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  本文關(guān)鍵詞： caveolin-1 弓形蟲 入侵力　出處：《廣東藥學院》2010年碩士論文　論文類型：學位論文

【摘要】： 剛地弓形蟲(Toxopalsma gondii)是一種單細胞原蟲,呈世界性分布,能侵犯包括人類在內(nèi)的幾乎所有溫血動物有核細胞,其所致的弓形蟲病是常見的人獸共患寄生蟲病。弓形蟲病能引起孕婦流產(chǎn)、新生兒畸形以及死胎等;同時作為一種機會性致病原蟲,弓形蟲還是引起艾滋病患者、器官移植和惡性腫瘤患者等免疫功能受損或缺陷者死亡的重要原因之一,如艾滋病病人中30-40%會發(fā)生弓形蟲性腦炎,2%會發(fā)生多器官弓形蟲病。隨著人們飲食習慣及行為方式的改變,弓形蟲感染率在我國正呈上升的趨勢,我國目前弓形蟲血清陽性率約為7.88%,其感染所帶來的危害正受到越來越多人的關(guān)注,但是至今仍無治療弓形蟲病安全有效的藥物。 入侵是弓形蟲首要的致病過程,弓形蟲入侵宿主細胞后,在納蟲泡里不斷增殖,增殖到一定數(shù)量后,納蟲泡破裂,散出的蟲體再侵入到新的細胞中,對其中任何一環(huán)節(jié)的阻斷都可以治療弓形蟲病。 弓形蟲對宿主細胞的入侵涉及多種蛋白的表達,近年來宿主細胞表面蛋白caveolin-1(窖蛋白1)在病原體內(nèi)在化過程中的作用越來越引起人們的重視。Caveolin-1是胞膜窖(caveolae)的重要結(jié)構(gòu)性蛋白,在人體多種組織和細胞中存在,且能與脂質(zhì)和蛋白的脂質(zhì)錨結(jié)構(gòu)相互作用,參與多種生命活動,如膽固醇運輸、細胞內(nèi)外信號傳遞等。在弓形蟲入侵宿主細胞形成納蟲泡的過程中,caveolin-1被納蟲泡膜排斥在外,是否caveolin-1參與了蟲體對宿主的入侵,目前尚未見相關(guān)報道。本論文通過研究弓形蟲入侵力與caveolin-1的表達變化之間的關(guān)系,探討在弓形蟲入侵過程中caveolin-1的作用,為深入研究弓形蟲入侵機制,開發(fā)新的藥物及發(fā)現(xiàn)新的藥物靶點奠定基礎(chǔ)。 研究目的: 探討在弓形蟲入侵過程中caveolin-1的作用,為深入研究弓形蟲入侵機制,開發(fā)新的藥物及發(fā)現(xiàn)新的藥物靶點奠定基礎(chǔ)。 研究方法: 1.弓形蟲入侵宿主細胞后caveolin-1的表達變化:將高、中、低三組濃度的弓形蟲分別感染HeLa細胞10min、30min和60min,顯微鏡下觀察蟲體入侵力改變并利用實時熒光定量PCR檢測caveolin-1的相對表達水平。 2.抑制caveolin-1基因表達后弓形蟲入侵率的變化:利用藥物英卡磷酸抑制宿主細胞中caveolin-1基因的表達,將106個的弓形蟲感染宿主細胞,檢測caveolin-1的抑制效果、計算弓形蟲入侵率的變化以及弓形蟲MIC2基因的表達 變化。 研究結(jié)果: 1.弓形蟲入侵宿主細胞后,caveolin-1的表達量增加,且與時間延長和濃度增加呈正相關(guān),以60min高濃度組的表達變化最為顯著,表達量約為正常細胞的3.2倍。 2.抑制caveolin-1基因的表達后,弓形蟲入侵力下降,弓形蟲MIC2蛋白的表達也相應(yīng)降低,60min時MIC2蛋白的表達量約為caveolin-1基因表達未抑制的50%。 結(jié)論: 弓形蟲入侵后宿主細胞caveolin-1的表達顯著升高,與弓形蟲的濃度呈正相關(guān),且抑制caveolin-1的表達弓形蟲的入侵率下降,入侵蛋白MIC2的表達也明顯降低。初步證實caveolin-1參與了弓形蟲入侵宿主細胞的過程,為進一步研究弓形蟲的入侵機制以及弓形蟲病的防治奠定基礎(chǔ)。
[Abstract]:Toxoplasma gondii (Toxopalsma gondii) is a unicellular protozoa, with worldwide distribution, almost all warm blooded animal invasion including human nucleated cells, which induced toxoplasmosis is a common zoonosis. Toxoplasmosis can cause miscarriage, stillbirth and neonatal malformations; at the same time as a opportunistic protozoan, Toxoplasma gondii is one of the important reasons caused by AIDS, organ transplantation and immune function in patients with malignant tumor is damaged or defective of death, such as 30-40% AIDS patients may result in Toxoplasma encephalitis, 2% may induce multiple organ toxoplasmosis. As people's eating habits and behavior changes, the infection rate of Toxoplasma gondii in there is a rising trend in China, at present the seropositive rate of Toxoplasma is about 7.88%, the harm caused by infection is drawing more and more attention, but so far There is no safe and effective drug treatment of toxoplasmosis.
Invasion is the primary pathogenic process of Toxoplasma gondii, Toxoplasma gondii invasion of host cells, in parasitophrous proliferation, proliferation to a certain number, parasitophorous vacuole rupture, worm out and invading new cells, blocking any one part of could treat toxoplasmosis.
