臺灣博碩士論文加值系統

腸道屏障由單層上皮細胞及細胞之間的緊密連結 (tight junctions) 組成，可防止腸腔面細菌及抗原產物進入漿膜面循環至全身。緊密連結蛋白包括occludin、claudin及zonula occluden (ZO) 等。臨床上，有報導指出在細菌性腸炎及發炎性腸疾中均有腸道屏障缺損之現象。

根據先前研究，利用高劑量革蘭氏陰性菌內毒素刺激腸道上皮細胞Caco-2之腸腔面，會引起細胞凋亡及增加細胞間通透性 (Yu et al., 2005 & 2006)。內毒素之受體複合物包括CD14、TLR-4及MD-2等在單核球及其分化細胞上可發現，然而，在腸道上皮細胞有不同的表現形式。因此，本篇研究目的為1) 探討內毒素引起緊密連結構造缺損是否為細胞凋亡途徑之下游反應，2) 檢測內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞的機制中，內毒素受體複合分子如CD14及TLR-4所扮演的角色，3) 尋找內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞所參與之細胞訊息傳遞途徑為何。

實驗中將人類大腸直腸癌上皮細胞株Caco-2細胞培養達全滿，以50 μg/mL之大腸桿菌內毒素刺激腸腔面24小時後，測量細胞凋亡程度。利用Hoechst及TUNEL染色法觀察發現內毒素刺激會引起染色質濃縮及DNA片段化現象。利用流式細胞技術以DiOC6(3) 染劑亦可偵測到內毒素刺激引起粒線體膜電位下降。預先投予caspase-3抑制劑 (z-DEVD-FMK) 能阻斷內毒素刺激所引起之上皮細胞凋亡。Caco-2之控制組細胞其緊密連結蛋白如：ZO-1、occludin在免疫螢光染色下呈現鐵絲網圍欄狀。而內毒素刺激會導致緊密連結蛋白結構破壞，預先投予caspase-3抑制劑則可阻斷此現象。除此之外，預先投予polymyxin B可阻斷內毒素刺激引起之細胞凋亡及緊密連結蛋白結構改變，表示細胞凋亡反應乃因內毒素之脂質A作用所造成，而非由於其他細菌組成成份污染所致。

本實驗更進一步瞭解受體複合物如：CD14及TLR-4在內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞中所參與之機制。以免疫螢光染色法及流式細胞技術可偵測到Caco-2上皮細胞中表現CD14，由共軛焦XZ連續堆疊影像顯示CD14表現在細胞頂面膜。利用三種TLR-4單株抗體 (clones HTA-125、76B357.1及lG11) 藉由免疫螢光染色法證實Caco-2細胞並無TLR-4表現，而陽性控制組T84細胞則可見TLR-4染色。在內毒素刺激下，CD14及TLR-4表現在上述細胞中並無明顯改變。此外，西方墨點法結果證實CD14 (分子量53∼55 kDa) 表現於Caco-2細胞。最後，結果顯示預先投予中和性抗CD14之抗體 (clone 134620) 能減少內毒素刺激引起之細胞凋亡及緊密連結蛋白結構之破壞，且呈現濃度依賴形式，然而，預先投予中和性抗TLR-4之抗體 (clone HTA-125) 則無作用。

過去針對單核球/巨噬細胞之研究顯示，內毒素刺激會經由TLR-4引起MyD88依賴性途徑，使得轉錄因子NF-κB發生核轉移及MAPK磷酸化現象。為進一步確認或排除已知的TLR-4媒介訊息參與之可能性，利用西方墨點法檢測NF-κB、 MyD88及MAPK在Caco-2細胞之表現。結果顯示內毒素刺激Caco-2細胞不會發生NF-κB核轉移、MyD88總量改變及JNK、p38之磷酸化現象，而陽性控制組THP-1細胞則可見。

綜合上述，這些結果顯示內毒素引起腸道上皮細胞Caco-2細胞凋亡會導致緊密連結蛋白結構之破壞及屏障缺損，且這些病理現象是經由CD14而非TLR-4所參與的。

Intestinal barrier is composed of a single layer of epithelial cells connected by tight junctions that prevents luminal bacteria and antigenic products from gaining access into the body proper. Tight junctional proteins include occludin, claudin, and zonula occluden (ZO) etc. Clinical manifestations of intestinal barrier defects were reported in bacterial enteritis and inflammatory bowel disease.

