本文選題：瘧疾　切入點：肝期瘧原蟲　出處：《第三軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：據(jù)統(tǒng)計,瘧疾仍然是世界上危害最為嚴(yán)重的傳染病之一。隨著近年來WHO提出在全球范圍內(nèi)消除瘧疾的宏偉目標(biāo),瘧疾的防治策略也逐漸由治療為主轉(zhuǎn)為預(yù)防為主。作為瘧疾的病原體,瘧原蟲在人體內(nèi)的發(fā)育包括紅內(nèi)期和紅外期。而紅外期不但是瘧原蟲感染的始動階段,而且也是導(dǎo)致間日瘧原蟲復(fù)發(fā)的重要時期。如果能有效地干預(yù)紅外期瘧原蟲(EEFs, exo-erythrocytic forms)的發(fā)育,則不僅能在源頭上阻斷瘧疾的感染,而且能防止瘧疾復(fù)發(fā),因此肝期是設(shè)計瘧疾預(yù)防手段的理想時期。 宿主細(xì)胞自噬是近年來發(fā)現(xiàn)非常重要的胞內(nèi)固有免疫應(yīng)答機(jī)制,然而其在紅外期瘧原蟲發(fā)育中的作用和機(jī)制尚未見報道。因此,本課題首先利用子孢子與肝細(xì)胞體外培養(yǎng)平臺,采用間接免疫熒光和激光共聚焦技術(shù)來觀察子孢子入侵小鼠肝細(xì)胞株后自噬發(fā)生情況；然后,通過定量PCR和計數(shù)存活EEFs的方法觀察分別加入自噬誘導(dǎo)劑或抑制劑后對紅外期發(fā)育的影響。實驗結(jié)果發(fā)現(xiàn),子孢子感染能誘導(dǎo)較低水平的肝細(xì)胞自噬瘧原蟲,加入自噬誘導(dǎo)劑雷帕霉素(Rapamycin, Rap)后則能促進(jìn)感染肝細(xì)胞自噬的發(fā)生,并自噬其中的瘧原蟲,而加入自噬抑制劑3-MA則能明顯抑制肝細(xì)胞對EEFs的自噬；有意思的是,誘導(dǎo)肝細(xì)胞自噬EEFs后不但不能清除瘧原蟲,反而能明顯促進(jìn)肝期瘧原蟲的發(fā)育。因此,本課題還對肝細(xì)胞自噬后肝期瘧原蟲存活以及促進(jìn)其發(fā)育的具體分子機(jī)制進(jìn)行了初步的探討。這有利于我們揭示新的肝期瘧原蟲免疫逃避機(jī)制,還可為設(shè)計新的紅外期干預(yù)措施和最終消除瘧疾,提供重要的理論依據(jù)和手段。 一、自噬促進(jìn)肝期瘧原蟲發(fā)育 1.P.y BY265-RFP子孢子和Hepa1-6細(xì)胞體外共培養(yǎng)平臺的建立：通過使用德國Whatman公司的DE52對解剖后的P.y BY265-RFP子孢子進(jìn)行過柱,這種方法能很好地去除雜質(zhì)和絕大多數(shù)細(xì)菌,然后再與Hepa1-6細(xì)胞進(jìn)行共培養(yǎng),以達(dá)到48h內(nèi)細(xì)胞狀態(tài)良好、無細(xì)菌污染的狀態(tài),并且子孢子入侵活力不受影響。 2.間接免疫熒光檢測子孢子入侵Hepa1-6后自噬發(fā)生情況：P.y BY265-RFP子孢子與Hepa1-6孵育6h、12h和24h,采用間接免疫熒光的方法標(biāo)記LC3,再用激光共聚焦觀察是否發(fā)生自噬,并統(tǒng)計出發(fā)生自噬的具體比例。結(jié)果發(fā)現(xiàn),子孢子感染后最早能在孵育后6h誘導(dǎo)肝細(xì)胞自噬,但在孵育后24h的自噬最為明顯,自噬發(fā)生率在20-30%。 3.自噬誘導(dǎo)劑和抑制劑對瘧原蟲紅外期的影響：在自噬發(fā)生率較高的24h這個時間點,使用自噬誘導(dǎo)劑雷帕霉素(Rapamycin, Rap)誘導(dǎo)自噬,結(jié)果顯示,誘導(dǎo)自噬能明顯增加肝細(xì)胞自噬EEFs的比例,以及含EEF自噬體與溶酶體結(jié)合的比例。然而,使用自噬的PI3K類抑制劑3-MA后,肝細(xì)胞自噬EEFs比例及其與溶酶體結(jié)合的比例顯著降低。 4.體外觀察肝細(xì)胞自噬對肝期瘧原蟲發(fā)育的影響：在P.yBY265子孢子和Hepa1-6共培養(yǎng)3h后,加入Rapamycin/3-MA后42h,收集細(xì)胞,采用定量RT-PCR檢測瘧原蟲蟲荷。結(jié)果提示,Rapamycin處理后能明顯地促進(jìn)EEFs的發(fā)育。反之,3-MA則能明顯地抑制EEFs的發(fā)育。 二、自噬促進(jìn)瘧原蟲肝期發(fā)育及機(jī)制研究 1. EEFs能在自噬體內(nèi)存活并增殖 為了證明誘導(dǎo)自噬能促進(jìn)肝期瘧原蟲發(fā)育,我們對自噬體中EEFs的形態(tài)及增殖能力進(jìn)行了觀察。子孢子和Hepal-6細(xì)胞共培養(yǎng)3h換液后,在雷帕霉素作用下培養(yǎng)至24h。然后采用間接免疫熒光的方法對LC3進(jìn)行標(biāo)記,并且使用DAPI或者EdU,(一種可以被整合進(jìn)入復(fù)制中DNA的胸腺嘧啶核苷類似物)觀察瘧原蟲增殖情況。實驗結(jié)果發(fā)現(xiàn)大多數(shù)(-70%)EEFs均處于分裂狀態(tài),這說明EEFs在自噬體中是存活的并處于增殖狀態(tài)。通過EdU進(jìn)一步檢測增殖情況表明,和宿主肝細(xì)胞細(xì)胞核一樣,EdU同樣被整合進(jìn)入了EEFs的細(xì)胞核,在被LC3包裹的自噬體中EEFs的核同樣標(biāo)記有EdU。因此,這些數(shù)據(jù)表明肝期瘧原蟲可以在自噬體中存活并分裂增殖。 2. EEFs抑制自噬溶酶體的酸化 被自噬的病原體大多數(shù)都會降解,而我們的結(jié)果卻表明瘧原蟲能夠存活其中,是否是因為包裹瘧原蟲的自噬溶酶體不具備降解能力?因此,為驗證包裹瘧原蟲的自噬溶酶體的降解功能,通過間接免疫熒光和共聚焦技術(shù)檢測溶酶體的酸化標(biāo)記CathD與EEFs結(jié)合情況,以此判斷瘧原蟲是否抑制了自噬溶酶體的酸化。結(jié)果顯示：正常入侵情況下,約30%的子孢子被自噬溶酶體包裹,但未被Cath D標(biāo)記包裹。而Rap誘導(dǎo)后,約80%的EEFs被自噬形成自噬溶酶體,但也未觀察到與Cath D聚集的情況。說明自噬溶酶體中溶酶體的酸化可能受到抑制。 本研究利用紅色熒光子孢子和肝細(xì)胞共培養(yǎng)平臺,采用間接免疫熒光技術(shù)證明了子孢子感染后Hepal-6對其發(fā)生自噬,加入自噬誘導(dǎo)劑Rap后則顯著促進(jìn)肝細(xì)胞自噬瘧原蟲,而加入自噬抑制劑3-MA明顯抑制肝細(xì)胞自噬瘧原蟲。RT-PCR及計數(shù)存活瘧原蟲結(jié)果顯示,誘導(dǎo)肝細(xì)胞自噬能明顯促進(jìn)肝期瘧原蟲的發(fā)育,其機(jī)制可能與瘧原蟲能抑制自噬溶酶體酸化密切相關(guān)。因此,本研究提示肝期瘧原蟲可利用肝細(xì)胞自噬促進(jìn)自身發(fā)育,這可能是肝期瘧原蟲的一種新的免疫逃避機(jī)制,可為設(shè)計新的紅外期干預(yù)措施提供新的靶點和思路。
