本文選題：剛地弓形蟲 + 納蟲泡膜��； 參考：《南方醫(yī)科大學》2013年碩士論文

【摘要】：剛地弓形蟲(Toxoplasma gondii)是細胞內(nèi)寄生原蟲,能感染多種有核細胞,全世界約有三分之一的人口隱性感染弓形蟲。弓形蟲感染人體后,在免疫正常人群可由急性感染快速地轉(zhuǎn)為隱性感染,因而危害不明顯；而在免疫抑制患者體內(nèi)會廣泛散播,寄生于有核細胞引起炎癥,弓形蟲主要損害腦、眼和淋巴結(jié),甚至會引起死亡；在孕婦感染后,弓形蟲可通過胎盤傳播給胎兒,造成流產(chǎn)、死產(chǎn)、死胎、畸胎或新生兒弓形蟲病。人類感染弓形蟲的途徑主要是通過誤食未煮熟的中間宿主的肉或被終宿主(貓)排泄的卵囊污染的食物。 弓形蟲感染宿主后會刺激機體產(chǎn)生免疫反應以抵抗弓形蟲感染,在感染早期,細胞免疫反應在機體內(nèi)發(fā)揮主要抗感染作用。免疫的形成雖可有效地限制感染的發(fā)展和新?lián)p傷的形成,但一般并不能消除弓形蟲感染。多數(shù)研究者認為,弓形蟲具有帶蟲免疫的特點。機體只在感染過程中才對再感染具有一定程度的抵抗力,而蟲體清除后該種免疫力也消失。弓形蟲可以在機體免疫反應環(huán)境中保持存活,主要是通過在弓形蟲納蟲泡內(nèi)保持良好增殖和干擾宿主細胞信號轉(zhuǎn)導的方式進行的,但宿主免疫反應只能被部分削弱,而不是完全被消除。弓形蟲通過在誘導和抑制宿主免疫反應過程中保持著精致平衡,來保證在宿主細胞內(nèi)寄生、增殖并有機會傳播。弓形蟲入侵和寄生過程中有許多宿主細胞信號通路的參與并出現(xiàn)廣泛變化,在弓形蟲入侵、寄生、增殖及與弓形蟲-宿主細胞相互關(guān)系中起著重要的作用。 弓形蟲感染宿主細胞的機制已被廣泛研究,在弓形蟲入侵時,速殖子是弓形蟲的主要致病蟲期,其在細胞內(nèi)寄生和迅速繁殖破壞宿主細胞,逸出后的裂殖子又侵襲鄰近的細胞,如此反復,刺激淋巴細胞、巨噬細胞的浸潤,導致組織的急性炎癥和壞死。弓形蟲侵入細胞的過程依次為：附著于宿主細胞；伸出類錐體；進入宿主細胞；蟲體向后移動的運動連接(moving junction, MJ)形成；微線體的分泌作用；棒狀體的分泌作用；蟲體滑入納蟲空泡,在10s內(nèi)完成整個侵入過程,蟲體進入胞漿后運動較為緩慢,并且有向核移動的特性。在弓形蟲侵入宿主細胞過程中會形成納蟲泡(parasitophorous vacuole, PV),在這個過程中,MJ會清除宿主細胞的一些組分(如跨膜蛋白),同時保留大部分的宿主細胞膜組分組成到納蟲泡膜(parasitophorous vacuole membrane, PVM)上,此外因宿主細胞膜中能與溶酶體和內(nèi)涵體融合的組分已被MJ過濾掉,使PV成為一個非融合小室,有助于其內(nèi)的速殖子抵抗宿主細胞內(nèi)含體的融合及溶酶體的酸化作用。 GTP酶(GTPase)是能夠與三磷酸鳥苷(guanine triphosphate, GTP)結(jié)合并在Mg2+作用下將其水解為二磷酸鳥苷(guanine diphosphate, GDP)的超蛋白家族。小分子GTP酶是相對分子質(zhì)量為20kDa-40kDa的單體調(diào)控性GTP酶,至少由5個家族組成,分別是:Ras、Rho、Rab、Sar1/Arf和Ran。目前研究較多的為Ras和Rho家族。其中Rho族蛋白主要包括Rho、Rac和Cdc42三個亞家族,這些蛋白最主要的功能是調(diào)節(jié)細胞骨架和肌動蛋白重組,其他功能包括調(diào)控基因轉(zhuǎn)錄、細胞周期和膜泡運輸?shù)取?Rho GTP酶最主要的功能是調(diào)節(jié)肌動蛋白和細胞骨架的重組,從而調(diào)節(jié)細胞的形態(tài)變化和運動。該族蛋白的活化能促使細胞外基質(zhì)的降解、破壞上皮層的結(jié)構(gòu)和增加細胞的運動性。Rho族蛋白的活化和失活在真核細胞的許多基礎(chǔ)性生命活動中都起著關(guān)鍵的作用。弓形蟲入侵宿主細胞的過程伴隨著宿主細胞結(jié)構(gòu)的重組,包括宿主細胞中心粒及高爾基復合體定位在納蟲泡上,線粒體、微管及溶酶體被納入了納蟲泡的邊緣。 弓形蟲感染宿主細胞時,宿主細胞由干擾素誘導產(chǎn)生的GTP酶(IFN-y-Inducible GTPase6, Irga6)和小分子GTP酶ARF6可聚集在弓形蟲PVM上,Irga6可以抵抗小鼠被弓形蟲感染,Irga6在未感染的細胞內(nèi)主要是以GDP非活化結(jié)合狀態(tài)存在,但當弓形蟲感染細胞后Irga6會聚集在PVM上并且轉(zhuǎn)化為GTP活化狀態(tài)存在,Irga6的這種聚集最終導致PVM的破裂。ARF6通過激活PI3K、PIP2和PIP3一起參與納蟲泡的形成,并且是通過聚集在PVM上起重要作用的。GTP酶在弓形蟲入侵宿主過程中起所起的重要作用促使我們進一步去探討,是否其它小分子GTP酶也同樣參與了弓形蟲感染宿主細胞的過程。 目的： 1.分析在弓形蟲感染宿主細胞時,宿主細胞的RhoA和Racl GTP酶是否組成到弓形蟲的PVM上。 2.了解RhoA和Rac1GTP酶負顯性突變體是否組成到弓形蟲的PVM上。 