本文選題：伯氏瘧原蟲 + NT1基因�。� 參考：《安徽大學(xué)》2017年碩士論文

【摘要】：瘧疾是世界三大傳染病之一,以按蚊為傳播媒介,致病病原體是瘧原蟲。目前,32億人處在瘧疾威脅之下,其中95%的瘧疾死亡是由于惡性瘧原蟲感染導(dǎo)致的。肺癌是全球最常見的惡性腫瘤之一,發(fā)病率和死亡率都位居癌癥新增發(fā)病和死亡率之首,傳統(tǒng)的手術(shù)、化療、放療及其聯(lián)合療法由于各自的局限性在控制肺癌的發(fā)展已經(jīng)遇到很大的瓶頸,例如缺乏特異性,生物療法越來越受到重視。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),瘧原蟲通過廣泛激活機(jī)體的天然免疫系統(tǒng)和誘導(dǎo)一定的針對(duì)腫瘤的特異性抗原,從而顯著地抑制小鼠Lewis肺癌的生長(zhǎng)和延長(zhǎng)荷瘤小鼠的生存期。但是瘧原蟲具有一定的毒副作用,我們擬對(duì)瘧原蟲進(jìn)行減毒,提高瘧原蟲治療肺癌的安全性。瘧原蟲本身沒有嘌呤的合成途徑,只能從宿主中獲得嘌呤堿或者嘌呤核苷,依賴補(bǔ)救合成途徑滿足瘧原蟲的裂體增殖需求。核苷轉(zhuǎn)運(yùn)蛋白1(NT1)是瘧原蟲合成嘌呤途徑的一個(gè)關(guān)鍵酶,負(fù)責(zé)將嘌呤核苷和嘌呤堿基轉(zhuǎn)運(yùn)到瘧原蟲的膜內(nèi)。在C57BL/6小鼠中Plasmodium berghei ANKA(P.b ANKA)是致死性的鼠瘧原蟲株,Pb.ANKA感染C57BL/6小鼠是研究減毒瘧原蟲的最佳模型。本研究首先構(gòu)建雙交換同源重組的質(zhì)粒載體pL0018-NT1,質(zhì)粒上含有GFP標(biāo)記基因和tgdhfr/ts抗性基因,使用電轉(zhuǎn)化方式將質(zhì)粒轉(zhuǎn)進(jìn)P.b ANKA瘧原蟲中,經(jīng)過乙胺嘧啶藥篩,第一次單克隆化得到GFP表達(dá)和位點(diǎn)特異性整合的2個(gè)克隆8#和9#,其中8#克隆經(jīng)過第二次單克隆化,得到穩(wěn)定NT1基因敲除和位點(diǎn)特異性整合的P.b ANKA-ΔNT1-8C1克隆。觀察發(fā)現(xiàn)野生型P.b ANKA感染小鼠后小鼠很快全部死亡,NT1敲除的P.b ANKA-ΔNT1蟲株在接種C57BL/6小鼠后,能形成高達(dá)85%的感染率的原蟲血癥,感染周期能達(dá)到80天,6只感染的小鼠有5只不死亡,總體生存期顯著延長(zhǎng)。在P.b ANKA-Δ NT1感染小鼠38天時(shí)間里,在100倍油鏡下觀察發(fā)現(xiàn)配子體數(shù)顯著降低,每100個(gè)白細(xì)胞最高只能觀察到48個(gè)配子體,在接種2～18天內(nèi),配子體數(shù)在0～25個(gè)數(shù)量范圍內(nèi)波動(dòng),遠(yuǎn)低于野生型最多時(shí)400個(gè)配子體的水平。瘧原蟲的配子體數(shù)目的降低有利于阻斷瘧原蟲在按蚊體內(nèi)的有性生殖,是藥物減毒和輻照減毒策略所不具有的安全性優(yōu)勢(shì),避免瘧原蟲通過按蚊在人群中大量傳播。我們之前研究發(fā)現(xiàn)瘧原蟲感染荷瘤小鼠時(shí)間越長(zhǎng),抑制腫瘤效果越好。P.b ANKA-ΔNT1減毒蟲株紅內(nèi)期感染長(zhǎng)達(dá)80天,比約氏瘧原蟲30天要更長(zhǎng)。P.b ANKA-ΔNT1比野生型P.b ANKA感染Lewis肺癌皮下接種C57BL/6小鼠更加顯著的抑制腫瘤生長(zhǎng)的效果,中位生存期也要延長(zhǎng)五天。NT1基因敲除的P.b ANKA-ΔNT1蟲株顯著降低瘧原蟲對(duì)小鼠的毒副作用,從致死型蟲株轉(zhuǎn)變?yōu)榉侵滤佬韵x株,具有更長(zhǎng)的感染周期,同時(shí)顯著降低外周血中的配子體,提供了更加有效和安全的減毒瘧原蟲,可以作為肺癌疫苗的腫瘤抗原表達(dá)載體,為腫瘤的生物免疫治療提供新的方法和思路,同時(shí)還可以作為減毒全蟲活疫苗,用于保護(hù)人類健康和消滅瘧疾。
[Abstract]:Malaria is one of the three major infectious diseases in the world. The Anopheles mosquito is the medium and the pathogenic pathogen is the Plasmodium. At present, 3 billion 200 million people are under the threat of malaria, of which 95% of the deaths are caused by the infection of Plasmodium falciparum. Lung cancer is one of the most common malignant tumors in the world. The incidence and mortality of the disease are all in the new disease and death of cancer. In the first place, traditional surgery, chemotherapy, radiotherapy and combined therapy have encountered great bottlenecks in controlling the development of lung cancer. For example, the lack of specificity, biologic therapy is becoming more and more important. The specific antigen of the tumor significantly inhibits the growth of Lewis lung cancer in mice and prolongs the survival period of the tumor bearing mice. However, the Plasmodium has a certain toxic side effect. We intend to reduce the toxicity of the Plasmodium to improve the safety of the Plasmodium in the treatment of lung cancer. The parasite itself has no synthetic route of the MTX, only the purine base is obtained from the host. Or purine nucleoside, dependent on the remedial synthesis pathway to meet the proliferation demand of the Plasmodium, a key enzyme for the Plasmodium purine