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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/15764

Title: 發展DNA疫苗抗幽門螺旋桿菌感染及藉由幾丁聚醣奈米粒做為口服傳遞系統
Development of DNA vaccine against Helicobacter pylori infection and using chitosan nanoparticles for oral delivery system
Authors: Yu-Pin Chao
趙育彬
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 幽門螺旋桿菌;尿素酶;幾丁聚醣;DNA疫苗
Helicobacter pylori;Urease;chitosan;DNA vaccine
Date: 2010
Issue Date: 2011-06-30T08:19:06Z
Abstract: 幽門螺旋桿菌可長期的附著在人類胃黏膜組織而進一步引發腸胃相關的疾病。例如:急慢性胃炎、胃及十二指腸潰瘍甚至是胃癌。根據統計全世界有百分之五十以上的人口受到感染。目前普遍的治療方法主要採用抗生素合併其他藥物進行治療,然而抗生素治療衍生出許多的缺點:增加幽門螺旋桿菌的抗藥性、產生副作用,造成臨床上治療失敗。因此本篇論文將發展有效DNA疫苗來達到預防幽門螺旋桿菌之感染,並利用幾丁聚醣作為非侵入性的口服疫苗之傳遞載體。體外實驗中,使用不同比例之幾丁聚醣-DNA複合體對幼倉鼠腎細胞進行轉染效率、細胞毒性分析及DNA結合效率分析,發現理想的DNA與幾丁聚醣最適比例為5。模擬腸胃道環境耐受性試驗方面,以電子顯微鏡觀察下,複合體在胃酸環境可維持穩定的奈米球型外觀。活體實驗中,口服傳遞pCMV-β DNA,五天後可在小鼠的胃黏膜及小腸組織中發現抗原之表現。建立小鼠感染模式,發現採用109 CFU/ml 之菌落數,餵食菌二次,每次間隔為二天,其感染的成功率可以達到 97-100%。組織病理切片結果發現,成功感染後的小鼠,胃黏膜中有發炎細胞浸潤及幽門螺旋桿菌附著的現象。最後在免疫試驗方面,使用可表現幽門螺旋桿菌尿素酶之DNA疫苗(pCJ-3/ure B),並以幾丁聚醣作為口服傳遞,可引發小鼠(C3H/HeN)血清及腸胃黏膜中產生專一的保護性抗體且免疫調節走向均為Th1及Th17所介導的宿主防禦。免疫後血清也可中和幽門螺旋桿菌,尿素酶活性降低80%。最後免疫保護效果評估上,DNA經口服傳遞、DNA經由肌肉注射及peptide口服傳遞免疫組,皆能降低小鼠胃中菌量及尿素酶活性,其小鼠保護率分別為70%、70%及60%。
Helicobacter pylori is discovered in 1983 years. It can adhered on human gastric mucosal and involved in the pathogenesis of human active chronic gastritis, peptic and duodenal ulcer diseases and gastric cancer. The treatment of H. pylori infection includes antibiotics and combination with inhibitors, but the emergence of antibiotic resistance and side effect limit the clinical therapy for H. pylori infection. This study was developed the appropriate strategies through DNA vaccination and using chitosan for oral delivery system. In vitro assay, transfection efficiency, cell viability and DNA binding capacity of different N/P ratio of chitosan-pDNA complex was analyzed. Results show that the optimal ratio of the chitosan-pDNA complex was N/P ratio 5. The nanoparticles at pH 1.2, 2, 2.5 were stable and showed a spherical shape . In vivo assay, chitosan-pCMV-β DNA was orally delivered to C3H/HeN mice. After three days, β-galactosidase were expression in gastric and intestinal mucosal of mice. In the H. pylori infected C3H/HeN mice model, mice were inoculated with 109 CFU/ml by oral gavage twice times during a 5-days period, we found that the successful infection rate was about 97%. From histopathology study, the gastric mucosal of C3H/HeN mice could detected the infiltration of mononuclear cells and the colonization of H. pylori. In immunization studies, we used the Helicobacter pylori urease B gene expressive plasmid DNA (pCJ-3/ureB) combined the chitosan nanoparticles for oral delivery system to test the protective immunity induced by oral immunization with C3H/HeN mice. Anti-urease antibody titres in prophylactically, vaccinated mice were markedly higher than non-immune mice. Vaccination of mice against H pylori induces a strong Th-1 and Th-17 response of the stomach to cause post-immunization gastritis to eradication of H pylori. The neutralizing antibodies in serum after immunization, that could reduce about 80% urease activity. The finally, seventy percent of mice with prophylactically oral immunization with chitosan-pCJ-3/urease B DNA vaccine could effectively against the infection of H. pylori.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M97320037
http://ntour.ntou.edu.tw/ir/handle/987654321/15764
Appears in Collections:[食品科學系] 博碩士論文

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