本文選題：噬菌體　切入點：單鏈抗體　出處：《東北師范大學(xué)》2017年碩士論文

【摘要】：在全球,侵襲性真菌感染已經(jīng)成為導(dǎo)致人類死亡的重要原因。近年來,免疫功能不全的人、HIV感染者、接受化療或大型手術(shù)的患者、接受過器官或者造血干細(xì)胞抑制的人在逐漸增加,隨之而來的是真菌感染的病人也在逐漸增加。白色念珠菌是常見的醫(yī)院內(nèi)感染疾病,是造成系統(tǒng)性念珠菌感染的重要原因,死亡率接近50%。眾所周知,系統(tǒng)性白色念珠菌感染治療的難度很大。目前,臨床上允許用于治療真菌感染的藥物主要有四類,分別是核苷酸類似物、唑類、多烯類和棘白菌素類。近年來,雖然抗真菌藥物在不斷開發(fā),但是真菌感染的發(fā)病率和死亡率仍然居高不下。同時不斷出現(xiàn)的耐藥性菌株也要求我們不斷開發(fā)新的治療策略。本研究以白色念珠菌細(xì)胞壁蛋白為靶點,研究與其特異性結(jié)合的噬菌體單鏈抗體對系統(tǒng)性白色念珠菌感染治療的作用。本研究的內(nèi)容主要是,利用人源噬菌體展示單鏈抗體文庫,篩選出與白色念珠菌孢子體細(xì)胞壁蛋白結(jié)合能力最強的噬菌體單鏈抗體,從體外和小鼠系統(tǒng)性白色念珠菌感染模型兩方面,評價噬菌體單鏈抗體的抗真菌作用效果。本研究的主要方法及結(jié)果如下:1.人源噬菌體單鏈抗體的篩選:進(jìn)行4輪篩選,得到5條與白色念珠菌細(xì)胞壁相結(jié)合的噬菌體單鏈抗體,命名為JC1、JC2、JC3、JC4、JC5。2.結(jié)合力分析:將5種體單鏈抗體進(jìn)行抗體效價分析、Western-blotting、免疫熒光實驗,分析其與白色念珠菌的結(jié)合能力。結(jié)果顯示,JC1與白色念珠菌的結(jié)合效果最好。3.體外分析JC1的治療效果:利用細(xì)胞活性檢測試劑CCK分析了JC1的最佳工作濃度,繪制白色念珠菌的生存曲線,進(jìn)一步探索JC1的最佳作用時間及溫度;利用流式細(xì)胞術(shù)分析了JC1對白色念珠菌細(xì)胞周期進(jìn)程的影響;利用Hoechst和PI雙染法分析了JC1對白色念珠菌細(xì)胞凋亡與壞死的影響;利用掃描電鏡分析了JC1對白色念珠菌細(xì)胞壁表面形態(tài)的影響。結(jié)果表明,1×109個/ml JC1,30℃下共孵育8小時時,能夠顯著的抑制白色念珠菌孢子體細(xì)胞的增殖,并且能顯著的阻滯白色念珠菌的細(xì)胞周期,促進(jìn)其凋亡與壞死,能夠使白色念珠菌的細(xì)胞壁發(fā)生皺縮。4.構(gòu)建小鼠系統(tǒng)性白色念珠菌感染模型,分析JC1對小鼠腎臟、肝臟、脾臟的白色念珠菌負(fù)荷量的影響以及JC1對小鼠存活時間的影響。實驗結(jié)果顯示,JC1能夠顯著的降低小鼠腎臟、肝臟、脾臟的真菌定植量,并且延長了小鼠的存活時間。綜上所述,噬菌體單鏈抗體JC1,結(jié)合了單鏈抗體與噬菌體的優(yōu)點,具有顯著的治療系統(tǒng)性白色念珠菌感染的效果。因此,JC1具有用于治療系統(tǒng)性白色念珠菌感染疾病的潛能。
[Abstract]:Worldwide, invasive fungal infections have become an important cause of death in humans.In recent years, the number of people with HIV infection, chemotherapy or major surgery, organ or hematopoietic stem cell inhibition has been increasing, followed by fungal infection.Candida albicans is a common nosocomial infection and an important cause of systemic Candida infection. The mortality rate is close to 50%.It is well known that systemic Candida albicans infection treatment is very difficult.At present, there are four kinds of drugs that can be used to treat fungal infection. They are nucleotide analogs, azoles, polyenes, and echinacetin.In recent years, although antifungal drugs are being developed, the morbidity and mortality of fungal infections remain high.At the same time, the emergence of drug-resistant strains also requires us to develop new treatment strategies.The purpose of this study was to investigate the effect of phage specific phage scFv on the treatment of systemic Candida albicans infection with Candida albicans cell wall protein as the target.The main content of this study was to screen the phage scFv with the strongest binding ability to the cell wall protein of Candida albicans spore by human phage display scFv library.The antifungal effects of phage scFv were evaluated in vitro and in mice with systemic Candida albicans infection model.The main methods and results of this study are as follows: 1.Screening of human phage scFv: through four rounds of screening, five phage scFv combined with cell wall of Candida albicans were obtained and named as JC1, JC2, JC2, JC3, JC4, JC5.2.Binding ability analysis: the antibody titers of five kinds of single-chain antibodies were analyzed by Western-blotting.Immunofluorescence assay was used to analyze their binding ability with Candida albicans.The results showed that JC1 had the best binding effect with Candida albicans.In vitro analysis of the therapeutic effect of JC1: the best working concentration of JC1 was analyzed by CCK, the survival curve of Candida albicans was drawn, and the optimum time and temperature of JC1 were further explored.The effects of JC1 on cell cycle progression of Candida albicans were analyzed by flow cytometry, and the effects of JC1 on apoptosis and necrosis of Candida albicans were analyzed by Hoechst and Pi double staining.The effect of JC1 on the surface morphology of Candida albicans cell wall was analyzed by SEM.The results showed that after incubated at 30 鈩，

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