约氏疟原虫红外期相关基因及宿主抗疟原虫子孢子感染免疫分子研究

发布时间：2018-10-15 19:34

【摘要】： 蚊媒传播的疟疾是一种严重危害人类健康的热带传染病,在全世界人群中具有很高的发病率和致死率,影响流行地区经济和社会发展,威胁着100多个国家近40%的世界人口,在热带和亚热带地区尤为严重。虽然经过疟疾工作者长期不懈的努力探索,但是目前全世界每年大约有3～5亿人感染,约1～3百万患者因疟疾感染而死亡,其中绝大多数是五岁以下的儿童。在我国南方地区,尤其是山区,在特定的环境下仍有大规模流行的可能。近年来,随着疟原虫多药抗性株的出现与迅速扩散以及蚊媒对杀虫剂耐受性的增加,而目前又无有效的抗疟疫苗,这给疟疾防治工作带来很大的困难。疟疾控制工作正面临巨大的挑战,寻求新型、高效、安全的疟疾控制策略已迫在眉睫。疟疾由感染性蚊媒叮咬传播,疟原虫子孢子进入血循环后可迅速粘附并侵入肝细胞,完成红外期增殖之后才侵入红细胞导致疟疾发作。所以干扰裂殖子在肝细胞内的发育是防止疟疾传播与临床症状出现的关键。因此研制靶向红外期疟原虫的疫苗不仅可防止红内期疟原虫导致的病理损伤,还可阻断配子体传播。然而宿主肝细胞、肝组织所产生的高背景干扰,以及难以获得足量高纯度肝期疟原虫的技术障碍严重阻碍了疟原虫红外期的研究。但随着分子生物学和生物化学技术的发展,疟原虫红外期方面的研究已经越来越受到人们关注。疟原虫抗原差异与变异十分普遍,并具有非常复杂的抗原结构和众多不同的抗原表位,多数抗原表位在生活史不同时期具有阶段特异性,此外疟原虫还存在种、株特异性及抗原性弱等特点,侵入宿主体后可引起复杂的免疫反应。T细胞免疫在疟原虫引起的免疫应答中具有至关重要的地位,因此,研究诱导T细胞活化的共刺激分子在红外期免疫中的作用是疟疾治疗及疫苗研制的基础。因此本课题以分离获得的约氏疟原虫子孢子经尾静脉注射感染大鼠,建立约氏疟原虫—斯氏按蚊—哺乳动物宿主模型,对红外期疟原虫在宿主体内发育变化以及宿主免疫系统接受疟原虫刺激后所产生免疫应答的两个方面进行研究:一方面应用差异显示PCR(differential display PCR,DD-PCR)技术筛选约氏疟原虫红外期cDNA,经比较分析获得的差异序列,克隆至TA载体,根据序列同源分析和功能预测结果,推测可能与疟原虫红外期发育相关的基因;另一方面采用RT-PCR与直接免疫荧光反应、激光共聚焦技术相结合的方法,从基因和共刺激分子表达情况对宿主针对疟原虫抗原刺激产生的免疫反应进行研究。实验内容和结果主要包括以下几个方面: 1.针对疟原虫基因A+T含量高(＞70%)的特点,本实验通过改变常规DD-PCR引物的A+T含量,设计能从肝组织和疟原虫混存样本中特异扩增疟原虫基因的A+T含量60%的引物,与DD-PCR技术相结合,绕过了纯化裂殖子的技术屏障,特异扩增疟原虫基因。将得到的核酸序列登陆GenBank数据库,通过Blast查询,并进行序列同源性比对、分析,结果显示在21个待测克隆中:有8个与约氏疟原虫红外期相关基因(PyHs4、PyHs5、PyHs6、PyHs7、PyHs8、PyHs9、PyHs10、PyHs11);其中PyHs4与约氏疟原虫乙酰葡萄糖胺磷酸变位酶(Phosphoacetylglucosamine mutase,AGM)具有相似性,PyHs5与约氏疟原虫红细胞膜蛋白3(Erythrocyte membrance prontein,EMP3)具有同源性,PyHs6、PyHs7、PyHs8、PyHs9、PyHs10、PyHs11为功能未知基因。发现、鉴定的疟原虫差异基因,为进一步研究疟原虫红外期生长发育的分子机制提供了理论基础。 2.采用RT-PCR方法对不同时相点宿主免疫相关基因B7.1、B7.2、TGF-β、IFN-γ进行定性、定量分析,探讨感染疟原虫后哺乳动物宿主体内免疫相关基因变化情况:在宿主体感染约氏疟原虫后2～72h,B7.1表达几乎没有变化;B7.2、TGF-β、IFN-γ转录水平显著上调(P＜0.05)。该结果显示疟原虫侵入可能引发哺乳动物机体免疫防御、免疫调控等一系列相关反应。将直接免疫荧光反应和激光共聚焦技术相结合,观察疟原虫感染后共刺激分子B7.1、B7.2的表达定位情况,实验发现:在所选择的时相区间内,B7.1、B7.2分子表达有升高趋势,其中B7.1分子上升速度较缓慢在72h后开始下降,而B7.2分子上调迅速在48h达到峰值后于72h出现回落;在疟原虫感染后的2～72h,B7.2的表达水平始终高于B7.1(P＜0.05)。此外,激光扫描共聚焦显微镜观察显示:疟原虫感染后,B7.1、B7.2分子在巨噬细胞的表达随着时间点推延从细胞浆、细胞核逐渐转移到细胞膜。根据该现象我们可以推测:接受疟原虫刺激后,共刺激分子开始活化,其合成、表达增加并可能与T细胞活化有关。综上所述,我们从寄生虫和宿主两方面对红外期疟原虫发育相关分子及宿主抗疟原虫感染的部分免疫分子进行了研究,较为系统地探讨疟原虫裂殖子在肝细胞内发育的分子机理以及所引起的免疫反应,并在基因和免疫分子表达水平初步探讨疟原虫感染后宿主免疫细胞、效应分子的作用机制,从而为新型抗疟药物和疫苗研制提供靶目标。
[Abstract]:Anopheles mosquito-borne malaria is a tropical infectious disease that seriously endangers human health. It has high morbidity and mortality in people around the world, affecting economic and social development in epidemic areas, threatening nearly 40% of the world's population in more than 100 countries, Especially in tropical and sub-tropical areas. Despite the long-term efforts of malaria workers to explore, there are currently about 3-500 million people worldwide, about 1-3 million people die due to malaria infection, most of whom are children under five years of age. In the southern region of our country, especially in mountainous areas, there is still a large-scale epidemic in a particular environment. In recent years, with