Expression of Toxoplasma gondii invasion to host cells involves a variety of proteins, in recent years the host cell surface protein caveolin-1 (caveolin 1) in vivo pathogenic role in the process of more and more people pay attention to.Caveolin-1 is caveolae (caveolae) is an important structural protein, exists in a variety of human tissues and cells, and the interaction with lipid and protein lipid anchor structure, participate in various life activities, such as cholesterol transport, cellular signal transmission and so on. In the process of Toxoplasma gondii invasion of host cell formation of parasitophorous vacuole in the parasitophorous vacuole membrane caveolin-1 was excluded, whether caveolin-1 is involved in the invasion of the body of the host, there is no the relationship between the expression of relevant reports. Through the research of Toxoplasma gondii invasion force and caveolin-1, of caveolin-1 in the process of Toxoplasma gondii invasion in the role for the in-depth research of Toxoplasma gondii in The mechanism of invasion, the development of new drugs and the discovery of new drug targets lay the foundation.
The purpose of the study is:
The role of caveolin-1 during the invasion of Toxoplasma gondii was discussed, which lay the foundation for the in-depth study of the invasion mechanism of Toxoplasma gondii, the development of new drugs and the discovery of new drug targets.
Research methods:
1., the expression of caveolin-1 in Toxoplasma gondii after invasion of host cells: HeLa, 10min, 30min and 60min were infected by Toxoplasma gondii at high, medium and low concentrations of three groups. The invasion of the cells was observed under microscope, and the relative expression level of caveolin-1 was detected by real-time fluorescence quantitative PCR.
2. changes after inhibition of caveolin-1 gene expression of Toxoplasma gondii invasion rate: drugs were used to inhibit the expression of phosphate host cell caveolin-1 gene, 106 of Toxoplasma infection in host cells, to detect the inhibitory effects of caveolin-1, the expression of Toxoplasma gondii invasion rate and calculation of arch insect MIC2 gene
Change.
The results of the study:
1., after the invasion of host cells by Toxoplasma gondii, the expression of caveolin-1 increased, and was positively correlated with the prolongation of time and the increase of concentration. The expression level of 60min in high concentration group was the most significant, and the expression level was about 3.2 times of that of normal cells.
2., after inhibiting the expression of caveolin-1 gene, the invasion of Toxoplasma gondii decreased, and the expression of MIC2 protein in Toxoplasma gondii decreased correspondingly. The expression of MIC2 protein at 60min was about 50%., which was not inhibited by caveolin-1 gene expression.
Conclusion:
The expression of host cell caveolin-1 of Toxoplasma gondii invasion increased significantly and was positively correlated with the concentration of Toxoplasma gondii, and inhibit the expression of caveolin-1 of Toxoplasma gondii invasion rate decreased, the expression of invasion protein MIC2 was also decreased. The preliminary confirmed that caveolin-1 is involved in the process of Toxoplasma gondii invasion of host cells, and lay the foundation for the further study of the mechanism of invasion of Toxoplasma gondii and the prevention and treatment of toxoplasmosis.

【學位授予單位】：廣東藥學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R382

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本文編號：1503276