發(fā)布時(shí)間：2018-03-26 15:15

  本文選題：甲基乙二醛　切入點(diǎn)：晚期糖化終產(chǎn)物　出處：《福建醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：甲基乙二醛（MGO）是葡萄糖活性代謝產(chǎn)物，晚期糖化終產(chǎn)物受體（RAGE）可抑制MGO代謝的關(guān)鍵酶乙二醛酶1（GLO-1）。MGO和晚期糖化終產(chǎn)物受體的水平在糖尿病人中明顯增高，與糖尿病加速動(dòng)脈粥樣硬化有關(guān)，但它們之間相互作用在糖尿病加速動(dòng)脈粥樣硬化中的意義仍不清楚。本課題組前期研究發(fā)現(xiàn)MGO誘導(dǎo)Jurkat細(xì)胞分泌炎癥因子TNF-α、IFN-γ。本實(shí)驗(yàn)將進(jìn)一步研究RAGE及其胞內(nèi)信號(hào)對(duì)MGO誘導(dǎo)的Jurkat細(xì)胞分泌TNF-α和IFN-γ的影響，為了解糖尿病加速動(dòng)脈粥樣硬化機(jī)制提供了實(shí)驗(yàn)基礎(chǔ)。 方法：①不同濃度的MGO（0、15、30、60μM）作用PHA（0.25μg/ml）預(yù)刺激的Jurkat細(xì)胞24h，Western blot檢測(cè)RAGE表達(dá)。②不同濃度MGO（0、15、30、60μM）作用于PHA預(yù)刺激的Jurkat細(xì)胞24h，ELISA檢測(cè)TNF-α和IFN-γ的分泌；部分實(shí)驗(yàn)加RAGE抗體（1μg/ml）預(yù)處理，同時(shí)設(shè)非特異抗體（1μg/ml）為對(duì)照。③不同濃度MGO（0、15、30、60μM）作用PHA預(yù)刺激的Jurkat細(xì)胞24h, Western blot測(cè)鈣調(diào)蛋白依賴性蛋白激酶IV（CaMKIV）的表達(dá)；部分實(shí)驗(yàn)加RAGE抗體（1μg/ml）預(yù)處理，同時(shí)設(shè)非特異抗體（1μg/ml）為對(duì)照。免疫熒光檢測(cè)30μM MGO作用0、15、30、60min后的Jurkat細(xì)胞內(nèi)CaMKIV轉(zhuǎn)核情況；部分實(shí)驗(yàn)加RAGE抗體（1μg/ml）預(yù)處理，同時(shí)設(shè)非特異抗體（1μg/ml）為對(duì)照。④30μM MGO作用PHA預(yù)刺激24h的Jurkat細(xì)胞0、15、30、60min，Western blot檢測(cè)p38MAPK磷酸化表達(dá)；部分實(shí)驗(yàn)加RAGE抗體（1μg/ml）預(yù)處理，同時(shí)設(shè)非特異抗體（1μg/ml）為對(duì)照。⑤30μMMGO作用于CaMKIV抑制劑KN62（10μM）、p38MAPK抑制劑SB203580（25μM）預(yù)處理的Jurkat24h，，ELISA檢測(cè)TNF-α和IFN-γ的分泌。 結(jié)果：①M(fèi)GO作用于PHA預(yù)刺激的Jurkat細(xì)胞24小時(shí)，Western Blot顯示Jurkat細(xì)胞表達(dá)RAGE，15、30、60μM MGO均上調(diào)RAGE的表達(dá)，與MGO未作用組比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）。②15、30、60μM MGO作用于Jurkat細(xì)胞24h可促使Jurkat細(xì)胞分泌TNF-α及IFN-γ，與MGO未作用組比較差異有統(tǒng)計(jì)學(xué)意義（p0.05）。RAGE抗體能抑制MGO誘導(dǎo)Jurkat細(xì)胞分泌TNF-α及IFN-γ（p0.05），而非特異抗體預(yù)處理對(duì)MGO誘導(dǎo)Jurkat細(xì)胞分泌TNF-α及IFN-γ無影響（p0.05）。③15-60μM MGO能增加CaMKIV的表達(dá)，與MGO未作用組比較差異有統(tǒng)計(jì)學(xué)意義（p0.05）。RAGE抗體抑制MGO誘導(dǎo)的CaMKIV的表達(dá)（p0.05）；而非特異抗體預(yù)處理對(duì)MGO誘導(dǎo)CaMKIV的表達(dá)無影響（p0.05）。免疫熒光顯示30μM MGO作用15-60min促進(jìn)Jurkat細(xì)胞CaMKIV核轉(zhuǎn)位；RAGE抗體抑制MGO誘導(dǎo)的CaMKIV蛋白核轉(zhuǎn)位，而非特異抗體預(yù)處理對(duì)MGO誘導(dǎo)CaMKIV蛋白核轉(zhuǎn)位無影響。④30μM MGO作用Jurkat細(xì)胞15-60min引起Jurkat細(xì)胞內(nèi)p38MAPK磷酸化表達(dá)增加（p0.05）；RAGE抗體抑制MGO引起的p38MAPK磷酸化（p0.05），而非特異抗體預(yù)處理對(duì)MGO誘導(dǎo)p38MAPK磷酸化無影響（p0.05）。⑤CaMKIV，p38MAPK通路抑制劑預(yù)處理抑制MGO誘導(dǎo)TNF-α和IFN-γ的分泌（p0.05）。 結(jié)論：MGO上調(diào)Jurkat細(xì)胞表面RAGE的表達(dá)，并通過RAGE引起CaMKIV表達(dá)增加、促進(jìn)CaMKIV核轉(zhuǎn)位以及p38MAPK磷酸化,進(jìn)一步促進(jìn)炎癥因子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- 偽 and IFN- 緯 by Jurkat cells. This study will further study RAGE and its intracellular signal response. MGO induced secretion of TNF- 偽 and IFN- 緯 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- 偽 and IFN- 緯 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- 偽 and IFN- 緯 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- 偽 and IFN- 緯 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- 偽 and IFN- 緯 in Jurkat cells for 24 h, and the antibody against MGO could inhibit MGO induced secretion of TNF- 偽 and IFN- 緯 -p0.05in Jurkat cells. Non-specific antibody pretreatment did not affect the secretion of TNF- 偽 and IFN- 緯 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- 偽 and IFN- 緯 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- 偽 and IFN- 緯.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 游捷;余洪根;彭雪峰;劉曉紅;劉禮斌;;甲基乙二醛對(duì)Jurkat細(xì)胞氧化應(yīng)激及分泌細(xì)胞因子的影響[J];中國(guó)免疫學(xué)雜志;2011年07期

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