本文選題：IL-33　切入點(diǎn)：TNF　出處：《華中科技大學(xué)》2013年碩士論文

【摘要】：目的：本實(shí)驗(yàn)通過研究TNF-α和LPS在體外刺激哮喘患者PBMC細(xì)胞24小時(shí)后，細(xì)胞內(nèi)IL-33的mRNA及上清中IL-33水平的改變來探討哮喘可能的發(fā)生機(jī)制，并研究可能影響IL-33分泌的幾種因素，最后通過地塞米松和p38、ERK通道阻滯劑干預(yù)來為哮喘的治療提供新的途徑。 方法：從同濟(jì)醫(yī)院門診收集20例哮喘患者外周靜脈血約15mL，通過人外周血淋巴細(xì)胞分離液分離出PBMC,并用在六孔板中用1640培養(yǎng)基進(jìn)行培養(yǎng)(每孔1*10^6個(gè)細(xì)胞)，本實(shí)驗(yàn)分為14個(gè)小組，給予不同刺激:（1）哮喘對(duì)照組；（2）哮喘+TNF-α組；（3）哮喘+LPS組；（4）哮喘+TNF-α+LPS組；（5）哮喘+DXM組；（6）哮喘+DXM+TNF-α組；（7）哮喘+DXM+LPS組；（8）哮喘+DXM+TNF-α+LPS組；（9）哮喘+PD98059+TNF-α組；（10）哮喘+PD98059+LPS組；（11）哮喘+PD98059+TNF-α+LPS組；12）哮喘+SB202190+TNF-α組；（13）哮喘+SB202190+LPS組；（14）哮喘+SB202190+TNF-α+LPS組。刺激24小時(shí)后，，每孔收集細(xì)胞和PBMCs培養(yǎng)后的上清。再用realtime-PCR的方法來檢測(cè)細(xì)胞內(nèi)IL-33mRNA表達(dá)的相對(duì)水平,用Elisa方法來測(cè)定培養(yǎng)后上清中IL-33蛋白的水平。 結(jié)果：(1)與哮喘對(duì)照組相比，TNF-α和LPS均能促進(jìn)IL-33mRNA的表達(dá)(P0.01)，兩者共同刺激后IL-33含量明顯增加(P0.01)；(2)與相應(yīng)對(duì)照組相比，加入地塞米松(DXM)均無法抑制TNF-α和LPS對(duì)PBMC中IL-33的促進(jìn)作用(P0.05)；（3）與相應(yīng)對(duì)照組相比,加入p38通道阻滯劑后，TNF-α對(duì)IL-33mRNA的升高作用明顯被抑制(P0.01)，但對(duì)LPS的上調(diào)作用無類似效果（P0.05）；(4）與相應(yīng)對(duì)照組相比,加入ERK通道阻滯劑后,均無法抑制TNF-α和LPS對(duì)PBMC中IL-33的促進(jìn)作用(P0.05)（；5）PBMC培養(yǎng)的上清中IL-33蛋白含量無法用Elisa檢測(cè)出來。 結(jié)論： TNF-α和LPS刺激后，哮喘患者PBMC中IL-33mRNA的表達(dá)明顯升高，兩者之間有協(xié)同作用，加入地塞米松后，上述促進(jìn)效應(yīng)未被明顯抑制。進(jìn)一步研究相關(guān)通路表明，TNF-α使IL-33mRNA表達(dá)升高，可能是與p38途徑有關(guān)，LPS使IL-33mRNA表達(dá)升高，可能并不是通過p38和ERK途徑。
[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- 偽 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- 偽 group 3) Asthma LPS group 4) Asthma TNF- 偽 LPS group 5) Asthma DXM TNF- 偽 group (TNF- 偽) Asthma DXM TNF- 偽 group (TNF- 偽) Asthma DXM LPS group (TNF- 偽 LPS group 9) Asthma PD98059 TNF- 偽 group 10) Asthma. PD98059 LPS group (n = 11) asthma PD98059 TNF- 偽 LPS group (n = 12) asthma SB202190 TNF- 偽 group (n = 13) asthma SB202190 LPS group (n = 14) asthma SB202190 TNF- 偽 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- 偽 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- 偽 and LPS on IL-33 in PBMC. After adding p38 channel blocker, the increase of IL-33mRNA was significantly inhibited by TNF- 偽, 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- 偽 and LPS could not inhibit the effect of TNF- 偽 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- 偽 could not be detected by Elisa. Conclusion: after stimulation of TNF- 偽 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- 偽 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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R562.25

【共引文獻(xiàn)】

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