發(fā)布時間：2019-03-06 10:31

【摘要】：目的：研究人永生化角膜上皮細(xì)胞(THCE)感染煙曲霉菌菌絲抗原后,THCE細(xì)胞表面Dectin-1和TLR2的表達(dá)情況,兩者在固有免疫階段的關(guān)系,及其對趨化因子的影響。 方法：常規(guī)體外培養(yǎng)的THCE細(xì)胞為空白對照組,實驗組又分為真菌刺激組和通路受體阻斷組,用煙曲霉菌菌絲抗原感染體外培養(yǎng)的THCE細(xì)胞,建立真菌感染的THCE細(xì)胞模型,此為真菌刺激組,通路受體阻斷組分別用Dectin-1阻斷劑(laminarin)和TLR2中和抗體預(yù)先處理THCE細(xì)胞30分鐘,后加入煙曲霉菌菌絲抗原刺激液,此為Dectin-1阻斷+真菌刺激組和TLR2阻斷+真菌刺激組,應(yīng)用實時熒光定量PCR(Real time-PCR)檢測4h、8h、16h THCE細(xì)胞Dectin-1、TLR2、CXCL1、 CXCL8mRNA的表達(dá)；于8h、16h、24h行Western blot檢測THCE細(xì)胞Dectin-1、 TLR2蛋白的表達(dá)。 結(jié)果：Dectin-1、TLR2mRNA在真菌菌絲抗原刺激THCE細(xì)胞后4h表達(dá)升高,8h達(dá)高峰,真菌菌絲抗原刺激后8h組與4h、16h組間比較,差異有統(tǒng)計學(xué)意義(P0.05)；在真菌菌絲抗原刺激THCE細(xì)胞后8h, Dectin-1、TLR2蛋白表達(dá)均升高,16h表達(dá)量達(dá)峰值。趨化因子(CXCL1、CXCL8)的升高趨勢與Dectin-1和TLR2的表達(dá)趨勢一致。laminarin阻斷Dectin-1后,真菌菌絲抗原刺激THCE細(xì)胞8h, TLR2mRNA表達(dá)明顯下降(P0.05),而TLR2蛋白在Dectin-1被阻斷后16h后,表達(dá)也相應(yīng)下降(P0.05); TLR2中和抗體阻斷TLR2后真菌菌絲抗原刺激THCE細(xì)胞8h, Dectin-1mRNA表達(dá)明顯下降(P0.05),其蛋白表達(dá)量在阻斷TLR2后真菌菌絲抗原刺激16h減少(P0.05)。阻斷Dectin-1與阻斷TLR2受體,趨化因子(CXCL1、 CXCL8)的表達(dá)量均受到明顯抑制(P0.05)。 結(jié)論：TLR2和Dectin-1共同參與了THCE細(xì)胞對煙曲霉菌菌絲抗原體的識別,兩者在人角膜上皮細(xì)胞抗真菌感染固有免疫階段存在相互作用,均引起下游趨化因子(CXCL1, CXCL8)的產(chǎn)生,介導(dǎo)角膜上皮細(xì)胞的炎癥反應(yīng)。
[Abstract]:Aim: to study the expression of Dectin-1 and TLR2 on the surface of human immortalized corneal epithelial cells (THCE) infected with Aspergillus fumigatus mycelium antigen, the relationship between them in the innate immune stage and their effects on chemokine. Methods: THCE cells were cultured in vitro as blank control group. The experimental group was divided into two groups: fungal stimulation group and pathway blocking group. The cultured THCE cells were infected with Aspergillus fumigatus mycelium antigen to establish the THCE cell model of fungal infection. THCE cells were pretreated with Dectin-1 blocker (laminarin) and TLR2 neutralizing antibody for 30 minutes, and then added Aspergillus fumigatus mycelium antigen stimulation solution. The expression of Dectin-1,TLR2,CXCL1, CXCL8mRNA in THCE cells was detected by real-time fluorescence quantitative PCR (Real time-PCR) at 4 h, 8 h and 16 h in Dectin-1-blocking fungal stimulation group and TLR2-blocking fungal stimulation group. The expression of Dectin-1, TLR2 protein in THCE cells was detected by Western blot at 8 h, 16 h and 24 h. Results: the expression of Dectin-1,TLR2mRNA increased at 4 h and reached a peak at 8 h after stimulation of fungal hyphal antigen in THCE cells. There was significant difference between 8 h group and 4 h group and 16 h group after stimulation of fungal hyphal antigen (P0.05). The expression of Dectin-1,TLR2 protein increased at 8 h after stimulation with fungal hyphae antigen in THCE cells, and reached its peak at 16 h. The increasing trend of chemokine (CXCL1,CXCL8) was consistent with the expression trend of Dectin-1 and TLR2. After Dectin-1 was blocked by laminarin, the expression of TLR2mRNA in THCE cells stimulated by fungal hyphal antigen decreased significantly (P0.05), while the expression of TLR2 protein was decreased 16 hours after Dectin-1 was blocked. The expression also decreased (P0.05). After TLR2 was blocked by TLR2 neutralizing antibody, the expression of Dectin-1mRNA in THCE cells stimulated by fungal hyphal antigen was significantly decreased (P0.05), and the protein expression was decreased at 16h after blocking TLR2 (P0.05). The expression of chemokine (CXCL1, CXCL8) was significantly inhibited by blocking Dectin-1 and TLR2 receptor (P0.05). Conclusion: both TLR2 and Dectin-1 are involved in the recognition of Aspergillus fumigatus mycelium by THCE cells. Both of them interact with each other in the innate immune stage of anti-fungal infection of human corneal epithelial cells, and both lead to the production of downstream chemokine (CXCL1, CXCL8). Mediating inflammation of corneal epithelial cells.
【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】：2014
【分類號】：R772.21

【參考文獻(xiàn)】

中國期刊全文數(shù)據(jù)庫 前5條

1 杜兆東;梁濤;趙桂秋;劉建偉;張麗娜;張振華;;真菌性角膜炎病原菌的流行病學(xué)調(diào)查[J];青島大學(xué)醫(yī)學(xué)院學(xué)報;2007年02期

2 ;The roles of surfactant protein D during Aspergillus fumigatus infection in human corneal epithelial cells[J];International Journal of Ophthalmology(English Edition);2012年01期

3 ;Nucleotide oligomerization domain 2 contributes to the innate immune response in THCE cells stimulated by Aspergillus fumigatus conidia[J];International Journal of Ophthalmology(English Edition);2012年04期

4 Cheng-Ye Che;Cui Li;Ang Gao;Jing Lin;Li-Li Zhang;Qiang Xu;Qian Wang;Gui-Qiu Zhao;;Dectin-1 expression at early period of Aspergillus fumigatus infection in rat's corneal epithelium[J];International Journal of Ophthalmology(English Edition);2013年01期

5 孫旭光,王智群,羅時運,金秀英,張文華,李然,武宇影;眼部真菌感染的病原學(xué)分析[J];中華眼科雜志;2002年07期

中國博士學(xué)位論文全文數(shù)據(jù)庫 前1條

1 任美玉;Toll樣受體4信號通路在棘阿米巴角膜炎發(fā)病過程中的作用研究[D];山東大學(xué);2010年

，

本文編號：2435451