pathway, which is responsible for transshipment of the purine nucleosides and purine bases into the Plasmodium Plasmodium membrane. In C57BL/6 mice, Plasmodium berghei ANKA (P.b ANKA) is fatal to the Plasmodium Plasmodium strain. Pb.ANKA infected C57BL/6 mice is the best model for the study of Plasmodium Plasmodium. First, we construct a double exchange homologous recombinant plasmid vector pL0018-NT1, which contains GFP marker gene and tgdhfr/ts resistance gene. The plasmid is transferred into the Plasmodium P.b ANKA by electric transformation. The first monoclonal antibody is obtained by ethamidine sieving. GFP expression and site specific integration of 2 clones 8# and 9#, in which 8# clones were clones of P.b ANKA- Delta NT1-8C1 that stabilized NT1 gene knockout and site specific integration. After that, the infection rate of protozoa could be up to 85%, the infection cycle could reach 80 days, 5 of the 6 infected mice were not dead, and the overall survival time was prolonged significantly. In the 38 days of P.b ANKA- Delta NT1 infected mice, the number of gametes decreased significantly under 100 times of oil microscope, and only 48 gametophytes could be observed at the highest level of every 100 white cells. Within 2~18 days, the number of gametes fluctuates in 0~25 quantities, far below the level of 400 gametophytes at the maximum of the wild type. The reduction of the number of gametophytes of the Plasmodium is beneficial to blocking the sexual reproduction of the Plasmodium in the Anopheles mosquitoes. It is the safety advantage that the drug reduction and radiation reduction strategies do not have, to avoid the passing of malaria parasites by the malaria parasite. The longer the mosquitoes spread in the population, the longer we found that the longer the infected mice were infected with Plasmodium, the better the effect of the tumor suppressing.P.b ANKA- Delta NT1 in the erythroid infection for up to 80 days, and the longer.P.b ANKA- Delta NT1 than Plasmodium komoya, which was more significant than that of the wild type P.b ANKA infected Lewis lung cancer subcutaneous inoculation C57BL/6 mice. The effect of the growth of the tumor, the median survival period also extended the five days of.NT1 gene knockout P.b ANKA- Delta NT1 to significantly reduce the toxic and side effects of the Plasmodium to mice, from the death type to the non lethal strain, with a longer period of infection, and a significant reduction in the gametophyte in the peripheral blood, providing a more effective and safe reduction of the toxicity. Plasmodium, which can be used as a tumor antigen expression vector for lung cancer vaccine, provides new methods and ideas for the biological immunotherapy of cancer, and can also be used as a live attenuated live vaccine to protect human health and eliminate malaria.

【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R73-36

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Antitumor activities of human autologous cytokineinduced killer(CIK)cells against hepatocellular carcinoma cells in vitro and in vivo[J];World Journal of Gastroenterology;2002年03期

，

本文編號(hào)：1829855