本文關(guān)鍵詞：MBL介導(dǎo)的人角膜上皮細(xì)胞對(duì)煙曲霉菌的固有免疫反應(yīng)　出處：《青島大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甘露糖結(jié)合凝集素 角膜上皮細(xì)胞 煙曲霉菌

【摘要】：目的研究甘露糖結(jié)合凝集素(mannan-binding lectin, MBL)在體外培養(yǎng)的人角膜上皮細(xì)胞(HCECs)中的表達(dá),及用自制煙曲霉菌孢子抗原刺激液刺激后的細(xì)胞中MBL及炎癥細(xì)胞因子的表達(dá)變化,分析MBL在角膜煙曲霉菌感染中的作用。 方法體外培養(yǎng)的HCECs細(xì)胞增殖達(dá)到80%-90%融合時(shí),棄掉培養(yǎng)基,更換為無(wú)血清培養(yǎng)基培養(yǎng)24h后,實(shí)驗(yàn)組加自制的滅活的煙曲霉菌孢子抗原刺激液,分別于刺激的0h、0.5h、1h、2h、4h、6h、8h收取細(xì)胞,逆轉(zhuǎn)錄RT-PCR檢測(cè)正常HCECs及煙曲霉菌孢子抗原刺激液刺激后的HCECs中MBL mRNA的表達(dá)；酶聯(lián)免疫吸附試驗(yàn)(ELISA)方法檢測(cè)正常HCECs及煙曲霉菌孢子抗原刺激液刺激后的HCECs中MBL的濃度及其HCECs培養(yǎng)上清中MBL、NF-κB、IL-1β、TNF-α、IL-6和IL-10的濃度,觀察刺激不同時(shí)間后各組HCECs培養(yǎng)上清中MBL、NF-κB、IL-1β、TNF-α、IL-6和IL-10的含量變化。 結(jié)果正常HCECs有微量MBL mRNA的表達(dá)。煙曲霉菌孢子抗原刺激HCECs后,MBL mRNA表達(dá)逐漸增多,在刺激2h時(shí)達(dá)高峰,之后逐漸下降,與正常HCECs比較差異具有顯著性(P0.05)；ELISA測(cè)得HCECs中MBL在刺激后2h含量達(dá)高峰,與正常HCECs比較差異具有顯著性(P0.05)；HCECs培養(yǎng)上清中MBL表達(dá)量逐漸下降,刺激后各時(shí)間點(diǎn)與刺激0.5h后HCECs比較差異具有顯著性(P0.05)；NF-κB、IL-1β、TNF-α、IL-6和IL-10分別在刺激2h、1h、2h、4h、6h后含量達(dá)高峰,各時(shí)間點(diǎn)與正常HCECs比較差異具有顯著性(P0.05)。 結(jié)論1.體外培養(yǎng)的人角膜上皮細(xì)胞可表達(dá)MBL。2.MBL參與了人角膜上皮細(xì)胞對(duì)煙曲霉菌孢子抗原的識(shí)別。3.MBL可介導(dǎo)人角膜上皮細(xì)胞NF-κB通路的活化并可誘導(dǎo)下游炎性細(xì)胞因子TNF-α、IL-β、IL-6和IL-10的表達(dá),提示MBL可在角膜抵御真菌感染早期固有免疫中起作用。
[Abstract]:Objective to investigate the expression of mannose binding lectin (MBL) in cultured human corneal epithelial cells (HCECs). The expression changes of MBL and inflammatory cytokines in the cells stimulated by self-made Aspergillus fumigatus spore antigen were analyzed to analyze the role of MBL in the infection of Aspergillus fumigatus. Methods when the proliferation of HCECs cells reached 80% -90% in vitro, the culture medium was abandoned and replaced with serum-free medium for 24 hours. In the experimental group, the inactivated aspergillus fumigatus spore antigen was added to the stimulation solution, and the cells were collected at 0. 5 h, 0. 5 h, 1 h, 2 h, 4 h, 4 h, 6 h and 8 h, respectively. The expression of MBL mRNA in normal HCECs and HCECs stimulated by Aspergillus fumigatus spore antigen was detected by reverse transcription RT-PCR. Enzyme linked immunosorbent assay (Elisa) was used to detect the concentration of MBL in normal HCECs and HCECs stimulated by Aspergillus fumigatus spore antigen and MBL in the supernatant of HCECs culture. The concentrations of TNF- 偽 -IL-6 and IL-10 in the supernatant of HCECs cultured in each group were observed after stimulation for different time, and the levels of IL-1 尾 in the supernatant of HCECs were observed. The contents of IL-6 and IL-10 in TNF- 偽 were changed. Results there was a small amount of MBL mRNA expression in normal HCECs, and the expression of mRNA increased gradually after HCECs was stimulated by Aspergillus fumigatus spore antigen, and reached its peak at 2h after stimulation. Then it decreased gradually, and the difference was significant compared with the normal HCECs (P 0.05). The content of MBL in HCECs reached its peak at 2 h after stimulation by ELISA, and the difference was significant compared with that of normal HCECs (P 0.05). The expression of MBL in the supernatant of HCECs decreased gradually, and there was a significant difference in HCECs between the two groups at each time point after stimulation and 0.5 h after stimulation (P 0.05). NF- 魏 B, IL-1 尾, TNF- 偽, IL-6 and IL-10 reached their peak levels at 2h, 1h, 2h, 2h, 4h and 6h, respectively. There was a significant difference in HCECs between each time point and normal group (P 0.05). Conclusion 1.Human corneal epithelial cells can express MBL.2.MBL in vitro and participate in the recognition of Aspergillus fumigatus spore antigen by human corneal epithelial cells. 3. MBL can mediate the expression of MBL.2.MBL in human corneal epithelial cells. Activation of 魏 B pathway can induce down-stream inflammatory cytokine TNF- 偽. The expression of IL-尾-6 and IL-10 suggests that MBL may play a role in the early innate immunity of cornea against fungal infection.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 金伯泉;;NLR:固有免疫模式識(shí)別受體中的一個(gè)重要家族[J];現(xiàn)代免疫學(xué);2006年03期

2 張彩;田志剛;;天然免疫識(shí)別機(jī)制及天然免疫受體的相互調(diào)節(jié)[J];生物化學(xué)與生物物理進(jìn)展;2008年02期

3 劉俊麗;曹誠(chéng);馬清鈞;;MBL調(diào)控MASP激活補(bǔ)體系統(tǒng)[J];生物技術(shù)通訊;2006年04期

4 白玲;趙桂臣;;甘露聚糖結(jié)合凝集素(MBL)的臨床研究進(jìn)展[J];新疆醫(yī)學(xué);2009年03期

5 王s，

本文編號(hào)：1419545