3.分析弓形蟲速殖子感染后宿主細胞內(nèi)源性RhoA和Rac1GTP酶的活化狀態(tài)。 4.探討RhoA GTP酶在弓形蟲PVM上聚集時哪些結(jié)構(gòu)域是必須的。 5.分析聚集在PVM上的RhoA和Rac1GTP酶的活性狀態(tài)是否依賴其本身的GTP酶活性。 6.探討細胞RhoA和Rac1GTP酶的缺失是否影響弓形蟲速殖子入侵宿主細胞效率。 方法： 1.轉(zhuǎn)染pECFP-RhoA-WT和pECFP-Rac1-WT質(zhì)粒于COS-7細胞,48hr后細胞分別用弓形蟲RH株和Pru株速殖子侵染2hr,4%多聚甲醛固定細胞30min后,用DAPI室溫下染色10mmin,熒光顯微鏡下觀察CFP標簽的RhoA和Rac1是否被組成到納蟲泡膜上。 2.弓形蟲RH速殖子感染16HBE細胞后,用間接免疫熒光法對內(nèi)源性RhoA和Rac1進行定位。 3.弓形蟲速殖子感染轉(zhuǎn)染了pECFP-RhoA-WT于COS-7細胞后,在感染5min后用共聚焦顯微法每10min實時觀察宿主細胞RhoA GTP酶在PVM上的聚集情況。 4.分別轉(zhuǎn)染RhoA和Racl負顯性突變體(pECFP-RhoA-N19和pECFP-Racl-N17)于COS-7細胞中,48hr后用速殖子侵染2hr,4%多聚甲醛固定細胞30mmin后,用DAPI室溫下染色10min,熒光顯微鏡下觀察非活化的RhoA和Rac1是否會聚集在納蟲泡膜上。 5.16HBE細胞用RH株速殖子感染后收集細胞,用RIPA試劑裂解,120ug總蛋白用于Rho GST pull down,檢測弓形蟲入侵是否激活RhoA和Rac1GTP酶。 6.以pECFP-RhoA-WT為親本質(zhì)粒,構(gòu)建RhoA每10個氨基酸缺失的突變體19個即M1-M19。將M1-M19轉(zhuǎn)化入COS-7細胞,48hr后用RH株速殖子侵染2hr,4%多聚甲醛固定細胞30min后,用DAPI室溫下染色10min,熒光顯微鏡下觀察各突變體RhoA在納蟲泡膜上的聚集情況。 7.COS-7細胞轉(zhuǎn)染pECFP-RhoA-WT后速殖子侵染2hr,100nM EGF加于六孔板一角,激活5min后用多聚甲醛固定細胞,DAPI室溫下染色,熒光顯微鏡下觀察RhoA的轉(zhuǎn)位情況。 8.宿主細胞RhoA和Racl的活性及存在與弓形蟲感染率關(guān)系的研究： a.正常COS-7細胞COS-7細胞分別轉(zhuǎn)染pECFP-RhoA-WI、pECFP-RhoA-N19和pECFP-Racl-WT、pECFP-Rael-N17,48hr后速殖子侵染2hr,多聚甲醛固定細胞,Giemsa染色,計算細胞感染率并進行統(tǒng)計學分析,比較正常細胞與轉(zhuǎn)染細胞的感染率是否有統(tǒng)計學差異。 b.正常16HBE細胞及單獨轉(zhuǎn)染RhoA-siRNA、Rac1-siRNA和共同RhoA-siRNA和Racl-siRNA轉(zhuǎn)染組的16HBE細胞,用速殖子侵染2hr,多聚甲醛固定細胞,Giemsa染色,計算細胞感染率并進行統(tǒng)計學分析,比較正常細胞與轉(zhuǎn)染細胞的感染率是否有統(tǒng)計學差異。 結(jié)果： 1.小分子GTP酶-RhoA和Racl在弓形蟲感染宿主細胞時會在PVM上聚集。 2. RhoA和Racl GTP酶在PVM上的聚集以GTP酶活性狀態(tài)存在。 3.弓形蟲速殖子的感染可以激活宿主細胞內(nèi)源性RhoA和Racl GTP酶。 4. RhoA GTP酶依賴不同的RhoA結(jié)構(gòu)域在弓形蟲PVM上聚集。 5.細胞RhoA和Racl GTP酶為弓形蟲速殖子有效入侵宿主細胞所必需。 結(jié)論： 1.無論弓形蟲弱毒株還是強毒株速殖子,宿主細胞RhoA和Racl GTP酶在弓形蟲速殖子入侵時都會聚集在PVM上,而且這種聚集依賴其完整的GTP酶活性。 2.弓形蟲速殖子感染細胞可以激活內(nèi)源性RhoA和Racl GTP酶,并且在弓形蟲剛開始入侵時RhoA和Racl GTP酶就會在PVM上聚集,聚集的RhoA和Racl GTP酶來自細胞膜或者細胞質(zhì)。 3.決定RhoA在PVM上聚集的結(jié)構(gòu)域有:GTP/Mg2+結(jié)合位點、Mdia效應器作用位點、G1box、G2box和G5box。 4.表皮生長因子(EGF)刺激感染的宿主細胞后,RhoA和Racl GTP酶在PVM上并沒有發(fā)生移位現(xiàn)象,這顯示RhoA和Racl可能以GTP活化形式存在于PVM上。 5.細胞過表達pECFP-RhoA-WT、pECFP-Rac1-WT、pECFP-RhoA-N19、 pECFP-Racl-N17和將細胞內(nèi)RhoA和Racl GTP酶基因沉默后對細胞感染率的統(tǒng)計數(shù)據(jù)顯示RhoA和Racl GTP酶為弓形蟲有效入侵所必需,RhoA和Racl GTP酶在PVM上聚集及其必須的生物活性都顯示RhoA和Racl GTP酶參與并作用于弓形蟲速殖子感染宿主細胞這一過程。