多重轉譯體分析微角塵蟎在上皮細胞內誘發的發炎反應

Multiplex transcriptome profile analysis for the crude allergens from Dermatophagoides microceras involved the inflammatory responses in the epithelium cell treatments

馮天浩(Tien-Hao Feng)

指導教授 ： 劉玉凡

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2021年)

https://doi.org/10.6834/csmu202100054

摘要

[Background]
家塵蟎 (House dust mites, HDMs) 是誘發氣喘的重要過敏原。台灣存在有3種常見的HDMs品種，包括: 微角塵蟎 (Dermatophagoides microceras, Der m)，屋塵蟎 (Dermatophagoides pteronyssinus, Der p)與粉塵蟎 (Dermatophagoides farinaea, Der f)。前期的研究顯示台灣中部地區的462位特異反應性 (atopy) 過敏幼童 (2-16歲)，有高達 80% 以上病患特別對微角塵蟎，具有致敏作用 (sensitization)，因此本研究專題將探討微角塵蟎致敏原，刺激人類上皮細胞的致敏機制，透過致敏機制的分析，藉此建立呼吸道修補 (Airway remodeling) 的療效評估。
[Methods]
採用多重轉譯體分析 (Multiplex transcriptional profile analysis)，分別進行Der p、Der f與Der m粗萃取液處理下，探討誘發人支氣管上皮細胞 (BEAS-2B) 的全轉錄基因圖譜。採取不同的時間 ( 4和24小時 ) 及不同處理條件下，共十六組樣品的RNA-seq資料，包括: 對照組、家塵蟎處理組、纖維蛋白原組 (fibrinogen, 重要的凝血因子 )、Der m 2組 ( 主要致敏原 )、合成皮質類固醇藥物處理組(Dexamethasone, Dexa)。其中分析三種粗萃過敏原蛋白粗萃物 (Der m、Der p和Der f)、Dexa + Der m治療組，及 Der m 作用於凝血因子，不同時間點的水解產物 (fibrinogen cleavage products, FCPs) 分別加入主要致敏原 Der m 2。採用次世代測序技術，使用Hisat2，featureCounts和DESeq等全轉錄基因的分析流程，並應用基因富集分析 (Gene Set Enrichment Analysis, GSEA) 配合京都基因與基因組百科全書 (Kyoto Encyclopedia of Genes and Genomes, KEGG) 與基因本體 (Gene Ontology, GO)資料庫的註解。透用Real-time PCR及Western blot分別針對目標基因表達與蛋白質層次的再驗證工作。
[Results]
使用三種粗萃過敏原蛋白提取物 (Der m、Der p和Der f) 處理的4小時組中的細胞因子 (IL-6和IL-8) 基因表現量高於24小時組的5-8倍，顯示致敏原誘發的發炎反應在4小時達到高點；首先應用GSEA於KEGG發現三種致敏原所誘發的共同富集路徑分別為Toll-like受體信號轉導通路 (P-value=0.0001)、Nod-like受體信號通路 (P-value =0.0007)、和Jak-Stat信號通路途徑 (P-value =0.0004)，並且利用Real-time PCR分別在驗證參與Toll-like受體信號轉導路徑的作用基因IL-6, IL-8, NFκB1, PIK3R1, TBK1與RNA-Seq分析的結果一致，並利用Western blot再探討蛋白質的作用機制，核轉錄因子NF-κB會在刺激後30min由細胞質進入細胞核，並啟動下游基因的調控。
其次分析Der m處理下的特有富集基因，是與第一型糖尿病的調控有關(P-value =0.0001)，其中上調基因有IL-12A，IL-1與HLA-II，並透過Real-time PCR再驗證了促T細胞增殖因子IL-12A為Der m誘發致敏作用的特有基因。
接著在Dexa + Der M治療組中，利用GO資料庫的4D scatter plot，顯示藥物治療所影響的特有基因，經富集後有纖毛組成(Cilium Organization, P-value =8.29x10-8)、運動纖毛 (Motile Cilia, P-value =1.05x10-7) 及微管細胞骨架 (Microtubule Cytoskeleton, P-value =3.41x10-6) 等分子功能。
最後針對主要過敏原作用的分析中，顯示1分鐘 FCPs加入 Der m 2組別中所誘發的發炎反應具協同性的加乘作用 (synergistically effect)，而利用富集分析的結果，顯示對細胞連結 (Adherens junction, (P-value =0.0003) 調控基因具有上調作用，說明FCPs透過細胞結合素(Nectins) 傳遞訊號，來重組肌動蛋白及細胞骨架改變細胞通透性來調節Adherens junction的形成，改變細胞間黏附的作用。
[Conclusion]
結果顯示，三種家塵蟎的過敏原蛋白萃取物，誘發上皮細胞的致敏機制是共同經由Toll-like受體信號轉導通路，來引起核轉錄因子NF-κB進入細胞核使促發炎細胞因子的上調作用。另一方面暴露於微角塵蟎的環境下，相較其他兩種家塵蟎的致敏作用更容易導致β-cell失去功能死亡而引發第一型糖尿病。在藥物治療組別中發現Dexa有調節纖毛組成、及微管細胞組成等分子功能。最後，經微角塵蟎處理後的 FCPs會導致Der m 2經由細胞結合素 (Nectins)傳遞訊號來調節Adherens junction的形成及改變細胞間的黏附作用，藉此增加主要過敏原Der m 2所誘發的發炎反應協同性的加乘作用。

