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標題: | 兩種腸道上皮細胞株在常態生長與細菌內毒素刺激下NF kappa B活化及細胞凋亡現象關聯性之探討 Differential Responses of NF kappa B Activation and Cell Apoptosis in Constitutional Growth and upon LPS Challenge in Two Intestinal Epithelial Cell Lines |
作者: | Yin-Chung Au 區曣中 |
指導教授: | 余佳慧 |
關鍵字: | 腸道上皮細胞,細菌內毒素,NFkappa B,細胞凋亡,TLR4,CD14, intestinal epithelial cells,LPS,NFκB,apoptosis,TLR4,CD14, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 單層的腸道上皮細胞不但具有消化、吸收、分泌的功能,更是面對腔管內有害刺激之第一道防線。腸道上皮細胞凋亡過多會導致防禦功能的喪失,若凋亡不足則與癌化有密切關係。過往利用腸道上皮細胞株之研究發現細菌內毒素(lipopolysaccharide, LPS)在腸腔面的刺激會增加細胞凋亡的現象並呈現時間與濃度之依賴性,另一方面亦有文獻指出LPS能活化細胞中促發炎反應之轉錄因子nuclear factor kappa B (NFκB)。而兩種反應之間的關聯性不明。至於NFκB之於腸道上皮細胞是促使其凋亡抑或存活,迄今尚未定論。本研究的第一個目標即為檢驗NFκB在兩種人類腸道上皮細胞株T84及Caco-2中調控細胞凋亡之角色;其次,欲比較兩種細胞株在遭受LPS刺激下,細胞凋亡與NFκB活化現象之發生,並探討不同的LPS受體複合物分子在各別細胞株之表現。
在第一部份研究,使用NFκB 抑制劑pyrrolidinedithiocarbamate (PDTC) (0-50μM)處理腸道癌上皮細胞株T84與Caco-2, 以檢測NFκB活化與細胞凋亡之關聯性。首先,PDTC處理細胞後,分離萃取其胞核蛋白進行NFκB p65次單元之核轉移偵測與p50次單元DNA結合能力之定量。西方墨點實驗結果發現兩者細胞株皆有常態性p65之核表現,而PDTC以時間依賴形式降低p65之核轉移。此外,PDTC處理使p50對DNA結合之活性降低。以Hoechst、TUNEL染色法與免疫酵素分析法偵測發現,PDTC以濃度依賴形式引發核濃縮、DNA斷裂等細胞凋亡表徵,以及細胞剝落。綜上,PDTC降低T84與Caco-2細胞株中之NFκB活化程度,並引起細胞凋亡,顯示常態性活化之NFκB在腸道上皮癌化細胞中具有促進細胞存活之功能。 研究之第二部分以大腸桿菌LPS (血清型O26:B6) (0-50 ug/ml) 經由腸腔面刺激T84與Caco-2細胞,於15和60分鐘後分析NFκB p65之核轉移量,並於24小時後檢視細胞凋亡之程度。實驗結果發現在Caco-2 細胞中,LPS刺激以濃度依賴形式導致細胞凋亡與剝落,但不增加NFκB之核轉移。而T84細胞則反之,LPS刺激會引起大量NFκB之活化,卻無細胞凋亡的現象。此外,免疫螢光染色顯示T84和Caco-2表現不同的LPS受體複合物分子。T84細胞表現Toll-like receptor (TLR)-4,但無CD14。Caco-2細胞上則只表現CD14,而無TLR4。過去許多文獻指出LPS處理引發之NFκB活化主要由TLR4所媒介作用,本研究利用抗體遏阻實驗發現LPS在Caco-2引起之細胞凋亡與CD14有關,而不經由TLR4。綜言之,兩種細胞株對LPS刺激之相異反應可能歸因於不同的LPS受體複合分子之表現。 The intestinal epithelial monolayer not only serves for digestive, absorptive and secretive functions, but also acts as the first line of defense against luminal noxious stimuli. Excessive epithelial apoptosis may compromise this barrier function, whereas delayed apoptosis is in part involved in intestinal carcinogenesis. Previous studies have documented that luminal exposure to bacterial lipopolysaccharide (LPS) augments enterocytic cell death in dose- and time-dependent manner, whereas others reported the activation of a pro-inflammatory transcription factor, i.e. nuclear factor kappa B (NFκB) upon LPS challenge, in intestinal epithelial cell lines. The relationship between the two phenomena is still not understood. It remains elusive whether NFκB is pro- or anti-apoptotic in enterocytes. The first aim of the study was to examine the role of NFκB in modulating apoptosis in two intestinal epithelial cell lines, i.e. T84 and Caco-2, in constitutional growth. The second aim was to characterize the phenomena of enhanced cell apoptosis and NFκB activation induced by LPS in these two cell lines. In the first part of the study, colonic carcinoma T84 and Caco-2 cell lines were treated with an NFκB inhibitor, pyrrolidinedithiocarbamate (PDTC), at 0-50μM for various time points to examine its effect on cell apoptosis. The effect of PDTC in modulating nuclear translocation and DNA binding activity of NFκB (heterodimer of p50 and p65 subunits) was first verified. Western blotting results revealed constitutive nuclear translocation of p65 in the two cell lines, which is reduced by PDTC in a time-dependent fashion. The DNA binding activity of p50 was also decreased in cells treated with PDTC. Our results showed that PDTC dose-dependently induced apoptosis, i.e. nuclear condensation and DNA fragmentation, in both T84 and Caco-2 cell as evidenced by Hoechst and TUNEL staining, as well as Cell death ELISA. These results indicated that constitutive NFκB activation plays a pro-survival role in intestinal epithelial carcinoma cell lines. In the second part of the study, confluent T84 and Caco-2 cells were luminally exposed to bacterial LPS (from E. coli, O26:B6 serotype) at 0-50 ug/ml. The level of NFκB activation was examined at 15 and 60 mins post challenge and cell death was examined at 24 hrs after LPS exposure. LPS dose-dependently triggered cell apoptosis, but failed to enhance nuclear translocation of NFκB in Caco-2 cells. In contrast, LPS exposure increased NFκB activation in T84 cells, whereas the level of apoptosis did not change compared to untreated controls. Moreover, immunofluorescent staining showed differential expression of LPS receptor components on the two cell lines. In line with previous reports, the expression of Toll-like receptor (TLR)-4 was detected in T84, but none was seen in Caco-2 cells. On the other hand, expression of CD14 was observed on Caco-2, but not T84 cells. Previous studies have demonstrated that TLR-4 is the main receptor component responsible for inducing downstream NFκB activation. Our current study using functional blocking antibodies revealed that CD14 is involved in the induction of apoptosis by LPS challenge in Caco-2 cells. Taken together, the distinct response in T84 and Caco-2 cells upon LPS challenge may be attributed to the differential expression of LPS receptor components. |
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