高糖对巨噬细胞TLR4信号转导的调节作用

发布时间：2018-09-05 17:45

【摘要】：目的:糖尿病肾病(Diabetic Nephropathy,DN)是糖尿病(Diabetes Mellitus,DM)造成的严重和危害性较大的一种慢性并发症。其发病机制与慢性炎症有关,动物实验和DN临床患者的研究中均发现肾脏组织大量浸润的炎症细胞以巨噬细胞为主。其浸润程度与肾组织的损害程度趋势一致呈正相关。因此,巨噬细胞介导的炎症反应是加快DN发展的重要原因之一。TLR4是巨噬细胞表面的模式识别受体,TLR4信号通路在DN的发病中的作用逐渐受到人们的关注,许多临床和实验均表明TLR4在高糖状态下表达增高。生理条件下LPS结合巨噬细胞和树突状细胞表面的TLR4可诱导促炎信号和抗炎信号的转导,分别产生促炎因子TNF-α、IL-β和抗炎因子IL-10,来维持炎症因子的平衡。那么,在糖尿病状态下巨噬细胞介导的TLR4的激活是否改变了这种平衡状态,导致DN的发生呢?目前还未见详细报道。因此,本课题依据前期结果和文献资料,在体外用高糖培养巨噬细胞,用TLR4的不同配体进行刺激,观察TLR4介导的促炎和抗炎信号转导分子的表达,探讨高糖对巨噬细胞TLR4信号转导的影响,明确巨噬细胞在DM和DN发生中的炎症机制。方法:1经PMA活化的THP-1细胞同时用正常培养基和高糖培养基培养24小时后,再用LPS刺激,在0、2、4、8、16、24小时收集细胞。用Real-time PCR检测炎症因子IL-10、IL-1β、TNF-αm RNA的表达水平。2经PMA活化的THP-1细胞同时用正常培养基和高糖培养基培养24小时后,再用LPS刺激,在0、2、4、8、16、24小时收集细胞上清;用HSP60刺激24h收集细胞上清用ELISA检测炎症因子IL-10、IL-1β、TNF-α的表达水平。3经PMA活化的THP-1细胞同时用正常培养基和高糖培养基培养24小时后,再用LPS刺激,在0、15、30、60、120、180min收集细胞。另一组用LPS和HSP60刺激,在0、30、60、180min收集细胞,用Western blot检测p110δ、P-NF-κB p65、NF-κB p65、P-AKT、AKT的表达水平。结果:1与空白对照组相比,IL-10m RNA正常组与高糖组在2h、4h时表达明显增高,IL-1βm RNA正常组与高糖组在2h、4h、8h时表达明显增高,TNF-αm RNA的正常组与高糖组在2h、4h、16h、24h时表达明显增高。对照组IL-10m RNA表达量的高峰值出现在LPS刺激后的2小时,高糖组出现在4小时,出现延迟现象。高糖组在4小时IL-1βm RNA表达量明显高于正常组。高糖组在2小时TNF-αm RNA表达量显著高于正常组。2上清ELISA结果,IL-10无明显变化,LPS刺激时,IL-1β和TNF-α的产生随着时间延长均逐渐升高,且高糖组在任一时间点均高于正常组。LPS和HSP60同时刺激24h时,LPS刺激下IL-1β、TNF-α变化显著,且高糖组高于正常组,HSP60刺激下变化不明显。3 Western blot结果显示:LPS刺激细胞0、15、30、60、120、180min,P-NF-κB p6515分钟时表达量增加,正常组60分钟,高糖组15分钟时时达到高峰,P-AKT60分钟时表达增高,高糖组在同一时间点总体高于正常组;LPS和HSP60刺激细胞0、30、60、180min,高糖状态下p110δ无论在LPS还是HSP60刺激下表达水平降低,P-NF-κB p65、P-AKT在LPS的作用下变化明显。结论:1在不同配体刺激下,促炎因子IL-1β、TNF-α无论在上清ELISA水平还是P-NF-κB p65,P-AKT的蛋白水平,总体上LPS较HSP60作用显著。2 p110δ在高糖状态下无论LPS还是HSP60刺激表达水平均降低。3 TLR4介导的促炎信号通路高糖状态下明显高于正常组。抗炎信号分子变化不明显。
[Abstract]:Objective: Diabetic nephropathy (DN) is a serious and harmful chronic complication caused by diabetes mellitus (DM). The pathogenesis of DN is related to chronic inflammation. Macrophages are found to be the main infiltrating inflammatory cells in renal tissues in animal experiments and clinical studies of DN patients. TLR4 is a pattern recognition receptor on the surface of macrophages. The role of TLR4 signaling pathway in the pathogenesis of DN has attracted more and more attention. Many clinical and experimental studies have shown that TLR4 is hyperglycemic. Under physiological conditions, LPS binds to TLR4 on the surface of macrophages and dendritic cells to induce the transduction of pro-inflammatory and anti-inflammatory signals, producing pro-inflammatory factors TNF-alpha, IL-beta and anti-inflammatory factors IL-10 to maintain the balance of inflammatory factors, respectively. So, according to the previous results and literature, macrophages were cultured with high glucose and stimulated with different ligands of TLR4 in vitro to observe the expression of TLR4-mediated pro-inflammatory and anti-inflammatory signal transduction molecules, and to explore the effect of high glucose on TLR4 signal transduction in macrophages. Methods: 1 THP-1 cells activated by PMA were cultured in