Su zhu kang jun tai Cathelicidin zai you men luo gan jun wei nei cun huo ji gan ran zhong zuo yong de yan jiu

幽門螺桿菌感染在世界範圍內普遍存在，超過50%的世界人口都曾被感染。幽門螺桿菌與胃炎，胃潰瘍，胃癌和其他胃內疾病的發生密切相關。盡管目前已有多種有效除菌的抗生素，但耐藥菌的出現仍然不可忽視。防止幽門螺桿菌感染能有效的減緩疾病進程及其相關疾病引發的死亡率。因此，新藥物或者新的藥物劑型的研發十分重要。Cathelicidin是一種宿主免疫防禦系統用於抵抗不同種類病原微生物感染的生物肽。然而，其在幽門螺桿菌感染引發的炎癥中的作用仍未被揭示。本研究旨在發現Cathelicidin在幽門螺桿菌體內及體外感染中的可能抗菌作用及其機制。為了研究不同劑量Cathelicidin對幽門螺桿菌的直接抗菌作用，我們主要觀察了細菌生長，生物膜形成及細菌形態的改變。實驗結果表明，Cathelicidin可有效的抑制幽門螺桿菌的生長，破壞其生物膜形成，及在細菌胞膜形成孔狀結構，以改變其正常的超微形態。在宿主抵禦幽門螺桿菌感染的機制中，自噬不僅具有抗菌活性，同時在清除胃上皮細胞內病原體的方面發揮著重要作用。然而，幽門螺桿菌則可能得益於自噬通路，並掌控自噬這一工具，進而幫助其自身的存活及感染。研究發現，維生素D於體內的活性形式1,25 - 二羥維生素D3（1,25D3）可促進Cathelicidin的合成及激活自噬通路，從而發揮自身免疫來殺死在胃上皮細胞內定植的幽門螺桿菌。此外，通過RNAi沈默技術，與對照基因沈默的細胞相比，LL-37在人胃上皮細胞（HEF-145）中表達被抑制後，細胞內幽門螺桿菌的存活數量顯著上升。同樣的結果也被發現於動物模型中，在急性及慢性幽門螺桿菌感染的小鼠模型中，CRAMP基因敲除小鼠胃內的幽門螺桿菌數量比野生型小鼠胃內更多。為了進一步研究Cathelicidin是否具有潛在的治療幽門螺桿菌感染的作用，本研究采用生物工程的方法將CRAMP轉入乳酸球菌中，再將這種分泌CRAMP型及對照組乳酸球菌餵給被幽門螺桿菌感染的小鼠。預防性和治療性的研究結果表明，這種能夠分泌CRAMP的益生菌可在胃黏膜表面存活和定植。更多的幽門螺桿菌能夠定植在CRAMP基因敲除小鼠的胃內，同時其胃內的促炎癥因子，IL-6，IL-1β及ICAM表達也高於野生型小鼠。此外，幽門螺桿菌感染上調了野生型小鼠胃上皮型來源的CRAMP表達，這一結果可部分解釋為什麽在野生型小鼠胃內只有少量幽門螺桿菌及輕度炎癥反應的原因。重要的是，預防性及治療性的實驗顯著的提高了兩種小鼠胃黏膜中抗菌肽的水平，並降低了幽門螺桿菌感染及促炎癥因子mRNA的表達。值得注意的是，預防性的給藥還促進了胃粘液層的合成及防止表皮細胞雕亡，從而加強胃黏膜屏障的保護作用。總結而言，本研究結果揭示Cathelicidin作為一種天然抗生素，在清除幽門螺桿菌及治療其引發的慢性胃炎中發揮重要的作用。分泌Cathelicidin型食用益生菌和幫助Cathelicidin內源性表達的1,25D3則有望發展成為新型的生物制劑用於防治動物和人體幽門螺桿菌感染及其引發的相關性胃炎。Helicobacter pylori (H. pylori) infection is one of the most prevalent infectious diseases, affecting more than 50% of the world’s population and responsible for gastritis, peptic ulcer, gastric cancer and other stomach disorders.Although there are antibiotics which are effective to eradicate the bacteria, the worldwide appearance of drug resistance to H. pylori is common. It is therefore needed to search for new therapeutic agents or establish a new form of drug delivery system to prevent H. pylori infection at the early stage in order to reduce the disease progression and its associated morbidities.Cathelicidin, a host defense antibacterial peptide in humans can eradicate different kinds of microbial infection. However, its roles in H. pylori infection and inflammation remain unexplored. This study sought to elucidate the possible actions and mechanisms for cathelicidin to protect against H. pylori infection and its associated inflammation both in vitro and in vivo.To examine the direct antimicrobial action of cathelicidin, H. pylori survival, biofilm formation and morphology change were determined after exposure to different doses of cathelicidin in vitro. Results showed that exogenous cathelicidin could affect H. pylori growth, destroy bacteria biofilm and cause pore formation in H. pylori membranes. With respect to the host defense against H. pylori infection, autophagy plays a crucial role in antimicrobial activity, and contributes to clearance of intracellular pathogens in gastric cells. In this regard, H. pylori might benefit from autophagy pathway, and subvert the autophagy machinery in favor of its survival and infectious process.The active form of vitamin D3, 1, 25-dihydroxyvitamin D3 (1, 25D3) activated LL-37, a human cathelicidin antimicrobial peptide and produced autophagy, which could contribute