DNMT1诱导SOCS1高甲基化在巨噬细胞活化中的作用及其部分机制研究

发布时间：2019-01-19 21:41

【摘要】：在关节炎、肝纤维化等炎症疾病的发生发展过程中，巨噬细胞活化分泌炎症因子是其发病的主要特征，而抑制巨噬细胞的活化是抗炎免疫治疗的关键环节。作为细胞因子转导的抑制分子—SOCS1，在组织损伤和炎症性疾病的抑制中起着重要作用。研究发现，在各种类型的肿瘤中，SOCS1的失活都与启动子区域高甲基化有关。为了更进一步研究DNA甲基化在巨噬细胞释放炎症因子中的作用机制，本实验通过离体观察SOCS1基因在小鼠腹腔巨噬细胞中的变化，及其对巨噬细胞分泌炎症因子的作用，研究其部分作用机制。本实验研究内容主要含有以下四个部分： 1.SOCS1在LPS诱导RAW264.7小鼠腹腔巨噬细胞中的表达及甲基化的状态。选用RAW264.7(小鼠巨噬细胞株)为研究对象，利用LPS1μg/ml诱导24h诱导其活化，RT-PCR方法检测SOCS1，TNF-α，，IL-6的mRNA表达变化；免疫印迹观察SOCS1蛋白的表达变化；Elisa方法检测细胞培养上清中炎症因子TNF-α，IL-6的表达变化。实验结果发现，巨噬细胞分泌炎症因子的过程中SOCS1的表达降低。利用甲基化特异性PCR（MSP-PCR）检测LPS诱导的RAW264.7小鼠巨噬细胞活化过程中SOCS1甲基化的状态，我们发现SOCS1基因发生了高甲基化。 2. SOCS1在DNA甲基化抑制剂作用下发生了去甲基化及自身表达的恢复，进而抑制了RAW264.7巨噬细胞炎症因子的分泌。以RAW264.7为研究对象，利用LPS1μg/ml诱导活化24h和5-azadC2μmol/l温育48h后，RT-PCR方法检测SOCS1，TNF-α，IL-6的mRNA表达变化；免疫印迹观察SOCS1蛋白的表达；Elisa方法检测细胞培养上清中炎症因子TNF-α，IL-6的表达变化。利用甲基化特异性PCR（MSP-PCR）检测LPS和5-azadC作用的RAW264.7小鼠腹腔巨噬细胞中SOCS1基因甲基化的状态。实验结果发现，5-azadC能使SOCS1去甲基化进而恢复SOCS1的表达，并抑制炎症因子TNF-α，IL-6的分泌。 3. DNMT1调控SOCS1的表达及其对甲基化的作用研究。根据DNMT1的碱基序列设计并合成小干扰RNA(small interfering RNA,siRNA)，通过脂质体LipofectamineTM2000转染到RAW264.7细胞内，荧光倒置显微镜观察细胞的转染效率。RT-PCR检测SOCS1，TNF-α，IL-6mRNA的表达;Western Blot检测DNMT1、SOCS1蛋白的表达；Elisa方法检测细胞培养上清中炎症因子TNF-α，IL-6的表达变化。MSP-PCR检测沉默DNMT1基因后SOCS1甲基化的状态。实验结果发现，沉默的DNMT1可以促进SOCS1的去甲基化及SOCS1的表达，进而抑制LPS诱导的RAW264.7中炎症因子TNF-α，IL-6的分泌即DNMT1通过介导SOCS1甲基化来抑制炎症因子的分泌。 4. DNA甲基化抑制剂及DNMT1沉默对SOCS1下游信号通路的影响。 Western blot检测发现，LPS诱导的小鼠腹腔巨噬细胞活化中p-JAK2，p-STAT3的表达升高，DNA甲基化抑制剂和DNMT1沉默可抑制p-JAK2，p-STAT3表达水平。此外，在封闭DNMT1的RAW264.7中沉默SOCS1可以减弱DNMT1对JAK2/STAT3的活化作用，以上结果表明SOCS1高甲基化活化了下游的信号通路，进而促进了巨噬细胞炎症因子的分泌。
[Abstract]:The activation and secretion of inflammatory factors by macrophages is the main characteristic of inflammatory diseases such as arthritis, liver fibrosis and other inflammatory diseases, and inhibiting the activation of macrophages is the key link of anti-inflammatory and immunotherapy. SOCS1, an inhibitor of cytokine transduction, plays an important role in the inhibition of tissue injury and inflammatory diseases. SOCS1 inactivation is associated with hypermethylation of the promoter region in all types of tumors. In order to further study the mechanism of DNA methylation in the release of inflammatory cytokines from macrophages, we observed the changes of SOCS1 gene in murine peritoneal macrophages in vitro and its effect on the secretion of inflammatory cytokines by macrophages. Part of its mechanism is studied. The main contents of this study are as follows: the expression and methylation of 1.SOCS1 in RAW264.7 mouse peritoneal macrophages induced by LPS. RAW264.7 (mouse macrophage cell line) was selected as the study object. The activation was induced by LPS1 渭 g/ml for 24 h, the mRNA expression of SOCS1,TNF- 伪 and IL-6 was detected by RT-PCR method, and the expression of SOCS1 protein was detected by Western blot. Elisa method was used to detect the expression of TNF- 伪 and IL-6 in the supernatant of cell culture. The results showed that the expression of SOCS1 decreased during the secretion of inflammatory cytokines by macrophages. Methylation-specific PCR (MSP-PCR) was used to detect the status of SOCS1 methylation during the activation of macrophages in RAW264.7 mice induced by LPS. We found that the SOCS1 gene was hypermethylated. 