[Abstract]:According to statistics, malaria is still the world's harm is one of the most serious infectious diseases in recent years. With the WHO set a grand goal to eliminate malaria worldwide, malaria prevention strategy gradually from treatment to prevention. As the causative agent of malaria, Plasmodium development in the human body including the erythrocytic and erythrocytic stage while not infrared Plasmodium infection but the initial stage, but also lead to an important period of Plasmodium vivax relapse. If the effective intervention of Plasmodium liver (EEFs, exo-erythrocytic forms) development, not only in the source of blocking malaria infection, but also can prevent the recurrence of malaria, so the liver stage is the ideal design period of malaria a means of prevention.
Autophagy in host cells is a newly discovered important intracellular mechanism of innate immune response, but its role and mechanism in the development of liver stage malaria has not been reported. Therefore, the subject of the first training platform by sporozoites and liver cells in vitro, to observe the occurrence of autophagy in sporozoite invasion mouse liver cells by indirect immunofluorescence immunofluorescence and confocal laser technology; then, through quantitative PCR and survival counting method to observe the EEFs were added to the effect on the infrared developing autophagy inducers or inhibitors. The experimental results show that the sporozoite infected liver cells can induce autophagy in low levels, adding autophagy inducerrapamycin (Rapamycin, Rap) after the infection can promote liver cell autophagy and autophagy occurs, the parasite, and adding autophagy inhibitor 3-MA can significantly inhibit autophagy of hepatic cells to EEFs; intentionally Thinking is that liver cells after EEFs induced autophagy can not only remove parasite, instead can obviously promote the liver stage Plasmodium development. Therefore, the task of liver cell autophagy after liver stage Plasmodium survival and promote the development of specific molecular mechanism was discussed. It is beneficial to us to reveal new liver stage Plasmodium immunity escape mechanism, can also design a new infrared intervention period and ultimately eliminate malaria, provide an important theoretical basis and means.
