利用小鼠動物模式探討由 high-mobility group box 1 (HMGB 1) 在慢性氣喘的角色

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Title:	利用小鼠動物模式探討由 high-mobility group box 1 (HMGB 1) 在慢性氣喘的角色 Blockage of high-mobiltiy group box 1 (HMGB 1) inhibited airway inflammation and remodeling in a murine model of chronic asthma
Authors:	賴郁婷;Lai, Ting-Yu
Contributors:	免疫學研究所碩士班
Keywords:	HMGB1;呼吸道發炎;呼吸道重塑
Date:	2012-07-31
Issue Date:	2012-08-31 16:31:16 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	損害相關分子模式(Damage-associated molecular patterns, DAMPs)，這群分子本來存在於細胞內，但在發炎或是細胞修復的時候就會釋放到細胞外的環境中。而高遷移率族蛋白1(high mobility group box 1, HMGB1)就是屬於這群分子中的一員，HMGB1在微環境中扮演兩種角色，一是DNA的結合蛋白，二是細胞激素。因為HMGB1會從壞死細胞(necrotic cell)以及活化的免疫細胞中從細胞核釋放到細胞外的環境，結合上特定的受體後就會啟動下游的免疫反應，這樣的角色就有似於細胞激素的功能。 HMGB1在許多研究中被指出其參與在急性和慢性肺部疾病中，且與組織的損傷與重塑都有關，所以在我的研究中想要探討到HMGB1在慢性氣喘中扮演的角色。以下是我們所建立的動物模式，小鼠們利用腹腔注射的方式在第0、10、20天時給予雞卵蛋白(ovalbumin)，接著在接下來的四周中，每周有五次的時間，將小鼠暴露在 5% 10 ml雞卵蛋白的環境，同時在這五天的時間裡的第1、3、5天利用氣管滴入的方式給予30μg/mouse中和性HMGB1抗體。第55天犧牲之後我們利用免疫組織化學(immunechistochemistry)染色的結果可以知道我們所使用的中和性HMGB1抗體的確可以專一性的抑制HMGB1蛋白質的表現。另外在OVA-immunized 組別的小鼠(positive control)，其主要浸潤到呼吸道的發炎細胞是以嗜中性白血球(neutrophil)為主，在組織切片中 positive control以及 isotype control 組別小鼠其膠原蛋白(collagen)沉積在呼吸道周邊程度以及呼吸道所產生黏液(mucus)的量都相較negative control組別的小鼠程度嚴重，而在給予中和性HMGB1抗體組別的小鼠其產生的膠原蛋白以及黏液都有降低的趨勢。另外，利用即時定量聚合?○s鎖反應(real-time polymerase chain reaction, RT-PCR)測定RAGE、TLR4、TLR2、膠原蛋白, MMP-9, MMP-2, pro-inflammatory cytokines- IL-17 A、IL-17 F和INF-γ這幾個蛋白的mRNA表現量也在positive control組別的小鼠表現量上升而在中和性 HMGB 1 抗體組別小鼠中下降。而利用酵素免疫連結吸附試驗(enzyme linked immunosorbent assay) 來測定小鼠肺部灌洗液以及肺組織均質液的細胞激素：IFN-γ、IL-4、IL-13、IL-17、TNF-α以及細胞趨化素KC、eotaxin，在 positive control 組別小鼠濃度有升高，而在給予中和性 HMGB 1抗體組別小鼠則有被抑制的現象。以流式細胞儀 (flow cytometry)觀察細胞內染色(intracellular stain)的結果來討論由HMGB1所參與的慢性氣喘，其中主要的輔助性T細胞subtype以Th17為主。從研究結果中可以得知 HMGB 1 參與的慢性氣喘其主要浸潤到呼吸道的免疫細胞以嗜中性白血球為主，而輔助性 T 細胞則是以 Th 17 為主。在中和掉 HMGB 1 蛋白之後呼吸道發炎所產生的細胞激素以及細胞趨化素都有被抑制，而與呼吸道重塑有關的膠原蛋白沉積與黏液的產生都有降低其程度以及與呼吸道重塑相關的指標性蛋白也有被抑制的現象。 Damage-associated molecular patterns (DAMPs) comprise intracellular molecules characterized by the ability to reach the extracellular environment, and they involved in inflammation and cell repair. The high-mobility group box 1 (HMGB1) is a prototypic DAMP. It has two roles in microenvironment, one is DNA-binding protein and the other is cytokine. HMGB1 release during cell necrosis, and it’s also release by activated immune cell. Increasing HMGB1 level has been found in acute and chronic lung inflammatory conditions characterized by tissue damage and remodeling. In our study, we focus on the role of HMGB1 in chronic asthma. In a animal model of chronic asthma, mice were intraperitoneal injected with ovalbumin on day 0, 10, 20 , and exposured with 5% ovalbumin five times a week for 4 weeks. After treated HMGB1 neutralization antibody 30μg/mouse by intratracheal injection three times a week for four weeks in OVA-immunized mice, HMGB1 treated mice showed decreasing of OVA-induced lung neutrophilia, mucus formation and collegen deposition in lung tissues by histology study and decreased lung HMGB1, HMGB1 receptors-RAGE, TLR-2 and TLR-4, collagen, MMP-9, MMP-2, pro-inflammatory cytokines- IL-17A, IL-17F, and INF-γ mRNA expression by quantitative PCR. We can observe that treat HMGB1 neutralized antibody can suppress the secretion of cytokine like IL-4, IL-17, TNF-α, IFN-γ, KC, eotaxin in BALF and lung homogenates. Using intracellular stain, we investigate Th 17 cell is the major T helper cell infiltration in chronic asthma model. Using masson’s trichrome stain we observe blocking HMGB1 can improve airway remodeling degree. In this study, we found that inhibition of HMGB1 decreased airway inflammation and remodeling in a murine model of chronic asthma. Therefore, from our study, HMGB1 might take an important part in pathological mechanisms of chroic asthma.
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