normal medium and high glucose medium for 24 hours, then stimulated by LPS and collected at 0, 2, 4, 8, 16 and 24 hours. The expression levels of inflammatory factors IL-10, IL-1beta and TNF-alpha m RNA were detected by Real-time PCR. Cell supernatant was collected at 0,2,4,8,16 and 24 hours after the cells were cultured in normal medium and high glucose medium for 24 hours. The supernatant was collected at 24 hours after the cells were stimulated by HSP60 and the expression levels of inflammatory factors IL-10, IL-1beta and TNF-alpha were detected by ELISA. 3 PMA-activated THP-1 cells were cultured in normal medium and high glucose medium for 24 hours. One group was stimulated by LPS for 0,15,30,60,120,180 minutes, and the other group was stimulated by LPS and HSP60 for 0,30,60,180 minutes. The expression of p110delta, P-NF-kappa B p65, NF-kappa B p65, P-AKT and AKT was detected by Western blot. Results: 1 Compared with the blank control group, the expression of IL-10m RNA in normal group and high glucose group was significantly increased at 2 h and 4 h. The expression of IL-10m RNA in normal group and high glucose group increased significantly at 2h, 4H and 8h, while that in normal group and high glucose group increased significantly at 2h, 4h, 16h and 24h. The peak expression of IL-10m RNA in control group was 2 hours after LPS stimulation, and that in high glucose group was 4 hours after LPS stimulation. The expression of TNF-alpha m RNA in the high glucose group was significantly higher than that in the normal group at 2 hours. The results of ELISA in the supernatant of 2 hours showed that IL-10 had no significant change. The production of IL-1beta and TNF-alpha increased gradually with the prolongation of LPS stimulation, and the expression of IL-1beta and TNF-alpha in the high glucose group was higher than that in the normal group at any time point. Western blot showed that the expression of P-NF-kappa B p65 15 minutes increased in LPS-stimulated cells at 0,15,30,60,120,180 minutes. The expression of P-NF-kappa B p65 15 minutes increased in normal group at 60 minutes. The expression of P-AKT reached its peak at 15 minutes in high glucose group and increased at 60 minutes in high glucose group. The expression levels of p110delta were decreased under the stimulation of LPS and HSP60, and the expression levels of P-NF-kappa B p65 and P-AKT were significantly changed under the stimulation of LPS and HSP60 at 0,30,60,180 min. CONCLUSION: 1 Under the stimulation of different ligands, the levels of pro-inflammatory factors IL-1beta and TNF-alpha in the supernatant, ELISA, P-NF-kappa B p65 and P-AKT were higher than those of HSP on the whole. The expression level of LPS and HSP60 was decreased under high glucose condition. 3 TLR4-mediated pro-inflammatory signaling pathway in high glucose condition was significantly higher than that in normal control group.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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