肝细胞自噬对肝期疟原虫发育的影响及其机制初探

发布时间：2018-03-21 07:22

本文选题：疟疾　切入点：肝期疟原虫　出处：《第三军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：据统计,疟疾仍然是世界上危害最为严重的传染病之一。随着近年来WHO提出在全球范围内消除疟疾的宏伟目标,疟疾的防治策略也逐渐由治疗为主转为预防为主。作为疟疾的病原体,疟原虫在人体内的发育包括红内期和红外期。而红外期不但是疟原虫感染的始动阶段,而且也是导致间日疟原虫复发的重要时期。如果能有效地干预红外期疟原虫(EEFs, exo-erythrocytic forms)的发育,则不仅能在源头上阻断疟疾的感染,而且能防止疟疾复发,因此肝期是设计疟疾预防手段的理想时期。宿主细胞自噬是近年来发现非常重要的胞内固有免疫应答机制,然而其在红外期疟原虫发育中的作用和机制尚未见报道。因此,本课题首先利用子孢子与肝细胞体外培养平台,采用间接免疫荧光和激光共聚焦技术来观察子孢子入侵小鼠肝细胞株后自噬发生情况；然后,通过定量PCR和计数存活EEFs的方法观察分别加入自噬诱导剂或抑制剂后对红外期发育的影响。实验结果发现,子孢子感染能诱导较低水平的肝细胞自噬疟原虫,加入自噬诱导剂雷帕霉素(Rapamycin, Rap)后则能促进感染肝细胞自噬的发生,并自噬其中的疟原虫,而加入自噬抑制剂3-MA则能明显抑制肝细胞对EEFs的自噬；有意思的是,诱导肝细胞自噬EEFs后不但不能清除疟原虫,反而能明显促进肝期疟原虫的发育。因此,本课题还对肝细胞自噬后肝期疟原虫存活以及促进其发育的具体分子机制进行了初步的探讨。这有利于我们揭示新的肝期疟原虫免疫逃避机制,还可为设计新的红外期干预措施和最终消除疟疾,提供重要的理论依据和手段。一、自噬促进肝期疟原虫发育 1.P.y BY265-RFP子孢子和Hepa1-6细胞体外共培养平台的建立：通过使用德国Whatman公司的DE52对解剖后的P.y BY265-RFP子孢子进行过柱,这种方法能很好地去除杂质和绝大多数细菌,然后再与Hepa1-6细胞进行共培养,以达到48h内细胞状态良好、无细菌污染的状态,并且子孢子入侵活力不受影响。 2.间接免疫荧光检测子孢子入侵Hepa1-6后自噬发生情况：P.y BY265-RFP子孢子与Hepa1-6孵育6h、12h和24h,采用间接免疫荧光的方法标记LC3,再用激光共聚焦观察是否发生自噬,并统计出发生自噬的具体比例。结果发现,子孢子感染后最早能在孵育后6h诱导肝细胞自噬,但在孵育后24h的自噬最为明显,自噬发生率在20-30%。 3.自噬诱导剂和抑制剂对疟原虫红外期的影响：在自噬发生率较高的24h这个时间点,使用自噬诱导剂雷帕霉素(Rapamycin, Rap)诱导自噬,结果显示,诱导自噬能明显增加肝细胞自噬EEFs的比例,以及含EEF自噬体与溶酶体结合的比例。然而,使用自噬的PI3K类抑制剂3-MA后,肝细胞自噬EEFs比例及其与溶酶体结合的比例显著降低。 4.体外观察肝细胞自噬对肝期疟原虫发育的影响：在P.yBY265子孢子和Hepa1-6共培养3h后,加入Rapamycin/3-MA后42h,收集细胞,采用定量RT-PCR检测疟原虫虫荷。结果提示,Rapamycin处理后能明显地促进EEFs的发育。反之,3-MA则能明显地抑制EEFs的发育。二、自噬促进疟原虫肝期发育及机制研究 1. EEFs能在自噬体内存活并增殖为了证明诱导自噬能促进肝期疟原虫发育,我们对自噬体中EEFs的形态及增殖能力进行了观察。子孢子和Hepal-6细胞共培养3h换液后,在雷帕霉素作用下培养至24h。然后采用间接免疫荧光的方法对LC3进行标记,并且使用DAPI或者EdU,(一种可以被整合进入复制中DNA的胸腺嘧啶核苷类似物)观察疟原虫增殖情况。实验结果发现大多数(-70%)EEFs均处于分裂状态,这说明EEFs在自噬体中是存活的并处于增殖状态。通过EdU进一步检测增殖情况表明,和宿主肝细胞细胞核一样,EdU同样被整合进入了EEFs的细胞核,在被LC3包裹的自噬体中EEFs的核同样标记有EdU。因此,这些数据表明肝期疟原虫可以在自噬体中存活并分裂增殖。 2. EEFs抑制自噬溶酶体的酸化被自噬的病原体大多数都会降解,而我们的结果却表明疟原虫能够存活其中,是否是因为包裹疟原虫的自噬溶酶体不具备降解能力?因此,为验证包裹疟原虫的自噬溶酶体的降解功能,通过间接免疫荧光和共聚焦技术检测溶酶体的酸化标记CathD与EEFs结合情况,以此判断疟原虫是否抑制了自噬溶酶体的酸化。结果显示：正常入侵情况下,约30%的子孢子被自噬溶酶体包裹,但未被Cath D标记包裹。而Rap诱导后,约80%的EEFs被自噬形成自噬溶酶体,但也未观察到与Cath D聚集的情况。说明自噬溶酶体中溶酶体的酸化可能受到抑制。本研究利用红色荧光子孢子和肝细胞共培养平台,采用间接免疫荧光技术证明了子孢子感染后Hepal-6对其发生自噬,加入自噬诱导剂Rap后则显著促进肝细胞自噬疟原虫,而加入自噬抑制剂3-MA明显抑制肝细胞自噬疟原虫。RT-PCR及计数存活疟原虫结果显示,诱导肝细胞自噬能明显促进肝期疟原虫的发育,其机制可能与疟原虫能抑制自噬溶酶体酸化密切相关。因此,本研究提示肝期疟原虫可利用肝细胞自噬促进自身发育,这可能是肝期疟原虫的一种新的免疫逃避机制,可为设计新的红外期干预措施提供新的靶点和思路。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R531.3

【共引文献】