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a parasitic protozoa in cells and can infect a variety of nucleated cells. About 1/3 of the population in the world recessive infection of Toxoplasma gondii. After Toxoplasma infection, the immune normal population can be rapidly transformed from acute infection to recessive infection, and the harm is not obvious. "Spread, parasitic on nuclear cells causing inflammation, and Toxoplasma mainly damage the brain, eyes and lymph nodes and even cause death; after infection, Toxoplasma can be transmitted to the fetus through the placenta, causing abortion, stillbirth, stillbirth, Teratosis, or neonatal toxoplasmosis. Human infection of Toxoplasma gondii is mainly through misfeeding of uncooked middle Lodge." Food contaminated by the main oocysts or the excretory oocysts of the final host (CAT).
When Toxoplasma infects the host, it can stimulate the body to produce immune response to resist Toxoplasma infection. In the early stage of the infection, the cellular immune response plays a major anti infection role in the body. Although the formation of immune formation can effectively restrict the development of infection and the formation of new injuries, the infection can not be eliminated. Most researchers believe that the toxoplasmosis is arcuate. The insect has the characteristics of immunization with insects. The body has a certain degree of resistance to reinfection only in the process of infection, and the immunity of the species is disappearing after the insect body is cleared. Toxoplasma can remain alive in the immune response environment of the body, mainly by maintaining good proliferation and interfering with the signal transduction of host cells in the Toxoplasma Nana vesicles. The host immune response can only be partially weakened, rather than completely eliminated. Toxoplasma maintains a delicate balance in the induction and suppression of host immune responses to ensure parasitism in the host cell, proliferate and spread organically. In the process of Toxoplasma invasion and parasitism, there are many host cell signaling pathways. And extensive changes play an important role in the invasion, parasitism, proliferation and Toxoplasma gondii host cell interactions.
