核苷酸组的瘤负荷(n=5,1.130±0.1678cm)较模型对照组(n=8,2.294±0.3638 cm)显著下降,差异有统计学意义(p= 0.0354);NF-κB反义寡聚核苷酸组的小鼠结肠上皮细胞中CYP24A1表达水平显著下降(1.400 ±0.2449 vs 2.500 ± 0.3273,p=0.0362)。TNF-α单克隆抗体组的瘤负荷(n=5,1.780±0.5142)较模型对照组(n=8,2.294±0.3638)有下降趋势,但差异无统计学意义(p= 0.4195);TNF-α单克隆抗体组CYP24A1表达水平显著下降(1.167 ±0.1667 vs 2.500 ±0.3273,p=0.0144)。结论:AOM/DSS诱导的UC癌变的小鼠模型中CYP24A1表达升高,抑制TNF-α、NF-κB可部分逆转该作用。
[Abstract]:Part one: CYP24A1 participates in the mechanism of Wnt signaling pathways involved in inflammatory factors: CYP24A1 encoding 1,25- dihydroxyvitamin D3 specific metabolic enzyme, which has a decisive influence on the local tissue concentration of 1,25- dihydroxyvitamin D3, and the latter can downregulate the activation degree of Wnt/ beta -catenin pathway in colon polyps and colon cancer. The SNP distribution is also associated with the pathogenesis of ulcerative colitis. The activation of cytokine IL-6 and TNF- alpha and the activation of NF- kappa B pathway play a key role in the process of the transformation of inflammatory cancer. In our previous study, we found that IL-6 and TNF- alpha could regulate the activation of Wnt/ beta -catenin pathway through NF- kappa B pathway. 4A1 transcription is increasing, but the effect on protein content is not clear, and there is no related study on whether the expression of CYP24A1 gene is involved in the regulation of the activation of Wnt/ beta -catenin through the NF- kappa B pathway. The purpose of this study is to explore the role of CYP24A1 in the regulation of IL-6 and TNF- alpha through the NF- kappa pathway. The relationship and specific mechanism between the oncogenesis and the specific mechanism of inhibiting the activity of CYP24A1 enzyme and increasing the sensitivity of colon tissue to the anti cancer effect of 1,25- dihydroxyvitamin D3 in the prevention of UC carcinogenesis. Methods: This study applied the cytokine IL-6 and TNF- alpha to the colon cancer cell line HCT-116, Caco-2, and Western Blot. The effect of detection on the expression of CYP24A1 protein was detected by using gel migration test (EMSA) to detect the effect of NF- kappa B pathway activation. The effect of PDTC on IL-6 and TNF- alpha induced CYP24A1 expression by NF- kappa B pathway inhibitor PDTC was detected by Western Blot. The changes in the regulation of Wnt signaling pathway and the use of Western Blot to detect the level of nuclear beta -catenin protein. Results: the effect of IL-6 and TNF- alpha on Caco-2 and HCT-116 cells can increase the amount of CYP24A1 expression, which is time and dose dependent. IL-6 50ng/ml, TNF- alpha 100ng/ml acts on 6h, NF- kappa B pathway activation, and NF- kappa B pathway specific inhibitor PDTC can significantly inhibit the induction of IL-6 and TNF- alpha on the expression. The luciferase activity Caco-2 cell line IL-6 3.297 + 0.1610 vs 2.527 + 0.03323, p=0.0221; TNF- alpha 4.130 + 0.4289 vs 2.780 + 0.3092, p=0.0249; HCT116 cell line IL-6 1.357 + 0.03422 vs1.086 + 0.07545, p=0.0048; TNF- alpha 2.374 The activation of NF- kappa B pathway can further promote the increase of CYP24A1 expression, promote the increase of beta -catenin in the nucleus and increase the activation degree of Wnt pathway. The second part: the relationship between the expression of CYP24A1 gene expression in the mice with ulcerative colitis canceration model and the relationship between the expression of the inflammatory factors in mice: this experiment was done by establishing the UC canceration model of AOM combined with DSS mice to block the inflammation. Factor TNF- alpha and NF- kappa B were treated to explore the effect of inflammation related pathways on the expression of CYP24A1 in UC carcinogenesis. Methods: C57BL/6 mice were randomly divided into 4 groups, the first groups were blank control group, and the 2-4 group established the UC cancerous mice model (peritoneal injection of 12.5mg/kg AOM, 1 weeks after the drinking water for 2.5%DSS, after 5 days, then converted into ordinary drinking water), among them, The second groups were model control group, and the third groups were given NF- kappa B antisense oligodeoxynucleotides once every two weeks. The fourth groups were given TNF- alpha monoclonal antibody to death mice every two weeks after intraperitoneal injection for.12 weeks. The tumor load, pathological evaluation, and immunohistochemical method were used to detect the CYP24A1 expression. Results: CYP24A1 expression in the model control group (n In =8,2.500 + 0.3273), the tumor load (n=5,1.130 + 0.1678cm) of.NF- kappa B antisense oligonucleotide group was significantly higher than that of the blank group (n=6,1.167 + 0.1667). The difference was significantly lower than that of the model control group (n=8,2.294 + 0.3638 cm). The difference was statistically significant (p= 0.0354); the expression level of the colon epithelial cells in the mice of NF- kappa B antisense oligodeoligonucleotide group The tumor load (n=5,1.780 + 0.5142) of.TNF- a monoclonal antibody group (1.400 + 0.2449 vs 2.500 + 0.3273, p=0.0362) decreased significantly compared with model control group (n=8,2.294 0.3638), but the difference was not statistically significant (p= 0.4195), and the level of CYP24A1 expression in TNF- a monoclonal antibody group decreased significantly (1.167 + 0.1667 vs 2.500 + 0.3273, p=0.01). 44) conclusion: the expression of CYP24A1 in AOM/DSS induced UC canceration mice is increased, and the inhibition of TNF- alpha and NF- kappa B partially reverses this effect.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R363

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