to host immune responses against intracellular survival of H. pylori in gastric cells. Additionally, we transfected gastric epithelial cells (HFE-145) with siRNA specific for LL-37 (siLL-37) to knockdown the expression of LL-37 in HFE-145 cells, which markedly increased the number of intracellular H. pylori when compared to cells transfected with a scrambled control siRNA (Csi). Consistent with these findings, cathelicidin knockout (Cnlp⁻/⁻) mice exhibited stronger H. pylori colonization in stomachs with acute and chronic H. pylori infection when compared to the stomachs in cathelicidin wild-type (Cnlp⁺/⁺) mice.To further examine whether cathelicidin could be used as a therapeutic agent for H. pylori infection, we replenished exogenous CRAMP in H. pylori infected Cnlp⁺/⁺ and Cnlp⁻/⁻ mice with a bioengineered CRAMP-secreting strain of Lactococcus lactis (L. lactis) in a cost-effective manner. To this end, Cnlp⁺/⁺and Cnlp⁻/⁻ mice were pre-treated or post-treated with the control plasmid-encoded L. lactis or CRAMP-encoded L. lactis in H. pylori infected mice. They were then assessed for H. pylori infection and inflammatory responses in stomachs. Results showed that the probiotic L. lactis could survive in the gastric mucosa. In the absence of CRAMP, Cnlp⁻/⁻ mice exhibited more H. pylori harboring in the stomach together with marked expressions of IL-6, IL-1β and ICAM in the gastric mucosa when compared to wild type mice. Furthermore, in Cnlp⁺/⁺ mice, H. pylori infection stimulated gastric epithelium-derived CRAMP production but not in the Cnlp⁻/⁻ mice. These findings could partially explain why there were less bacterial infection and inflammatory responses in the wild type animals. Importantly, pre-treatment and post-treatment with CRAMP-encoded L. lactis significantly increased mucosal CRAMP level in both types of animals and reduced H. pylori infection and also pro-inflammatory cytokines mRNA expressions in these stomachs. It was noteworthy that delivering CRAMP intragastrically before H. pylori challenge strengthened the mucosal barrier by stimulating mucus layer synthesis and preventing epithelial cell apoptosis.Collectively, these findings indicate that cathelicidin plays a significant role as a potential natural antibiotic for H. pylori clearance and a therapeutic agent for chronic gastritis. The increase of cathelicidin expression in the gastric mucosa either by the food-grade probiotic encoded with cathelicidin or the active form of vitamin D, could be promising biological preparations for the treatment of H. pylori infection and its associated gastritis in animals and perhaps also in humans.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Zhang, Lin.Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.Includes bibliographical references (leaves 144-170).Abstract also in Chinese.ABSTRACT --- p.I中文摘要 --- p.VDECLARATION --- p.VIIACKNOWLEDGEMENTS --- p.VIIIPUBLICATIONS --- p.XIVLIST OF ILLUSTRATIONS --- p.XIXINTRODUCTION --- p.1Chapter 1.1 --- Helicobacter pylori --- p.1Chapter 1.1.1 --- Overview --- p.1Chapter 1.1.2 --- Epidemiology of H. pylori infection --- p.2Chapter 1.1.3 --- Diagnosis and treatment strategies for H. pylori-induced diseases --- p.2Chapter 1.1.4 --- Bacteria autophagy: restriction or persistence of infection? --- p.3Chapter 1.1.5 --- Virulence factors of H. pylori related to autophagy --- p.8Chapter 1.1.6 --- Future research directions concerning H. pylori and autophagy --- p.11Chapter 1.2 --- Cathelicidins --- p.11Chapter 1.2.1 --- Overview --- p.11Chapter 1.2.2 --- Cathelicidin and its antimicrobial action and possible mechanisms --- p.12Chapter 1.2.3 --- Mouse cathelicidin deficient model --- p.16Chapter 1.2.4 --- Multiple receptors enable diversified activities of cathelicidins --- p.17Chapter 1.2.5 --- Endogenous cathelicidin induction --- p.23Chapter 1.2.5 --- New uses for old drugs --- p.26METHODS --- p.29Chapter 2.1 --- General Materials --- p.29Chapter 2.1.1 --- Chemicals and reagents --- p.29Chapter 2.1.2 --- Antibodies --- p.33Chapter 2.1.3 --- Commercial Kits --- p.34Chapter 2.1.4 --- Bacteria and culture conditions --- p.35Chapter 2.1.5 --- Animals --- p.36Chapter 2.1.6 --- Cell Line --- p.36Chapter 2.2 --- Experimental Designs --- p.37Chapter 2.2.1 --- In vitro studies --- p.37Chapter 2.2.2 --- In vivo studies --- p.44Chapter 2.3 --- Statistical analysis --- p.52RESULTS AND DISCUSSION --- p.53Chapter 3.1 --- Antimicrobial activity of cathelicidin on H. pylori in vitro --- p.53Chapter 3.2 --- Anti-biofilm formation activity of cathelicidin on H. pylori in vitro --- p.58Chapter 3.3 --- Manipulation of autophagy by H. pylori for their survival --- p.62Chapter 3.3.1 --- H. pylori stimulated dysfunctional autophagy --- p.62Chapter 3.3.2 --- H. pylori compromised the autophagic flux in cells and thereby promoting self-multiplication --- p.68Chapter 3.3.3 --- Autophagy is a host defense process in controlling intracellular survival of H. pylori --- p.71Chapter 3.4 --- Vitamin D3 inhibited H. pylori infection through the induction of autophagy --- p.76Chapter 3.4.1 --- 1,25D3 triggered the formation of autophagosomes and autophagolysosomes in gastric epithelial cells --- p.76Chapter 3.4.2 --- 1,25D3 treatment inhibited intracellular H. pylori survival through induction of autophagy by cathelicidin --- p.79Chapter 3.5 --- Discussion --- p.86Chapter 3.6 --- Elucidation of the role of endogenous and exogenous cathelicidin in H. pylori colonization and the associated gastritis --- p.94Chapter 3.6.1 --- H. pylori SS1 colonized in Cnlp⁺/⁺ and Cnlp⁻/⁻ mouse gastric epithelium --- p.94Chapter 3.6.2 --- Endogenous cathelicidin protects against H. pylori SS1 colonization in vitro and in vivo --- p.96Chapter 3.6.3 --- Endogenous cathelicidin protects against drug-resistant H. pylori 10783 colonization --- p.100Chapter 3.6.4 --- The bioengineered L. lactis encoded with CRAMP could localize in mouse stomachs and express CRAMP mRNA --- p.104Chapter 3.6.5 --- Effects of CRAMP secreting bioengineered L. lactis on H. pylori growth in vitro --- p.106Chapter 3.6.6 --- Post-treatment of CRAMP-encoded L.lactis on H. pylori colonization and its associated gastritis --- p.108Chapter 3.6.7 --- Pre-treatment of CRAMP-encoded L.lactis on H. pylori colonization and its associated gastritis --- p.118Chapter 3.7 --- Discussion --- p.129SUMMARY AND FUTURE PERSPECTIVES --- p.140REFERENCES --- p.14