Previous studies utilizing intestinal epithelial Caco-2 cell culture have demonstrated that luminal exposure to high dose of Gram (-) bacterial LPS induced cell apoptosis and increased paracellular permeability (Yu et al., 2005 & 2006). LPS receptor complexes including CD14, TLR-4 and MD-2 were identified on monocytic lineage cells. However, distinct expression patterns on intestinal epithelial cells have been reported. The aim of the current study was to 1) examine whether LPS-induced tight junctional destruction is downstream of apoptotic pathway, 2) investigate the role of LPS receptor components, i.e. CD14 and TLR-4, in the mechanism of LPS-induced intestinal epithelial apoptosis and tight junctional destruction, 3) explore the cell signaling pathway involved in the LPS-induced intestinal epithelial apoptosis and tight junctional destruction.

Colorectal adenocarcinoma Caco-2 cells grown to confluence were luminally exposed to E.coli LPS at 50 μg/ml for 24 hrs and examined for cell apoptosis. Hoechst staining and TUNEL assay showed chromatin condensation and DNA fragmentation in cells following LPS exposure. Flow cytometric analysis of DiOC6(3) staining exhibited mitochondrial membrane potential collapse after LPS challenge. Pretreatment with caspase-3 inhibitor, z-DEVD-FMK, prevented the epithelial cell apoptosis induced by LPS. Immunostaining revealed chicken-wire pattern of tight junctional proteins, i.e. ZO-1 and occludin, on untreated Caco-2 cell monolayer en face. LPS caused destruction of ZO-1 and occludin in which the effect was abolished by pretreatment with caspase-3 inhibitor. In addition, polymyxin B blocked the apoptotic phenomenon and tight junctional reorganization, verifying the specificity of the lipid A moiety of LPS.

The role of LPS receptor components, e.g. CD14 and TLR-4, was further assessed in the mechanism. Presence of CD14, but not TLR-4, protein expression was identified on Caco-2 epithelial cells demonstrated by immunofluorescent staining and flow cytometry. The expression of CD14 on apical membrane of cells was evidenced by confocal XZ serial imaging. The absence of TLR-4 expression on Caco-2 cells was verified using three different monoclonal antibodies (clones HTA-125, 76B357.1, and 1G11), whereas positive TLR-4 staining was showed on T84 cells. After LPS challenge, the cellular expression of CD14 and TLR-4 did not change. Moreover, western blotting results confirmed the presence of CD14 (M.W. = 53~55 kDa) on Caco-2 cells. Furthermore, our results showed that neutralizing anti-CD14 antibody (clone 134620) reduced the level of LPS-induced apoptosis and tight junctional destruction in a dose-dependent manner, whereas anti-TLR-4 (clone HTA-125) had no effect.

Classical LPS/TLR-4 signaling pathway induced nuclear translocation of NF-κB and phosphorylation of MAPK by a MyD88-dependent manner. In order to confirm or rule out the involvement of TLR-4-mediated signaling pathway, western blotting was utilized to examine the expression of NF-κB, MyD88 & phosphorylation level of MAPK in intestinal epithelial Caco-2 cells. Our results showed that there are no nuclear translocation of NF-κB, phosphorylation of JNK and p38, nor augment of MyD88 in LPS challenged Caco-2 cells, in contrast to the positive control THP-1 cells.

Taken together, these findings suggest that LPS-induced cell apoptosis leads to tight junctional disruption and barrier defects in intestinal epithelial Caco-2 cells. These pathological effects on enterocytes were mediated via epithelial CD14, whereas TLR-4-mediated signaling pathway did not play a role.

