HMGB1 對於人類肺部纖維母細胞之轉移與增生的影響

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Title:	HMGB1 對於人類肺部纖維母細胞之轉移與增生的影響 Effects of Human High Mobility Group Box 1 on Human Lung Fibroblasts Migration and Proliferation
Authors:	蔡宜儒;Tsai, Yi-Ru
Contributors:	基礎醫學研究所碩士班
Keywords:	high mobility group box 1;人類肺部纖維母細胞;細胞外基質;移行;基質金屬蛋白?-9;human lung fibroblast;extracellular matrix;migration;matrix metalloproteinase-9
Date:	2012-03-01
Issue Date:	2012-08-31 16:33:47 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	背景　　呼吸道重塑為慢性氣喘中很重要的病理現象之一，而在呼吸道重塑下造成的下上皮纖維化機制目前尚未被完整發現，在下上皮纖維化中纖維母細胞因為細胞外基質像是膠原蛋白，fibronectins與Laminins的沉積； High mobility group box 1 (HMGB1)在早期被發現是涉及DNA與染色體調節與轉錄的細胞核蛋白。在近年被發現到HMGB1亦屬於一種細胞激素，其可以活化免疫細胞如單核球造成免疫反應的擴大。研究動機　　在本研究中，我們想要去調查HMGB1在人類肺部纖維母細胞WI-38在細胞移行及細胞增生的影響。方法與結果　　在利用不同濃度的HMGB1去刺激WI-38 24小時後以MTT assay及BrdU cell proliferation assay我們並沒有發現到明顯的細胞毒殺反應及增生反應；結論：而在傷口癒合實驗中，我們發現到HMGB1在濃度變化刺激下發現了WI-38的細胞移行量呈現濃度梯度變化。我們利用不同蛋白質激酶抑制劑觀察到MAP kinases 的ERK, JNK, p38及NF-B受到抑制後HMGB1刺激WI-38的細胞移行程度被抑制的結果，在西方墨點法發現了MAP kinases在HMGB1最高作用濃度 100ng/ml刺激後活化的結果，而在細胞核蛋白中看見了p65的表現量增加。我們在明膠電泳中觀察到了HMGB1在濃度變化刺激WI-38中MMP-9的酵素活性增加，在即時聚合酶連鎖反應及西方墨點法都看到了MMP-9的mRNA及蛋白質表現量的增加；此外我們也發現了PI3 kinase/Akt受到抑制的情況下HMGB1刺激WI-38的細胞移行程度有抑制的現象，及我們看到在細胞核內β-catenin在一小時後表現量增加，接著我們發現到了HMGB1最高濃度刺激下WI-38的細胞表面受器TLR2, TLR4及RAGE的mRNA表現量皆有上升的趨勢。結論　　我們發現到HMGB1刺激人類肺部纖維母細胞WI-38的細胞移行是透過MMP-9活化及PI3 kinase/Akt、MAPKs與β-catenin的訊息傳遞路徑。 Background Airway remodeling is an important characteristic in severe asthma. Subepithelial fibrosis is one of the features of airway remodeling and the mechanism is still unclear. Fibroblasts is the main resource of extracellular matrix including collagens, fibronectin and laminins which cause airway remodeling. High mobility group box 1(HMGB1) is a nuclear protein that involves the interactions with DNA and chromatin regulation and transcription. HMGB1 is also a cytokine that can activate monocytes neutrophils involving in inflammation. Statement of purpose In this study, we want to investigate the role of HMGB1 on cell migration on human fibroblast cell line –WI-38. Methods and results After treated with HMGB1 1, 10, 100 ng/ml for 24hrs, we did not found obviously cytoxicity and proliferation effect on WI-38 cells by MTT and BrdU incorporation assay, respectively. We found that HMGB1 induced cell migration on WI38 by dose-dependent manner. Using zymography to investigate matrix metalloproteinase (MMP) activity, we found that HMGB1 induced MMP9 activation but not MMP2. Using specific inhibitor of protein kinases, we observed that extracellular signal related kinase (ERK) inhibitor- PD98059(10μM), c-Jun N-terminal kinase (JNK) inhibitor - SB203580(5μM), p38 mitogen activated protein kinase (p38) inhibitor-SP600125(5μM), phosphoinositide 3-kinase (PI3 kinase) inhibitor- LY294002(5μM), and NF-κB inhibitor-Bay117082(10μM) all showed the inhibition effect on HMGB1 induced WI-38 cells migration. We also found that HMGB1 induced ERK, JNK and p38 activated and increased the nucleus p65 and β-catenin expression by time-dependently through Western blotting. Last we observed the WI-38 increased RAGE, toll-like receptor 2 (TLR2) and 4 (TLR4) mRNA and protein expression on Western blotting and Real-time PCR through HMGB1 treated. Conclusion We found that HMGB1-induced cell migration on WI-38 through MMP9 activation, PI3 kinas/AKT, MAPKs, and β-catenin signaling.
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