1. Autophagy promotes the development of Plasmodium in the liver
Co culture of 1.P.y BY265-RFP sporozoites and Hepa1-6 cells in vitro: establishment of the platform by using the German company Whatman DE52 on P.y BY265-RFP column sub dissected spores, this method can effectively remove impurities and most of bacteria, and then the Hepa1-6 cells were co cultured, in order to achieve the status of cells in 48h, no bacterial contamination, and sporozoite invasion activity was not affected.
2. indirect immunofluorescence detection of sporozoite invasion after Hepa1-6 autophagy: P.y BY265-RFP sporozoites incubated with Hepa1-6 6h, 12h and 24h, LC3 labeling methods by indirect immunofluorescence, and confocal laser to observe whether autophagy, autophagy and the statistics of the specific proportion. It is found that the sporozoite infection after the earliest could induce autophagy in liver cells after incubation with 6h, but in 24h after incubation of autophagy is most obvious, the incidence of autophagy in 20-30%.
3. autophagy inducer effects and inhibitors on Plasmodium infrared period: autophagy in higher rates of 24h at this time, the use of autophagy inducer rapamycin (Rapamycin, Rap) induced autophagy, results show that the induction of autophagy could increase hepatic autophagy EEFs ratio, and the ratio of EEF and lysosome combination. However PI3K, 3-MA using inhibitors of autophagy, autophagy and liver EEFs ratio and lysosomal combination significantly reduced.
4. in vitro to observe the effect of autophagy on the development of liver cells in the liver stage Plasmodium: 3H co cultured in P.yBY265 sporozoites and Hepa1-6, collected after joining Rapamycin/3-MA 42h cells was detected by quantitative RT-PCR parasite. Malaria bearing results suggest that Rapamycin treatment could significantly promote the development of EEFs. On the other hand, 3-MA can significantly inhibit the development of EEFs.
Two, autophagy promotes the development and mechanism of Plasmodium falciparum in the liver
1. EEFs can survive and proliferate in autophagy
In order to prove the induction of autophagy can promote the development of liver stage Plasmodium, we EEFs on autophagy in morphology and proliferation were observed. The co culture of sporozoites and Hepal-6 cells 3H was changed after the method in rapamycin culture to 24h. and by indirect immunofluorescence of LC3 in marker, and use the DAPI or EdU. (a can be integrated into the thymidine analogue replication DNA) observation of Plasmodium proliferation. Experimental results show that the majority of (-70%) EEFs were disunited, indicating that EEFs is alive and in the proliferation in autophagy. Detect the proliferation that further by EdU, and the host liver cell nuclei. EdU is also integrated into the EEFs cell by EEFs LC3 wrapped in autophagosome also marked nuclear EdU.. Therefore, these data suggest that the liver stage Plasmodium in autophagosomes in Survive and divide and proliferate.
Inhibition of autophagic lysosome acidification by 2. EEFs
Is the pathogen most autophagic degradation, and we showed that the parasite can survive in it, whether it is because the package does not have the lysosomal degradation ability of Plasmodium? Therefore, the degradation function of the validation package autolysosome with Plasmodium, by acidification markers CathD and EEFs indirect immunofluorescence and confocal detection of lysosomes in order to determine whether inhibition of Plasmodium, acidification of autophagy lysosome. The results showed that the normal intrusion situation, about 30% of the sporozoites are autolysosome package, but not the Cath D tag package. And after the induction of Rap, about 80% of the EEFs is the formation of autophagy lysosomal, but also not observed in the case of D and Cath together that autophagolysosomes in lysosomal acidification may be inhibited.
The research of co culture platform using the red fluorescent sporozoites and liver cells by indirect immunofluorescence technique to prove that the sporozoite infection after Hepal-6 on autophagy, adding inducer of autophagy after Rap significantly promote liver cell autophagy Plasmodium, while adding autophagy inhibitor 3-MA significantly inhibited the liver cell autophagy and survival of Plasmodium Plasmodium.RT-PCR counting results showed that autophagy induced by liver cells could promote liver stage Plasmodium development, its mechanism may be related to Plasmodium could inhibit autophagy lysosomal acidification are closely related. Therefore, this study suggests that the liver stage Plasmodium available to promote their own development with liver cell autophagy, which may be a new immune liver stage Plasmodium escaping mechanism, can provide a new target the intervention measures and ideas for the design of new infrared phase.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R531.3

【共引文獻(xiàn)】

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本文編號：1642837