本文關(guān)鍵詞： IL-32 TSLP 角膜上皮 caspase-1 NF-κB　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討白介素-32(Interleukin-32,IL-32)和胸腺基質(zhì)淋巴細(xì)胞生成素(thymic stromal lymphopoietin,TSLP)在人角膜上皮細(xì)胞炎癥反應(yīng)中的作用及調(diào)控機(jī)制。方法:(1)不同濃度(2、10、50ng/ml)的IL-32刺激人角膜上皮細(xì)胞,應(yīng)用熒光定量PCR和ELISA檢測HCECs中TSLP、IL-6、TNFαm RNA和蛋白的表達(dá)變化,同時用免疫組化技術(shù)定位TSLP蛋白在人角膜上皮組織中的表達(dá);(2)應(yīng)用Western blot檢測上述各組角膜上皮細(xì)胞中caspase-1的活性,加入caspase-1抑制劑VX-765后分別應(yīng)用Western blot、熒光定量PCR和ELISA檢測caspase-1活性、TSLP m RNA和蛋白水平的表達(dá)變化;(3)用同種型Ig G抗體(5μg/m L)、TSLP抗體(5μg/m L)、NF-k B抑制劑喹唑啉(NF-k B-I,10μM)以及caspase-1抑制劑VX-765預(yù)處理人角膜上皮細(xì)胞1小時,然后用IL-32(10ng/m L)刺激HCECs4小時后應(yīng)用熒光定量PCR檢測IL-6和TNFαm RNA水平的表達(dá),刺激48小時后用ELISA檢測IL-6和TNFα蛋白水平的表達(dá)。結(jié)果:(1)IL-32刺激人角膜上皮細(xì)胞后可引起TSLP、IL-6、TNFαm RNA和蛋白水平表達(dá)升高,呈一定的時間和濃度依賴性,差異有統(tǒng)計學(xué)意義(p0.05),TSLP蛋白在未經(jīng)處理的角膜組織細(xì)胞質(zhì)中表達(dá),而在經(jīng)IL-32(50 ng/ml)處理48小時后的供體角膜上皮組織中,TSLP在角膜各層中的表達(dá)均顯著增強(qiáng);(2)IL-32促進(jìn)caspase-1的活化,并且具有濃度依賴性,加入caspase-1抑制劑后,caspase-1的活性明顯受到抑制,TSLP m RNA和蛋白水平的表達(dá)也明顯減少,差異有統(tǒng)計學(xué)意義(p0.05);(3)TSLP抗體、NF-κB抑制劑喹唑啉和caspase-1抑制劑VX-765能夠顯著抑制IL-32介導(dǎo)的促炎因子(TNFα和IL-6)m RNA和蛋白的表達(dá),而同種型Ig G抗體對TNFα和IL-6的表達(dá)無明顯的抑制作用,差異有統(tǒng)計學(xué)意義(p0.05)。結(jié)論:IL-32和TSLP是參與人類角膜上皮炎癥反應(yīng)的重要的細(xì)胞因子,IL-32通過caspase-1信號通路上調(diào)TSLP的表達(dá);IL-32通過caspase-1/TSLP/NF-κB信號通路介導(dǎo)角膜上皮的炎癥反應(yīng)。
[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 偽 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 偽 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 偽 protein was detected by ELISA. Results the expression of TSLP IL-6 TNF- 偽 m RNA and TNF- 偽 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- 偽 and IL-6)m RNA and protein mediated by IL-32, but the expression of TNF 偽 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R77

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本文編號：1495303