the emergence and rapid diffusion of P-P resistant strains and the increase of mosquito-mediated resistance to insecticides, there is no effective antimalarial vaccine, which brings great difficulties to the prevention and treatment of malaria. Malaria control is facing enormous challenges and seeks new, efficient and safe malaria control strategies. Malaria is transmitted by infectious mosquito bites. After the sporozoites of Plasmodium vivax enter the blood circulation, they can quickly adhere to and invade the liver cells. After the proliferation of the infrared period, the invasion of red blood cells leads to malaria. The development of interfering merozoites in hepatocytes is the prevention of malaria transmission and clinical symptoms. Therefore, the development of vaccine targeting Plasmodium vivax can not only prevent the pathological damage caused by Plasmodium vivax, but also block the gamete. However, the high background interference caused by the host hepatocytes, liver tissue, However, with the development of molecular biology and biochemical technology, the research on the infrared period of P. P. has become more and more affected The antigenic difference and variation of P. falciparum are very common, and have very complex antigenic structure and many different antigen table positions. Most of the antigen table bits have stage specificity in different periods, and there are also species and strains of P. falciparum. has the characteristics of weak antigenicity and the like, and can be caused by invading the host body. Complex immune response. T-cell immunity plays an important role in the immune response caused by P. falciparum. Therefore, the role of co-stimulatory molecules inducing T-cell activation in infrared immunity is malaria therapy. On the basis of the development of vaccine, this topic was used to isolate the sporozoites of Plasmodium yoelii from the tail vein to infect the rats. According to the mammalian host model of Anopheles sinensis, two aspects of the immune response produced by Plasmodium vivax in the host body and the host immune system following the stimulation of Plasmodium vivax were studied: On the one hand, the technique of differential display PCR (DD-PCR) was applied to screen. The cDNA of Plasmodium vivax IR was cloned to TA vector by comparative analysis. According to the result of sequence homology analysis and function prediction, the gene related to the development of Plasmodium vivax was estimated. On the other hand, RT-PCR was used to react with direct immunofluorescence. Method of co-focusing technique, from gene and co-stimulatory molecule expression to host to Plasmodium vivax stimulation The immune response produced was studied. The experimental content and the knot The fruit mainly comprises the following aspects: 1. Aiming at the characteristics of high content of A + T (> 70%) of Plasmodium gene A + T, this experiment