2011-05-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658322摘要：在正常的生理狀態下，小腸腸道中的上皮細胞可藉由細胞間的cadherin junctions 和tight junctions 緊密結合，形成一個物理性的屏障。當這個物理性的屏障出現破裂時，會導致腸道中的抗原和微生物進入到生物體中。而梨型鞭毛蟲病是一種常見的腸道腹瀉型疾病，大多是因飲用了受汙染的水而感染。在先前的研究中有指出，這些寄生在腸道的梨型鞭毛蟲會產生一些物質，進而提升小腸上皮細胞的通透性病且降低上皮細胞單層膜的完整性。但是，梨型鞭毛蟲造成小腸上皮細胞屏障受損的下游機制卻尚未明瞭。目前已知梨型鞭毛蟲會破壞小腸上皮細胞屏障，造成細胞間cadherin junctions 和tight junctions 的瓦解，但這之間的訊息傳遞路徑和活化的途徑仍是未知的。其中一個可能的假說是，在受梨型鞭毛蟲感染的腸道中，MMP-7 和Matriptase 會共同表現，因而破壞cadherin junctions 和tight junctions。利用GSM 這株梨型鞭毛蟲進行感染的實驗，我們發現，這會導致MMP-7 的表現量提升並活化Matriptase。因此，cadherin junctions的瓦解可借由MMP-7 切割Syndecan-1 來釋出 Syndecan-1 胞膜外片段(ECTO-SYNDECAN-1)((圖一)。在Syndecan-1 被切割之後，其具有硫酸乙醯肝素(GAGs)的細胞外片段會被釋出，它可以抑制許多會跟醣胺多醣(GAGs)結合的細胞激素，例如IL-8、IL-4、Midikine…等，因此它可使腸道中受到梨型鞭毛蟲感染的區域產生免疫或發炎反應。另外我們發現，在in vivo 的情況下讓GSM 梨型鞭毛蟲感染宿主，在宿主腸黏膜微絨毛被shedding 和priming 較顯著的區域中，GSM 這株梨型鞭毛蟲的表面會與MMP-7 結合，而當梨型鞭毛蟲的表面與宿主的腸道中的MMP-7 結合時可促使它侵入宿主體內。在先前的報導中有提到，LPS 會促使MMP-7 的過量表現，而這表示TLR-4 的訊息傳遞路徑可能就是調控在GSM 梨型鞭毛蟲感染時MMP-7 表現量的上游路徑。在此計劃中，會以腸道上皮細胞株，例如T84細胞、Caco-2細胞、MMP-7基因剔除梨型鞭毛蟲感染模式鼠最為實驗材料，以探討梨型鞭毛蟲寄生和細菌入侵時對上皮細胞屏障破壞現象的影響，同時分析在不同型況下MMP-7的表現量變化。在研發IO-IBS的治療藥物這部分，我們以中草藥唐古特大黃為首要目標。唐古特大黃具有β-穀甾醇和大黃酚兩種活性成分。在先前的文獻報導中已顯示β-穀甾醇具有抑制基質金屬蛋白酶活性的功能，且可能具有抗前發炎反應的作用；而大黃酚則是具有抑制TLR-4下游NF kappa B的訊息傳遞路徑。我們會測驗此兩種分子抑制MMP-7酵素活性和其表現量的效果。另外，我們會利用重組的MMP-7蛋白去純化中草藥唐古特大黃和茯苓菌中與MMP-7有交互作用的分子；同時，亦可藉由DNA親和力分析的方式去測試萃取物與MMP-7啟動子DNA序列的親和性。其中，與MMP-7蛋白有直接交互作用的活性成分可經由MMP-7蛋白親和性純化的方式萃取出來，並進行MMP-7 CM-轉鐵蛋白酵素電泳法分析。而藉由MMP-7蛋白親和性純化萃取出來的分子將可進行細胞與動物實驗，以測試這些分子對於上皮細胞屏障破壞和腸黏膜共生菌叢變化現象的抑制效果。此外，我們會利用MMP-7啟動子進行冷光分析，以測試這些分子與MMP-7啟動子間的交互作用。唐古特大黃與茯苓菌的中藥萃取物治療急躁性結腸症病患的臨床試驗由台北市立仁愛醫院方志男醫師進行。(子計劃三的共同主持人)截至目前為止，市面上並沒有治療急躁性結腸症的有效用藥。因此，為了進行此病症的藥物研發，我們建立了Caco-2細胞株和MMP-7的基因轉殖鼠等實驗材料，以進行中草藥混合萃取物(唐古特大黃與茯苓菌)對於抑制MMP-7酵素活性、調整上皮細胞接合、神經肌肉接合等現象之分子機制的探討。而了解唐古特大黃與茯苓菌萃取物的分子作用機制將有利於將此中藥萃取物應用於後感染急躁性結腸症的治療上。<br> Abstract: The intestinal barrier constitutes a single layer of intestinal epithelial cells that are linked byparacellular cadherin and tight junctions. The breach of epithelial barrier may lead to influx ofantigen products and microorganisms located in the intestinal lumen. Giardiasis is one of themost commonly identified waterborne intestinal diarrheal diseases. The Giardia parasriticproducts enhance monolayer permeability and decrease of transepithelial electrical resistancewere documented. However, the downstream signaling that cause intestinal epithelial barrierdefects is unclear.The downstream signaling or activation pathways responsible for Giardia duodenalis-induced epithelial barrier defects associated with cleavage of cadherin and tight junctions areunknown. One of the possible hypothesis is that con-junction of MMP-7 and matriptaseco-expression could lead to cadherin junction and tight junction destruction during the Giardiasonicate exposure. The association of up regulation of MMP-7 expression and activation ofmatriptase are associated with Giardia duodenalis GSM infections. Consequently, thecadherin-junction destruction associated MMP-7-mediated Syndecan-1 shedding could takeplace. The shedding of syndecan-1 could potentially release the soluble heparin sulfatecontaining ecto domain, which can sequester many glycosaminoglycan binding cytokine, suchas IL-8, IL-4, midikine, etc. and potentially build up the local immune or inflammatory stormin response to Giardia duodenalis infection. The Giardia duodenalis GSM surface binding ofMMP-7 were observed in the area where the active