2. SOCS1 induced demethylation and self-expression recovery under the action of DNA methylation inhibitor, which inhibited the secretion of inflammatory factors in RAW264.7 macrophages. RAW264.7 was used to induce activation for 24 h and 5-azadC2 渭 mol/l for 48 h. MRNA expression of SOCS1,TNF- 伪 and IL-6 was detected by RT-PCR method, SOCS1 protein was detected by Western blot. Elisa method was used to detect the expression of TNF- 伪 and IL-6 in the supernatant of cell culture. Methylation-specific PCR (MSP-PCR) was used to detect the methylation of SOCS1 gene in the peritoneal macrophages of RAW264.7 mice treated with LPS and 5-azadC. The results showed that 5-azadC could induce SOCS1 demethylation and restore the expression of SOCS1, and inhibit the secretion of TNF- 伪 and IL-6. 3. DNMT1 regulates the expression of SOCS1 and its effect on methylation. Small interfering RNA (small interfering RNA,siRNA was designed and synthesized according to the base sequence of DNMT1. The transfection efficiency of RAW264.7 cells was observed by using liposome LipofectamineTM2000. The transfection efficiency was observed by fluorescence inverted microscope. The expression of SOCS1,TNF- 伪 and IL-6mRNA was detected by RT-PCR. The expression of DNMT1,SOCS1 protein was detected by Western Blot. Elisa method was used to detect the expression of inflammatory factor TNF- 伪 and IL-6 in the supernatant of cell culture, and MSP-PCR was used to detect the methylation of SOCS1 after silencing DNMT1 gene. The results showed that silencing DNMT1 could promote the demethylation of SOCS1 and the expression of SOCS1, and then inhibit the secretion of TNF- 伪 and IL-6 in RAW264.7 induced by LPS, that is, DNMT1 mediated SOCS1 methylation to inhibit the secretion of inflammatory factors. 4. Effects of DNA methylation inhibitor and DNMT1 silencing on downstream signaling pathway of SOCS1. Western blot assay showed that the expression of p-JAK _ 2 and p-STAT3 was increased during the activation of murine peritoneal macrophages induced by LPS, and DNA methylation inhibitor and DNMT1 silencing could inhibit the expression of p-JAK _ 2 and p-STAT3. In addition, silencing SOCS1 in RAW264.7 blocking DNMT1 can attenuate the activation of JAK2/STAT3 by DNMT1. These results suggest that SOCS1 hypermethylation activates the downstream signaling pathway and promotes the secretion of inflammatory cytokines in macrophages.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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