歐室塵蟎過敏原Der p 2 誘發人類支氣管上皮細胞Beas-2B細胞凋亡機制之研究

Investigating apoptosis of human bronchial epithelial cell Beas-2B induced by a major allergen Der p 2

洪玉全(Yu-Chuan Hong)

指導教授 ： 高紹軒

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2012年)

https://doi.org/10.6834/CSMU.2012.00143

摘要

細胞凋亡 (Apoptosis)是一種細胞正常程序性的死亡。在生理上，細胞凋亡可以剔除不需要的細胞。然而，在肺部細胞凋亡的作用更是可以汰換掉不要的細胞，填補新的上皮細胞已保持上皮細胞的完整。然而，當上皮細胞遭受到壓力，像ROS、NO、Cytokine或病毒的感染等，一旦反覆持續刺激，就會使上皮細胞導致過渡的細胞凋亡，新生填補的作用來不及去平衡過渡的細胞凋亡，肺部上皮細胞開始不完整，最後導致肺部纖維化或呼吸道重塑（airway remodeling）。這種現象也與許多肺部的疾病有關，如：氣喘、急性肺損傷、急性呼吸窘迫症、慢性肺阻塞等。Der p 2 (DP2) 是歐室塵蟎（Dermatophagoides pteronyssinus, DP）的主要過敏原，雖不具蛋白水解酶的特性，但仍會引起過敏性氣喘。過去研究證實DP2會透過TLR4 (toll-like receptor 4)去誘發發炎反應，促使轉錄因子NF-κB與MAPK pathway ( mitogen-activated protein kinase) 活化，導致支氣管上皮細胞釋放細胞激素（cytokine）與趨化激素 (chemokine)。另外，研究也證實DP2能使呼吸道上皮細胞間的tight junction protein 如 occludin、ZO-1表現量改變，讓呼吸道上皮層通透性增加，使DP2更易穿越上皮細胞屏障。由於目前DP2如何導致呼吸道重塑（airway remodeling）的機制仍不是很清楚。在本實驗中，特別針對DP2如何引起呼吸道上皮細胞走向細胞凋亡，讓上皮屏障瓦解作為研究目標。實驗發現DP2對於人類呼吸道上皮細胞由MTT assay結果顯示，DP2會使Beas-2B細胞死亡，再利用TUNEL assay與Flow cytometry更進一步確定死亡的細胞是由細胞凋亡所造成的。另外DP2會活化MAPK pathyway，使得Bcl-2蛋白表現量減少，Bax蛋白表現量增加。DP2也會去增加ROS的產生，誘發細胞凋亡內源路徑的活化，促使粒腺體釋放cytochrome c活化caspase-9。另外，更進一步由RT-PCR發現DP2會使BEAS-2B 中Fas、FasL、FADD mRNA表現量增加。過去研究指出DP2會活化Akt使GSK3β磷酸化，讓GSK3β失去活性（inactive）。然而實驗發現DP2會讓GSK3β下游的抗細胞凋亡蛋白c-FLIP表現量下降，c-FLIP能抑制caspase-8的活化，DP2使c-FLIP表現量下降更進一步的去活化細胞凋亡外在路徑。上述實驗結果顯示歐室塵蟎過敏原DP2有誘發上皮細胞細胞凋亡的能力，一旦反覆持續的刺激上皮細胞，會使上皮屏障受損，更容易造成呼吸道重塑（airway remodeling）。

關鍵字

歐室塵蟎過敏原

並列摘要

Excessive apoptosis in airway has been reported to trigger airway remodeling and inhibit airway epithelium repair which highly associates with development of asthma and chronic obstructive pulmonary disease (COPD). Der p 2 (DP2) is a major allergen derived from house dust mites, Dermatophagoides pteronyssinus and is a major cause of allergic asthma. Although DP2 has been reported to trigger airway hypersensitivity and inflammatory responses through Th2 and Th1 signaling, effects of DP2 on airway remodeling remain unclear. Thus we aimed to investigate effects of DP2 on airway remodeling with emphasis on apoptosis of epithelial cells and the consequent mechanisms. Our findings showed that DP2 inhibited cell viability and induced apoptosis of human bronchial epithelial cell Beas-2B cells by using MTT assay and TUNEL assay. Moreover, DP2 could induce FAS, FasL and FADD mRNA expression and promote c-FLIP, an anti-apoptotic protein degradation through Akt-dependent inhibition of GSK3β. Further investigation revealed that DP2 activated caspase-3, caspase-8 and caspase-9, which may attribute to increased level of and cytosolic cytochrome c. In parallel, DP2 significantly decreased level of anti-apoptotic protein, Bcl-2 and increased level of pro-apoptotic protein, Bax. Taken together, these findings indicate that DP2 diminished cell viability and trigger apoptosis of Beas-2B cells, which may attribute to activation of intrinsic and extrinsic pathway.

並列關鍵字

Der p 2

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