intestinal mucosa microvillus sheddingand priming during the in vivo Giardia duodenalis GSM challenges and the Giardia surfacebinding of host MMP-7 could facilitate its invasiveness. LPS inducing the over expression ofMMP-7 were documented and this indicate that TLR-4 pathway could well be up streamsignaling regulation for modulating MMP-7 expression during the Gierdia duodenalis GSMchallenge.The general aim of the project is to understanding the mechanism of Giardiasis duodenalis-induced barrier defects and the role of matrix metalloprotease-7 in the downstream signaling pathways. Furthermore, to investigate the effects of chinese herb mixture extracts (唐古特大黃Rheum tanguticum Maxim. Plus 茯苓菌Poria cocos(Schw.)Wolf) on the MMP-7 induced barrier destruction and alteration of the intestinal mucosa comensal bacteria flora during the IBS.The effects of Giardiasis parasites and sonicate on bacterial internalization, and bacterial-induced epithelial adherent junction destruction and MMP-7 expression will be analyzed in intestinal epithelial cell cultures, e.g. T84 and Caco-2 cells and in MMP-7 knockout mice Giardiasis infection model.In the case of drug discovery for treating IO-IBS, we focus on the Chinese herb, Rheum tanguticum Maxim. Containing the both active compounds, -sitosteral and chrysothanol, which were documented that the former one inhibit matrix metalloprotease activities and a potent anti pro inflammatory effects and later can inhibit the TLR-4 down stream NF kappa B pathway. We will validate both compounds effects in habiting MMP-7 activities and expression. We will also further purify the MMP-7 interacting compounds by using the recombinant MMP-7 affinity /or MMP-7 promoter DNA sequence affinity chromatography from 唐古特大黃Rheum tanguticum Maxim. and 茯苓菌Poria cocos(Schw.)Wolf. The direct active compounds interacting with MMP-7 will be purified by application of MMP-7 affinity chromatography purification followed by MMP-7 CM-Transferrin zymography analysis. The MMP-7 interacting compounds will be further applied to the T84 and Caco-2 cells and in MMP-7 knockout mice v.s. wild type mice Giardiasis infection model to investigate the inhibitory effects of the compounds in the epithelial barrier destruction and the species of the conmensal bacteria flora. The underlying mechanisms will be revealed. The MMP-7 lucifersae promoter analysis will be used to characterize those compounds can interact MMP-7 promoter.Clinical trial of the唐古特大黃Rheum tanguticum Maxim. Plus 茯苓菌Poriacocos(Schw.)Wolf treatment for IBS patients performed by Taipei city Ren-Ai Hospital alternative medicine doctor Dr. Fan Chi Nan ( Co-PI in sub project 3)There is no effective drug for treating IBS in the market, we establish Caco-2 cell based and MMP-7 transgenic mice to test the molecular mechanism underlying the chinese herb mixture extract (唐古特大黃Rheum tanguticum Maxim. Plus 茯苓菌Poria cocos(Schw.)Wolf) on inhibit MMP-7 activities and modulating the epithelial junction and neuron mascular junction. To investigate the molecular mechanism of the chinese herb mixture extract (唐古特大黃Rheum tanguticum Maxim. Plus 茯苓菌Poria cocos(Schw.)Wolf) can be a possible stratagem for marketing this effective Chinese herb extracts for treating post infectious irritable bowl dieases.梨型鯾毛蟲唐古特大黃茯苓菌matrix metalloprotease-7腸躁症Giardiasis duodenalisEMTPI-IBSMMP-7Rheum tanguticum Maxim. PlusPoria cocos(Schw.)