Caveolin-1的表达变化与弓形虫入侵力的相互关系

发布时间：2018-02-11 14:27

本文关键词： caveolin-1 弓形虫入侵力　出处：《广东药学院》2010年硕士论文　论文类型：学位论文

【摘要】： 刚地弓形虫(Toxopalsma gondii)是一种单细胞原虫,呈世界性分布,能侵犯包括人类在内的几乎所有温血动物有核细胞,其所致的弓形虫病是常见的人兽共患寄生虫病。弓形虫病能引起孕妇流产、新生儿畸形以及死胎等;同时作为一种机会性致病原虫,弓形虫还是引起艾滋病患者、器官移植和恶性肿瘤患者等免疫功能受损或缺陷者死亡的重要原因之一,如艾滋病病人中30-40%会发生弓形虫性脑炎,2%会发生多器官弓形虫病。随着人们饮食习惯及行为方式的改变,弓形虫感染率在我国正呈上升的趋势,我国目前弓形虫血清阳性率约为7.88%,其感染所带来的危害正受到越来越多人的关注,但是至今仍无治疗弓形虫病安全有效的药物。入侵是弓形虫首要的致病过程,弓形虫入侵宿主细胞后,在纳虫泡里不断增殖,增殖到一定数量后,纳虫泡破裂,散出的虫体再侵入到新的细胞中,对其中任何一环节的阻断都可以治疗弓形虫病。弓形虫对宿主细胞的入侵涉及多种蛋白的表达,近年来宿主细胞表面蛋白caveolin-1(窖蛋白1)在病原体内在化过程中的作用越来越引起人们的重视。Caveolin-1是胞膜窖(caveolae)的重要结构性蛋白,在人体多种组织和细胞中存在,且能与脂质和蛋白的脂质锚结构相互作用,参与多种生命活动,如胆固醇运输、细胞内外信号传递等。在弓形虫入侵宿主细胞形成纳虫泡的过程中,caveolin-1被纳虫泡膜排斥在外,是否caveolin-1参与了虫体对宿主的入侵,目前尚未见相关报道。本论文通过研究弓形虫入侵力与caveolin-1的表达变化之间的关系,探讨在弓形虫入侵过程中caveolin-1的作用,为深入研究弓形虫入侵机制,开发新的药物及发现新的药物靶点奠定基础。研究目的: 探讨在弓形虫入侵过程中caveolin-1的作用,为深入研究弓形虫入侵机制,开发新的药物及发现新的药物靶点奠定基础。研究方法: 1.弓形虫入侵宿主细胞后caveolin-1的表达变化:将高、中、低三组浓度的弓形虫分别感染HeLa细胞10min、30min和60min,显微镜下观察虫体入侵力改变并利用实时荧光定量PCR检测caveolin-1的相对表达水平。 2.抑制caveolin-1基因表达后弓形虫入侵率的变化:利用药物英卡磷酸抑制宿主细胞中caveolin-1基因的表达,将106个的弓形虫感染宿主细胞,检测caveolin-1的抑制效果、计算弓形虫入侵率的变化以及弓形虫MIC2基因的表达变化。研究结果: 1.弓形虫入侵宿主细胞后,caveolin-1的表达量增加,且与时间延长和浓度增加呈正相关,以60min高浓度组的表达变化最为显著,表达量约为正常细胞的3.2倍。 2.抑制caveolin-1基因的表达后,弓形虫入侵力下降,弓形虫MIC2蛋白的表达也相应降低,60min时MIC2蛋白的表达量约为caveolin-1基因表达未抑制的50%。结论: 弓形虫入侵后宿主细胞caveolin-1的表达显著升高,与弓形虫的浓度呈正相关,且抑制caveolin-1的表达弓形虫的入侵率下降,入侵蛋白MIC2的表达也明显降低。初步证实caveolin-1参与了弓形虫入侵宿主细胞的过程,为进一步研究弓形虫的入侵机制以及弓形虫病的防治奠定基础。
[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.

【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R382

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