Caspase-8參與人類嗜伊紅血球陽離子蛋白所引起人類呼吸道支氣管上皮BEAS-2B細胞之凋亡

Eosinophil Cationic Protein-induced Apoptosis is Mediated by Caspase-8 in BEAS-2B Cells

張昆哲(Kun-Che Chang)

指導教授 ： 黎耀基 ； 張大慈

國立清華大學/生命科學院/生物科技研究所/碩士(2008年)

摘要

嗜伊紅血球在人類免疫系統中扮演很重要的角色，當嗜伊紅血球被激發時，許多的細胞激素與具有細胞毒性的嗜伊紅血球陽離子蛋白(eosinophil cationic protein, ECP)將被釋放出來。ECP是一種具有低活性的核醣核酸酶，目前已經被廣泛的使用在氣喘疾病的偵測上，此蛋白是一個很好的氣喘指標。ECP已知會抑制細胞生長，然而它所引起的細胞死亡的機制仍尚未被討論過。在本實驗中有了新的發現，我們發現重組ECP(recombinant ECP, rECP)會導致細胞凋零並且是透過Caspase-8的途徑。首先，我們先確認rECP造成人類支氣管表皮細胞(Beas-2B)的影響，並且測出抑制一半生長的濃度(IC50)為21.03 uM. 我們利用檢視細胞核的萎縮、PARP的裂解、Sub G1與Annexin V來判斷此細胞是經由細胞凋零而死亡。為了找出是透過哪一種細胞凋零途徑，Caspase的廣泛抑制劑(Z-VAD-FMK)與Caspase-8的抑制劑(Z-IETD-FMK)將用來證明其的路徑，更進一步的發現顯示當細胞在rECP 的處理之下，Caspase-8會裂解而被活化。雖然細胞經歷細胞凋零，但是粒線體與內質網所引起的細胞凋零途徑似乎沒有明顯的參予於其中。我們亦發現了當細胞在rECP處理下，會釋放出細胞素－腫瘤壞死因子(TNF-a?於培養液中，因此我們推測Caspase-8 所參予的細胞凋零途徑是被功能如同自主免疫的TNF-?所激發。我們同時也推測各種核醣核酸酶如ECP、EDN與RNase A之細胞毒性是依據核醣核酸本生的等位點而不同。最後，我們發現rECP亦會引起肺表皮癌細胞(A549)產生細胞凋零，此發現將對癌細胞的治療方針上有幫助。

關鍵字

嗜伊紅血球陽離子蛋白 ； 細胞凋零 ； 人類支氣管表皮細胞

並列摘要

The purpose of this study is to investigate the effects of eosinophil cationic protein (ECP) on human bronchial epithelial cells (Beas-2B cells). Eosinophilic granulocytes are important for immune system in human body. Many derived cationic proteins with cytotoxic activities such as ECP and eosinophil derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, has been widely used to be a biomarker for asthma. ECP is reported to inhibit cell viability, but it is never fully verified its mechanism of cell death. In this study, it was the novel discovery that rECP could cause cell apoptosis and involve in caspase-8 pathway in Beas-2B cells. First, we found that rECP could inhibit the cell viability in Beas-2B cells with IC50 of 21.03 uM. The cell death undergoing apoptotic pathway was investigated by chromatin condensation, cleavage of PARP, sub-G1 of cell cycle and Annexin V. To elucidate the specific apoptotic pathway, the general caspase inhibitor (Z-VAD-FMK) and caspase-8 inhibitor (Z-IETD-FMK) were used to prove the caspase and caspase-8 dependent apoptosis. Further, the cleavage of caspase 8 was also detected after rECP treatment. Although apoptosis occurred, both mitochondrial membrane potential (MMP) and endoplasmic reticulum (ER) response were not obviously involved in the rECP-induced apoptosis. We also found that TNF-? was released from Beas-2B cells into medium during rECP-induced apoptosis. Therefore, we hypothesized that caspase-8 mediated the specific apoptosis pathway which was trigger by TNF-a functioned as autocrine. We also assumed that the cytotoxicites of different RNases such as ECP, EDN and RNase A depended on their isoelectric pH (pI). Finally, we found that rECP also induced apoptosis in lung carcinoma cells (A549 cells).

並列關鍵字

Eosinophil cationic protein ； Apoptosis ； Beas-2B cells

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