The mechanism of Toxoplasma infection in host cells has been widely studied. At the time of the invasion of Toxoplasma gondii, tachyonus is the main pathogenic stage of Toxoplasma gondii. The parasitic and rapid propagation of the parasitic and rapid propagation of the host cells, the escape of the merozoites and the invasion of adjacent cells, so repeated, stimulating gonadal cells, macrophage infiltration, causing acute inflammation of the tissue. Disease and necrosis. The process of toxoplasmosis invades the cells in sequence: attaching to the host cell; extending the cones; entering the host cell; the movement of the moving junction, MJ; the secretory action of the micro body; the secretory effect of the rod; the insect body slips into the vacuoles of the NETA, and completes the whole invasion process within 10s. When the body enters the cytoplasm, it moves slowly and has the characteristics of moving to the nucleus. In the process of Toxoplasma invasion, parasitophorous vacuole (PV) can be formed. In this process, MJ will remove some components of the host cell (such as transmembrane protein), and retain most of the host cell membrane components to form the nanofilm membrane (PA Rasitophorous vacuole membrane, PVM), in addition, the components that can be fused with lysosomes and endosomes in the host cell membrane have been filtered out by MJ, making PV a non fusion chamber, helping the tachyonus to resist the fusion of the host cell and the acidification of the lysosome.
GTP enzyme (GTPase) is a superprotein family that combines with guanine triphosphate (guanine triphosphate, GTP) and hydrolyze it into guanosine two phosphate (guanine diphosphate, GDP) under the action of Mg2+. The small molecule GTP enzyme is a monomeric regulative GTP enzyme with relative molecular mass as 20kDa-40kDa, at least 5 families. Ras and Rho families, which are currently studied by Ran., are mainly composed of three subfamilies of Rho, Rac and Cdc42. The main functions of these proteins are the regulation of cytoskeleton and actin, and other functions include regulation of gene transcription, cell cycle and vesicle transport.
The main function of Rho GTP enzyme is to regulate the reorganization of actin and cytoskeleton, thereby regulating the morphological changes and movement of cells. The activation of this protein can induce the degradation of the extracellular matrix, destroy the structure of the epithelial layer and increase the activation and inactivation of the motile.Rho protein of the cell in many basic life activities of the eukaryotic cells. The process of the invasion of the host cell of Toxoplasma gondii is accompanied by the reorganization of the host cell structure, including the host cell centroid and the Golgi complex located on the nanoscale vesicles, and the mitochondria, microtubules and lysosomes are incorporated into the edge of the nanoscale vesicles.
When Toxoplasma infects host cells, the GTP enzyme (IFN-y-Inducible GTPase6, Irga6) and small molecule GTP enzyme ARF6 induced by interferon can accumulate on the Toxoplasma PVM. Irga6 can resist the infection of mice by Toxoplasma gondii. Irga6 is mainly in the non activated binding state of GDP in the uninfected cells, but when Toxoplasma infected cells are infected. Irga6 will accumulate on PVM and convert to GTP activation state. This aggregation of Irga6 eventually leads to the disruption of PVM by.ARF6 by activating PI3K, PIP2 and PIP3 to participate in the formation of Nana vesicles, and is an important role of the.GTP enzyme that plays an important role in the PVM and plays an important role in the invasion of Toxoplasma gondii. One step is to explore whether other small GTP enzymes are also involved in the process of Toxoplasma infection in host cells.
Objective:
1. to analyze whether the RhoA and Racl GTP enzymes of the host cells are on the PVM of Toxoplasma gondii.
2. understand whether RhoA and Rac1GTP negative dominant mutants are on the PVM of Toxoplasma gondii.
3. to analyze the activation of endogenous RhoA and Rac1GTP enzymes in Toxoplasma gondii infected tachyhost.
4. to explore which domains are necessary for RhoA GTP enzyme to accumulate on PVM of Toxoplasma gondii.
5. analyze whether the activity of RhoA and Rac1GTP enzymes aggregated on PVM depends on their GTP enzyme activity.
6. to investigate whether the deletion of RhoA and Rac1GTP enzymes affects the efficiency of Toxoplasma gondii in the invasion of host cells.
Method錛，

本文編號：1973731