探討塵螨過敏原Der f 2誘發上皮細胞免疫反應特性與促進上皮間質轉換之分子機制

Investigation of the characteristics of epithelial immune response and the molecular mechanism of epithelial-mesenchymal transition induced by House dust mite allergen Der f 2

林暉翰(Hui-Han Lin)

指導教授 ： 高紹軒

中山醫學大學/醫學院/生化微生物免疫研究所/博士(2019年)

https://doi.org/10.6834/CSMU201900039

摘要

本研究以酵母菌Pichia表現與親和層析法純化出重組歐洲室塵螨過敏原蛋白Der f 2 (rDF2)，並以非癌化人類之氣喘上皮細胞BEAS-2B以及肺癌細胞A549為細胞模式，探討rDF2誘發上皮細胞的免疫反應特性與分子機轉，以及rDF2是否可以誘發上皮細胞產生上皮間質轉換。實驗結果顯示，低濃度的rDF2對於BEAS-2B細胞活率並無影響，但是高濃度的rDF2則會造成其存活率下降。進一步發現低濃度的rDF2可以誘發BEAS-2B細胞中IL-6與GM-CSF基因表現並促進其分泌。有趣的是rDF2並不會影響IL-8的表現與分泌。接著探討rDF2誘發IL-6與GM-CSF基因表現的分子機制，發現rDF2會造成p38MAPK與JNK的活化，但是只有p38MAPK在IL-6與GM-CSF基因表現上扮演主要的角色。此外，rDF2也會誘發TSLP與TARC/CCL17的基因表現，而且這兩種基因表現上升主要也與p38MAPK的活化有關。在探討rDF2與上皮間質轉換的部分，實驗結果顯示rDF2 會抑制A549的細胞黏附並促進其細胞脫附。進一步分析發現rDF2 會下調E-cadherin的表現並提升vimentin的表現。此外，rDF2 也會提升Snail與Slug的蛋白質量。這些 結果顯示rDF2 可能會誘發上皮間質轉換。最後，我們進一步建立致敏動物模式探討rDF2對呼吸道上皮組織的影響。經過連續10天的氣管內投予rDF2蛋白，再休息2天後犧牲致敏小鼠，並收集其肺部沖洗液、血清、以及肺部組織進行相關分析。分析結果顯示吸入rDF2確實會造成小鼠肺部組織的E-cadherin 表現量的下降，以及vimentin與-catenin表現量的增加。綜合以上所述，我們認為rDF2 具有誘發呼吸道上皮細胞產生過敏性免疫介導因子IL-6與GM-CSF，以及促進呼吸道發生上皮間質轉換的潛在能力。

關鍵字

塵蟎上皮-間質轉換與呼吸道重塑

並列摘要

The present study was aimed to investigate the immunological characteristics and epithelial-mesenchymal transition (EMT)-inducing ability of a major house dust mite allergen, Def f 2 from Dermatophagoides farinae. We expressed the recombinant Der f 2 (rDF2) using Pichia yeast expression system and purified the rDF2 by the affinity chromatography using specific anti-Der f 2 antibody. Non-tumorigenic human bronchial cell BEAS-2B and lung carcinoma cell A549 were used as cell model. Our findings showed that rDF2 insignificantly influenced the cell viability of BEAS-2B under lower concentrations (<20 g/mL). We then used the lower doses of rDF2 (< 10 g/mL) for the following analyses. We observed that rDF2 was able to induce the mRNA expression of IL-6 and GM-CSF but insignificantly affected the mRNA expression of IL-8. These observations were further demonstrated by using quantitative real-time PCR (qPCR) and ELISA analysis, and the results were consistent. We then investigated the signal cascades that associate with the rDF2-induced expression of IL-6 and GM-CSF. Our results showed that rDF2 induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), and only p38 MAPK activation was involved in the rDF2-induced expression of IL-6 and GM-CSF. Moreover, rDF2 also induced the mRNA expression of thymic stromal lymphopoietin (TSLP) and thymus- and activation-regulated chemokine (TARC). We also demonstrated that p38 MAPK plays an important role in the rDF2-induced TSLP and TARC expression. Epithelial-mesenchymal transition (EMT) has been known to involve in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). Accordingly, we next investigated whether rDF2 induced EMT of airway epithelial cells. Our findings revealed that rDF2 promoted cell dissociation while reducing cell adhesion of lung carcinoma cell A549. Further investigation showed that rDF2 decreased epithelial E-cadherin expression but increased mesenchymal vimentin expression of A549 cell. In addition, rDF2 upregulated the level of Snail and Slug, the EMT inducers in A549 cell. Moreover, we established rDF2-sensitized mice model to investigate the in vivo effects of rDF2 on airway epithelium, and the results revealed that rDF2 downregulated E-cadherin level but upregulated vimentin and b-catenin level in the lung tissue of rDF2-sensitized mice. In conclusion, our findings demonstrate that rDF2 can induce important allergic and inflammatory cytokine expression in airway epithelial cells and may trigger EMT of airway epithelium.

並列關鍵字

Dermatophagoides farinae ； Epithelial-Mesenchymal Transition

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