RAGE在MGO诱导Jurkat细胞分泌炎症因子TNF-α和IFN-γ中的作用

发布时间：2018-03-26 15:15

  本文选题：甲基乙二醛　切入点：晚期糖化终产物　出处：《福建医科大学》2012年硕士论文

【摘要】：目的：甲基乙二醛（MGO）是葡萄糖活性代谢产物，晚期糖化终产物受体（RAGE）可抑制MGO代谢的关键酶乙二醛酶1（GLO-1）。MGO和晚期糖化终产物受体的水平在糖尿病人中明显增高，与糖尿病加速动脉粥样硬化有关，但它们之间相互作用在糖尿病加速动脉粥样硬化中的意义仍不清楚。本课题组前期研究发现MGO诱导Jurkat细胞分泌炎症因子TNF-α、IFN-γ。本实验将进一步研究RAGE及其胞内信号对MGO诱导的Jurkat细胞分泌TNF-α和IFN-γ的影响，为了解糖尿病加速动脉粥样硬化机制提供了实验基础。 方法：①不同浓度的MGO（0、15、30、60μM）作用PHA（0.25μg/ml）预刺激的Jurkat细胞24h，Western blot检测RAGE表达。②不同浓度MGO（0、15、30、60μM）作用于PHA预刺激的Jurkat细胞24h，ELISA检测TNF-α和IFN-γ的分泌；部分实验加RAGE抗体（1μg/ml）预处理，同时设非特异抗体（1μg/ml）为对照。③不同浓度MGO（0、15、30、60μM）作用PHA预刺激的Jurkat细胞24h, Western blot测钙调蛋白依赖性蛋白激酶IV（CaMKIV）的表达；部分实验加RAGE抗体（1μg/ml）预处理，同时设非特异抗体（1μg/ml）为对照。免疫荧光检测30μM MGO作用0、15、30、60min后的Jurkat细胞内CaMKIV转核情况；部分实验加RAGE抗体（1μg/ml）预处理，同时设非特异抗体（1μg/ml）为对照。④30μM MGO作用PHA预刺激24h的Jurkat细胞0、15、30、60min，Western blot检测p38MAPK磷酸化表达；部分实验加RAGE抗体（1μg/ml）预处理，同时设非特异抗体（1μg/ml）为对照。⑤30μMMGO作用于CaMKIV抑制剂KN62（10μM）、p38MAPK抑制剂SB203580（25μM）预处理的Jurkat24h，，ELISA检测TNF-α和IFN-γ的分泌。 结果：①MGO作用于PHA预刺激的Jurkat细胞24小时，Western Blot显示Jurkat细胞表达RAGE，15、30、60μM MGO均上调RAGE的表达，与MGO未作用组比较差异有统计学意义（P0.05）。②15、30、60μM MGO作用于Jurkat细胞24h可促使Jurkat细胞分泌TNF-α及IFN-γ，与MGO未作用组比较差异有统计学意义（p0.05）。RAGE抗体能抑制MGO诱导Jurkat细胞分泌TNF-α及IFN-γ（p0.05），而非特异抗体预处理对MGO诱导Jurkat细胞分泌TNF-α及IFN-γ无影响（p0.05）。③15-60μM MGO能增加CaMKIV的表达，与MGO未作用组比较差异有统计学意义（p0.05）。RAGE抗体抑制MGO诱导的CaMKIV的表达（p0.05）；而非特异抗体预处理对MGO诱导CaMKIV的表达无影响（p0.05）。免疫荧光显示30μM MGO作用15-60min促进Jurkat细胞CaMKIV核转位；RAGE抗体抑制MGO诱导的CaMKIV蛋白核转位，而非特异抗体预处理对MGO诱导CaMKIV蛋白核转位无影响。④30μM MGO作用Jurkat细胞15-60min引起Jurkat细胞内p38MAPK磷酸化表达增加（p0.05）；RAGE抗体抑制MGO引起的p38MAPK磷酸化（p0.05），而非特异抗体预处理对MGO诱导p38MAPK磷酸化无影响（p0.05）。⑤CaMKIV，p38MAPK通路抑制剂预处理抑制MGO诱导TNF-α和IFN-γ的分泌（p0.05）。 结论：MGO上调Jurkat细胞表面RAGE的表达，并通过RAGE引起CaMKIV表达增加、促进CaMKIV核转位以及p38MAPK磷酸化,进一步促进炎症因子TNF-α及IFN-γ分泌。
[Abstract]:Objective: methyl Glyoxal (MGO) is an active glucose metabolite. The level of Glyoxalase 1(GLO-1).MGO and late glycosylated end product receptor (1(GLO-1).MGO), the key enzyme of MGO metabolism, can be significantly increased in diabetic patients by advanced glycosylation end product receptor (RAGEG). Associated with diabetes accelerated atherosclerosis, However, the significance of their interaction in diabetic accelerated atherosclerosis is still unclear. Our previous study found that MGO induces the secretion of inflammatory factor TNF- 伪 and IFN- 纬 by Jurkat cells. This study will further study RAGE and its intracellular signal response. MGO induced secretion of TNF- 伪 and IFN- 纬 in Jurkat cells. It provides experimental basis for understanding the mechanism of accelerated atherosclerosis in diabetes mellitus. Methods the expression of RAGE was detected by 24 h Western blot of Jurkat cells prestimulated with PHA(0.25 渭 g / ml (PHA(0.25 渭 g / ml) at different concentrations of MGO015 (30 ~ 60 渭 M). The expression of RAGE was detected by Western blot. The expression of RAGE was detected by 24 h Elisa in PHA prestimulated Jurkat cells. The secretion of TNF- 伪 and IFN- 纬 in Jurkat cells was detected by Elisa for 24 h. Some experiments were pretreated with 1 渭 g / ml of RAGE antibody, and then treated with 1 渭 g / ml of RAGE antibody, and the expression of TNF- 伪 and IFN- 纬 was detected by Elisa. At the same time, the expression of calmodulin dependent protein