關鍵字

家塵螨 ； 微角塵螨 ； 次世代定序 ； 全轉錄體分析 ； 氣喘 ； 第一型糖尿病 ； 凝血纖維蛋白

並列摘要

[Background]
House dust mites (HDMs) are commonly known allergens to cause asthma in the subtropical area. There are three local breed HDMs in Taiwan including Dermatophagoides microceras (Der m), Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinaea (Der f). From the previous studies, more than 80% allergies sensitization rate exposure by the Der M allergens for children in the central Taiwan. Explore the sensitization mechanism of HDMs allergens to stimulate human epithelial cells, and evaluate the efficacy of airway remodeling from allergic symptoms
[Methods]
We proposed the multiplex transcriptional profile analysis approach to treatment of Der p, Der f and Der m crude extracts, explore the sensitization mechanism of human bronchial epithelial cells (BEAS-2B) we constructed different time (4 and 24 hours) and different processing conditions, a total of 16 sets of RNA-seq data including a control group, HDMs test groups, fibrinogen (an important blood coagulation factor produced), Der m 2 group (the major allergen), drug treatment group (Dexamethasone, Dexa, a synthetic corticosteroid to current treat allergies, asthma, rheumatic diseases, and skin diseases), three crude allergens protein extracts (such as Der m, Der p and Der f), in addition to Der m crude with fibrinogen, process different time points to obtain fibrinogen cleavage products (FCPs) and adding with Der m 2. we offered the next-generation sequencing (NGS) technology, and Hisat2, featureCounts and DESeq public packages for RNA-seq analysis. Gene Set Enrichment Analysis (GSEA) in Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database. Finally, Real-time PCR and Western blot were used to verify the results of target gene expression and protein level.
[Results]
The gene expression of cytokines (IL-6 and IL-8) in the 4-hour group treated with three crude allergen protein extracts (Der m, Der p and Der f) was 5-8 folds change higher than that in the 24-hour group, showing that the allergen-induced inflammatory response reached a high point in 4 hours. First, GSEA was used in KEGG to find that the common enrichment pathways induced by the three allergens were Toll-like receptor signaling pathway (P=0.0001): Nod-like receptor signaling pathway (P=0.0007) and Jak-Stat receptor signaling pathway (P=0.0004). In addition, Real-time PCR was used to verify that the genes IL-6, IL-8, NFκB1, PIK3R1, and TBK1 involved in the signal transduction pathway of Toll-like receptors are consistent with the results of RNA-Seq analysis, and the results were verified by Western blot. Protein mechanism, nuclear transcription factor NF-κB enters the nucleus from the cytoplasm, thereby initiating the regulation of downstream genes. Secondly, analyze the unique enriched genes under Der m treatment, which are related to the regulation of type 1 diabetes (P=0.0001). Among them, the up-regulated genes are IL-12A, IL-1 and HLA-II, and they are reproduced by Real-time PCR. It is verified that IL-12A (a T cell proliferation factor) is a unique gene for Der m to induce sensitization. Third, in the Dexa + Der m treatment group, the 4D scatter plot of the GO database was used to show the unique functions affected by the ineffective drug treatment. After gene enrichment analysis, there was Cilium Organization(P=8.29x10-8), Motile Cilia(P=1.05x10-7), and Microtubule Cytoskeleton(P=3.41x10-6) and other molecular functions. Finally, in the analysis of the effects of major allergens, it was shown that the inflammatory response induced by the addition of FCPs in the Der m 2 group for 1 minute had a synergistically effect. The gene enrichment analysis showed that it has an up-regulation effect on cell connection regulatory genes (Adherens junction, P=0.04). Its show that FCPs transmit Nectin signals through Nectins to reorganize actin and cytoskeleton to change cell permeability, regulate the formation of Adherens junction, and change the role of cell adhesion.
[Conclusion]
The results show that the allergen protein extracts of the three HDMs induce the sensitization mechanism of epithelial cells through the Toll-like receptor to cause the nuclear transcription factor NF-κB to enter the nucleus and promote the inflammation of cytokines upregulation. On the other hand, exposure to the environment of Der m is more likely to cause β-cell loss of function and death than Der p and Der f, leading to type 1 diabetes. In the drug treatment group, it was found that the Dexa has regulate Cilium Organization, Microtubule Cytoskeleton and other molecular functions. Finally, the FCPs treated by Der m will cause Der m 2 to transmit signals through Nectin to regulate the formation of Adherens junction and change the adhesion between cells, thereby increasing the inflammation response induced by the major allergen Der m 2, it had a synergistically effect.

並列關鍵字

house dust mite ； Next-Generation Sequencing ； Der m ； Rna-Seq ； Asthma ； Type I diabetes ； Fibrinogen

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