has been changed. According to the A + T content of the DD-PCR primer, a primer capable of specifically amplifying the A + T content of the Plasmodium gene from the sample of liver tissue and Plasmodium vivax is designed, the primer is combined with the DD-PCR technology, and the purified merozoite is bypassed. According to the technical barrier, the Plasmodium falciparum gene is specifically amplified. The obtained nucleic acid sequence is accessed into the GenBank database, and the sequence homology comparison is carried out through the Blast query, and the results are shown in 21 clones to be tested: 8 genes related to the infrared period of Plasmodium yoelii (PyHs7, PyHs5, PyHs6, PyHs7, PyHs8, PyHs9 PyHs10, PyHs11), in which PyHs5 is similar to Plasmodium vivax glucosamine phosphate utase (AGM), PyHs5 has homology with Plasmodium falciparum erythrocyte membrane protein 3 (EMP3), PyHs6, PyHs7, PyHs8, PyHs9, PyHs1 0. PyHs11 is a functionally unknown gene. The molecular mechanism of long development provides a theoretical foundation. By using the RT-PCR method, we can qualitatively and quantitatively analyze the host immune-related genes B7.1, B7.2, CD28-7721, IFN-, and explore the change of immune-related genes in mammalian hosts after Plasmodium infection. Conditions: There was little change in expression of B7.1 in the host body after infection of Plasmodium yoelii. The transcription level of P <0.05 was significantly increased (P <0.05). By combining direct immunofluorescence and laser co-focusing, the expression and localization of B7. 1 and B7.2 of the co-stimulatory molecules after Plasmodium infection were observed. The molecular expression of B7.1 and B7.2 was increased, in which the rising rate of B7.1 molecules began to decrease after 72h, while the up-regulation of B7.2 molecule increased rapidly after 48h reached the peak value, and then fell back at 72h; in the 2-72h after plasvax infection, the table of B7.2 In addition, laser scanning confocal microscopy showed that after Plasmodium infection, the expression of B7.1, B7.2 molecules in macrophages increased with time. The point is delayed from the cytoplasm and the nucleus gradually transferred to the cell membrane. According to this phenomenon, we can speculate that after the stimulation of P. P., the co-stimulatory molecule begins to activate. In conclusion, we studied some immune molecules of Plasmodium vivax development-related molecule and host anti-malaria parasite from parasites and hosts, and systematically discussed the merozoite of Plasmodium vivax. Molecular mechanism of sub-cell development and immune response induced by Plasmodium vivax, and preliminary study on the host immune cells and effector molecules after Plasmodium infection in the expression level of genes and immune molecules
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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