kinase (IVCaMKIV) was measured by Western blot in Jurkat cells prestimulated with PHA for 24 h, and was pretreated with RAGE antibody (1 渭 g / ml). At the same time, nonspecific antibody (1 渭 g / ml) was used as control. The CaMKIV transnucleation of Jurkat cells was detected by immunofluorescence after 30 minutes of exposure to 30 渭 M MGO, partial experiment was pretreated with 1 渭 g / ml of RAGE antibody, and 1 渭 g / ml of RAGE antibody was pretreated with 30 渭 m MGO. At the same time, the phosphorylation of p38MAPK was detected by Western blot after Jurkat cells were preincubated with PHA for 24 h after treatment with 1 渭 g / ml of nonspecific antibody 1 渭 g / ml. Some of the experiments were pretreated with 1 渭 g / ml of RAGE antibody. Non-specific antibody 1 渭 g / ml was used as control. 530 渭 MMGO was used to detect the secretion of TNF- 伪 and IFN- 纬 by Jurkat 24h Elisa pretreated with CaMKIV inhibitor KN62(10 渭 MM-p38MAPK inhibitor SB203580(25 渭 M. Results the expression of RAGE in Jurkat cells was upregulated by 60 渭 M MGO, which was induced by 1 MGO on Jurkat cells prestimulated by PHA for 24 h. The results showed that the expression of RAGE was up-regulated in Jurkat cells. Compared with the non-treated group of MGO, there was a significant difference between the two groups. P0.05N 路215A3060 渭 M MGO could induce the secretion of TNF- 伪 and IFN- 纬 in Jurkat cells for 24 h, and the antibody against MGO could inhibit MGO induced secretion of TNF- 伪 and IFN- 纬 -p0.05in Jurkat cells. Non-specific antibody pretreatment did not affect the secretion of TNF- 伪 and IFN- 纬 in Jurkat cells induced by MGO. 315-60 渭 M MGO could increase the expression of CaMKIV. Compared with the non-treated group of MGO, there was a significant difference between the two groups. The antibody p0.05 路rage inhibited the expression of CaMKIV induced by MGO, while the non-specific antibody pretreatment had no effect on the expression of CaMKIV induced by MGO. Immunofluorescence showed that 30 渭 M MGO enhanced the CaMKIV nuclear transformation of Jurkat cells induced by 15-60min. The nucleotide translocation of CaMKIV protein induced by MGO was inhibited by rage antibody. However, pretreatment with non-specific antibody had no effect on the nuclear translocation of CaMKIV protein induced by MGO. 430 渭 M MGO increased the expression of p38MAPK phosphorylation in Jurkat cells induced by 15-60min in Jurkat cells. P0.05 rage antibody inhibited p38MAPK phosphorylation induced by MGO, while non-specific antibody pretreatment induced MGO. The phosphorylation of p38MAPK did not affect the pretreatment of p0.05n.5CaMKIVP p38 MAPK pathway inhibitor, which inhibited the secretion of TNF- 伪 and IFN- 纬 by MGO. Conclusion: MGO up-regulates the expression of RAGE on the surface of Jurkat cells, increases the expression of CaMKIV through RAGE, promotes the nuclear translocation of CaMKIV and the phosphorylation of p38MAPK, and further promotes the secretion of inflammatory cytokines TNF- 伪 and IFN- 纬.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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1 游捷;余洪根;彭雪峰;刘晓红;刘礼斌;;甲基乙二醛对Jurkat细胞氧化应激及分泌细胞因子的影响[J];中国免疫学杂志;2011年07期

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