TNF-α和LPS对哮喘患者外周血单个核细胞IL-33分泌的影响

发布时间：2018-03-31 00:02

本文选题：IL-33　切入点：TNF　出处：《华中科技大学》2013年硕士论文

【摘要】：目的：本实验通过研究TNF-α和LPS在体外刺激哮喘患者PBMC细胞24小时后，细胞内IL-33的mRNA及上清中IL-33水平的改变来探讨哮喘可能的发生机制，并研究可能影响IL-33分泌的几种因素，最后通过地塞米松和p38、ERK通道阻滞剂干预来为哮喘的治疗提供新的途径。方法：从同济医院门诊收集20例哮喘患者外周静脉血约15mL，通过人外周血淋巴细胞分离液分离出PBMC,并用在六孔板中用1640培养基进行培养(每孔1*10^6个细胞)，本实验分为14个小组，给予不同刺激:（1）哮喘对照组；（2）哮喘+TNF-α组；（3）哮喘+LPS组；（4）哮喘+TNF-α+LPS组；（5）哮喘+DXM组；（6）哮喘+DXM+TNF-α组；（7）哮喘+DXM+LPS组；（8）哮喘+DXM+TNF-α+LPS组；（9）哮喘+PD98059+TNF-α组；（10）哮喘+PD98059+LPS组；（11）哮喘+PD98059+TNF-α+LPS组；12）哮喘+SB202190+TNF-α组；（13）哮喘+SB202190+LPS组；（14）哮喘+SB202190+TNF-α+LPS组。刺激24小时后，，每孔收集细胞和PBMCs培养后的上清。再用realtime-PCR的方法来检测细胞内IL-33mRNA表达的相对水平,用Elisa方法来测定培养后上清中IL-33蛋白的水平。结果：(1)与哮喘对照组相比，TNF-α和LPS均能促进IL-33mRNA的表达(P0.01)，两者共同刺激后IL-33含量明显增加(P0.01)；(2)与相应对照组相比，加入地塞米松(DXM)均无法抑制TNF-α和LPS对PBMC中IL-33的促进作用(P0.05)；（3）与相应对照组相比,加入p38通道阻滞剂后，TNF-α对IL-33mRNA的升高作用明显被抑制(P0.01)，但对LPS的上调作用无类似效果（P0.05）；(4）与相应对照组相比,加入ERK通道阻滞剂后,均无法抑制TNF-α和LPS对PBMC中IL-33的促进作用(P0.05)（；5）PBMC培养的上清中IL-33蛋白含量无法用Elisa检测出来。结论： TNF-α和LPS刺激后，哮喘患者PBMC中IL-33mRNA的表达明显升高，两者之间有协同作用，加入地塞米松后，上述促进效应未被明显抑制。进一步研究相关通路表明，TNF-α使IL-33mRNA表达升高，可能是与p38途径有关，LPS使IL-33mRNA表达升高，可能并不是通过p38和ERK途径。
[Abstract]:Objective: to explore the possible mechanism of asthma by studying the changes of mRNA and IL-33 levels in IL-33 and supernatant of PBMC cells stimulated with TNF- 伪 and LPS for 24 hours in vitro, and to study several factors that may affect the secretion of IL-33. Finally, dexamethasone and p38 ERK channel blockers were used to provide a new approach to the treatment of asthma. Methods: peripheral venous blood was collected from 20 asthmatic patients in Tongji Hospital. PBMCs were isolated from human peripheral blood lymphocytes and cultured in 1640 culture medium (10 ^ 6 cells per hole). The experiment was divided into 14 groups. Different stimuli 1) Asthma control group 2) Asthma TNF- 伪 group 3) Asthma LPS group 4) Asthma TNF- 伪 LPS group 5) Asthma DXM TNF- 伪 group (TNF- 伪) Asthma DXM TNF- 伪 group (TNF- 伪) Asthma DXM LPS group (TNF- 伪 LPS group 9) Asthma PD98059 TNF- 伪 group 10) Asthma. PD98059 LPS group (n = 11) asthma PD98059 TNF- 伪 LPS group (n = 12) asthma SB202190 TNF- 伪 group (n = 13) asthma SB202190 LPS group (n = 14) asthma SB202190 TNF- 伪 LPS group. The relative level of IL-33mRNA expression in cells was detected by realtime-PCR method and the level of IL-33 protein in supernatant was determined by Elisa method. Results compared with the asthmatic control group, both TNF- 伪 and LPS could promote the expression of IL-33mRNA, and the content of IL-33 increased significantly after the stimulation of both groups. DXM could not inhibit the effect of TNF- 伪 and LPS on IL-33 in PBMC. After adding p38 channel blocker, the increase of IL-33mRNA was significantly inhibited by TNF- 伪, but there was no similar effect on the up-regulation of LPS. Compared with the control group, the increase of ERK channel blocker was not similar to that of the control group. Both TNF- 伪 and LPS could not inhibit the effect of TNF- 伪 and LPS on the promotion of IL-33 in PBMC. The IL-33 protein content in the supernatant of PBMC cultured with P0. 05 and TNF- 伪 could not be detected by Elisa. Conclusion: after stimulation of TNF- 伪 and LPS, the expression of IL-33mRNA in PBMC of asthmatic patients was significantly increased, and there was a synergistic effect between them. After adding dexamethasone, the above effects were not significantly inhibited. Further study on the related pathways showed that TNF- 伪 could increase the expression of IL-33mRNA. The expression of IL-33mRNA may be related to p38 pathway, but it may not be through p38 and ERK pathway.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25

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