口試委員會審定書 I
謝辭 II
中文摘要 III
英文摘要 V
中英文縮寫名詞對照表 VII
一、前言 1
1、腸道管壁之組織結構 1
2、腸道上皮細胞之生理功能 2
2.1消化、吸收、分泌 2
2.1.1 消化 2
2.1.2 吸收 2
2.1.3 分泌 3
2.2屏障 3
2.2.1物理性屏障 3
2.2.2化學性屏障 5
2.2.3免疫性屏障 6
2.2.4屏障缺損 7
3、腸道上皮細胞之凋亡 8
3.1細胞凋亡概論 8
3.2腺窩-絨毛軸 11
3.3病原產物與上皮細胞凋亡 11
4、內毒素受體複合分子－TLR-4、CD14、MD-2及其訊息途徑 12
4.1 內毒素 (lipopolysaccharide, LPS) 12
4.2 內毒素受體複合分子 13
4.3訊息途徑 15
5、目的 17
二、材料與方法 19
1、細胞培養 19
1.1人類大腸直腸癌之腸道上皮細胞株Caco-2 19
1.2人類大腸直腸癌之腸道上皮細胞株T84 19
1.3人類急性單核球白血病細胞株THP-1 20
2、藥劑處理 20
2.1大腸桿菌內毒素 (lipopolysaccharide, LPS) 20
2.2 喜樹鹼 (camptothecin, CPT) 21
2.3 caspase-3抑制劑 (z-DEVD-FMK) 21
2.4多黏桿菌素B (polymyxin B, PMB) 21
2.5 中和性抗CD14、TLR-4抗體 22
3、Hoechst螢光染色法 (Hoechst staining) 22
4、缺口末端標記技術 (TdT-mediated dUTP-biotin nick end labeling, TUNEL) 23
5、流式細胞技術 (flow cytometry) 24
5.1 粒線體膜電位偵測 25
5.2 內毒素受體複合物分子CD14之表現 26
6、細胞免疫螢光染色 (immunocytochemistry) 27
6.1 緊密連結蛋白ZO-1及occludin結構偵測 27
6.2 內毒素受體複合物分子CD14、TLR-4之表現 28
7、NP-40可溶性蛋白質萃取 (extraction of NP-40 soluble proteins) 30
8、細胞核與細胞質蛋白質之萃取 (fractionation of nuclear and cytoplasmic proteins) 31
9、西方墨點法 (western blotting) 31
10. 統計分析 33
三、結果 34
1、內毒素刺激引起人類腸道上皮細胞株Caco-2之細胞凋亡，而此現象可藉由預先投予caspase-3抑制劑而阻斷 34
1.1 內毒素刺激引起人類腸道上皮細胞株Caco-2之染色質濃縮 34
1.2 內毒素刺激引起人類腸道上皮細胞株Caco-2之DNA片段化 34
1.3 內毒素刺激引起人類腸道上皮細胞株Caco-2之粒線體膜電位下降 35
2、內毒素刺激破壞人類腸道上皮細胞株Caco-2之緊密連結蛋白結構，而此現象可藉由預先投予caspase-3抑制劑而阻斷 35
2.1 內毒素刺激破壞人類腸道上皮細胞株Caco-2之緊密連結蛋白ZO-1及occludin結構為caspase-3依賴性 35
3、預先投予polymyxin B可阻斷內毒素刺激引起人類腸道上皮細胞株Caco-2之細胞凋亡及緊密連結蛋白結構之破壞 36
3.1 預先投予polymyxin B (PMB) 可阻斷內毒素刺激引起人類腸道上皮細胞株Caco-2之DNA片段化 36
3.2 預先投予polymyxin B可阻斷內毒素刺激引起人類腸道上皮細胞株Caco-2之緊密連結蛋白ZO-1及occludin結構破壞 37
4、內毒素受體複合物分子CD14在人類腸道上皮細胞株Caco-2之表現 37
4.1利用免疫螢光染色法偵測到內毒素受體複合物分子CD14在常態人類腸道上皮細胞株Caco-2上有表現 37
4.2利用流式細胞技術偵測內毒素受體複合物分子CD14在常態人類腸道上皮細胞株Caco-2上有表現 38
4.3 利用西方墨點法偵測到內毒素受體複合物分子CD14在常態人類腸道上皮細胞株Caco-2上有表現 38
5、內毒素受體複合物分子TLR-4在人類腸道上皮細胞株Caco-2之表現 39
5.1 利用免疫螢光染色法並未偵測到Caco-2上TLR-4之表現 39
6、預先投予中和性抗CD14之抗體可抑制內毒素刺激引起腸道上皮細胞株Caco-2之細胞凋亡及緊密連結蛋白結構之破壞；而中和性抗TLR-4之抗體則否 39
6.1 預先投予中和性抗CD14之抗體可抑制內毒素刺激引起腸道上皮細胞株Caco-2之DNA片段化 40
6.2 預先投予中和性抗CD14之抗體可抑制內毒素刺激引起腸道上皮細胞株Caco-2之緊密連結蛋白結構之破壞 40
6.3 預先投予中和性抗TLR-4之抗體無法抑制內毒素刺激引起腸道上皮細胞株Caco-2之DNA片段化、緊密連結蛋白結構之破壞 41
7、內毒素刺激腸道上皮細胞株Caco-2不會引起NF-κB之核轉移現象、MyD88總量之改變、JNK及p38之磷酸化 41
7.1內毒素刺激腸道上皮細胞株Caco-2不會引起NF-κB之核轉移現象 42
7.2內毒素刺激腸道上皮細胞株Caco-2不會引起MyD88總量之改變、JNK及p38之磷酸化 42
四、討論 43
五、圖表 51
六、參考文獻 79

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