IL-32诱导TSLP在角膜上皮细胞免疫炎症反应中的作用

发布时间：2018-02-08 11:08

本文关键词： IL-32 TSLP 角膜上皮 caspase-1 NF-κB　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨白介素-32(Interleukin-32,IL-32)和胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)在人角膜上皮细胞炎症反应中的作用及调控机制。方法:(1)不同浓度(2、10、50ng/ml)的IL-32刺激人角膜上皮细胞,应用荧光定量PCR和ELISA检测HCECs中TSLP、IL-6、TNFαm RNA和蛋白的表达变化,同时用免疫组化技术定位TSLP蛋白在人角膜上皮组织中的表达;(2)应用Western blot检测上述各组角膜上皮细胞中caspase-1的活性,加入caspase-1抑制剂VX-765后分别应用Western blot、荧光定量PCR和ELISA检测caspase-1活性、TSLP m RNA和蛋白水平的表达变化;(3)用同种型Ig G抗体(5μg/m L)、TSLP抗体(5μg/m L)、NF-k B抑制剂喹唑啉(NF-k B-I,10μM)以及caspase-1抑制剂VX-765预处理人角膜上皮细胞1小时,然后用IL-32(10ng/m L)刺激HCECs4小时后应用荧光定量PCR检测IL-6和TNFαm RNA水平的表达,刺激48小时后用ELISA检测IL-6和TNFα蛋白水平的表达。结果:(1)IL-32刺激人角膜上皮细胞后可引起TSLP、IL-6、TNFαm RNA和蛋白水平表达升高,呈一定的时间和浓度依赖性,差异有统计学意义(p0.05),TSLP蛋白在未经处理的角膜组织细胞质中表达,而在经IL-32(50 ng/ml)处理48小时后的供体角膜上皮组织中,TSLP在角膜各层中的表达均显著增强;(2)IL-32促进caspase-1的活化,并且具有浓度依赖性,加入caspase-1抑制剂后,caspase-1的活性明显受到抑制,TSLP m RNA和蛋白水平的表达也明显减少,差异有统计学意义(p0.05);(3)TSLP抗体、NF-κB抑制剂喹唑啉和caspase-1抑制剂VX-765能够显著抑制IL-32介导的促炎因子(TNFα和IL-6)m RNA和蛋白的表达,而同种型Ig G抗体对TNFα和IL-6的表达无明显的抑制作用,差异有统计学意义(p0.05)。结论:IL-32和TSLP是参与人类角膜上皮炎症反应的重要的细胞因子,IL-32通过caspase-1信号通路上调TSLP的表达;IL-32通过caspase-1/TSLP/NF-κB信号通路介导角膜上皮的炎症反应。
[Abstract]:Objective: to investigate the role and mechanism of interleukin-32 Interleukin-32 (IL-32) and thymic stromal lymphopoietin (TSLP) in the inflammatory response of human corneal epithelial cells. Fluorescence quantitative PCR and ELISA were used to detect the expression of TNF 伪 m RNA and protein in HCECs. Meanwhile, the expression of TSLP protein in human corneal epithelium was detected by immunohistochemistry. The activity of caspase-1 in corneal epithelial cells was detected by Western blot. After adding caspase-1 inhibitor VX-765, Western blotts, fluorescence quantitative PCR and ELISA were used to detect the expression of caspase-1 active TSLP m RNA and protein. The expression of caspase-1 active TSLP m RNA and protein were detected by Western blot, respectively.) the isotype IgG antibody was 5 渭 g / m L ~ (5 渭 g / m) TSLP antibody and 5 渭 g / m / m ~ (1) NF-K / B inhibitor quinazoline NF-k B-I10 渭 M respectively) and caspase-1 inhibitor VX-765 was used to detect the expression of NF-k B-I10 渭 M) and caspase-1 inhibitor VX-765. Human corneal epithelial cells were pretreated for 1 hour. The expression of IL-6 and TNF 伪 m RNA were detected by fluorescence quantitative PCR after HCECs4 stimulation with IL-32(10ng/m L for hours. After 48 hours of stimulation, the expression of IL-6 and TNF 伪 protein was detected by ELISA. Results the expression of TSLP IL-6 TNF- 伪 m RNA and TNF- 伪 m protein was increased in a time-and concentration-dependent manner after stimulation of IL-32 in human corneal epithelial cells. There was a significant difference in the expression of TSLP protein in the cytoplasm of untreated corneal tissue, while in donor corneal epithelium treated with IL-32(50 ng / ml for 48 hours, the expression of TSLP in all layers of cornea significantly enhanced the activation of caspase-1. In a dose-dependent manner, the activity of caspase-1 was significantly inhibited by the addition of caspase-1 inhibitor, and the expression of TSLP m RNA and protein was also significantly decreased. The difference was statistically significant (P < 0.05). Quinazoline, an NF- 魏 B inhibitor, and VX-765, an inhibitor of caspase-1, could significantly inhibit the expression of TNF- 伪 and IL-6)m RNA and protein mediated by IL-32, but the expression of TNF 伪 and IL-6 was not significantly inhibited by homotypic IgG antibody. Conclusion IL-32 and TSLP are important cytokines involved in the inflammation of human corneal epithelium. IL-32 up-regulates the expression of TSLP through the caspase-1 signaling pathway. IL-32 mediates the inflammatory response of corneal epithelium through caspase-1 / TSLP / NF- 魏 B signaling pathway.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R77

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