【作者】 蕳雪静；

【导师】 吴欣怡；

【作者基本信息】 山东大学 ， 临床医学， 2012， 博士

【摘要】 研究背景弓形虫是一种呈世界性分布的细胞内寄生原虫。目前世界范围内将近十亿人感染弓形虫。根据感染途径,分为获得性感染和先天性感染两大类。获得性感染常通过食入未煮熟的含有包囊的动物肉类、生奶、生蛋或误食了猫粪便中释放的卵囊而感染。近年研究显示,越来越多的弓形虫眼病为获得性感染,获得性弓形虫眼部感染通过免疫介导,引起全色素膜炎或坏死性视网膜炎,重症的色素膜炎可导致视网膜脱离、青光眼及眼内炎而失明,随着弓形虫感染率的不断增高,弓形虫眼病的发病机制及有效治疗已引起了广泛关注。弓形虫眼病对患者视功能损伤严重,弓形虫感染导致弓形虫眼病的发病机制尚不十分清楚。国内对该病的认识较少,对弓形虫眼病的基础研究更是罕见。为了进一步研究弓形虫眼病的发病机制,首先需要建立弓形虫眼病模型。小鼠对弓形虫RH强毒株极为敏感,经腹腔或皮下接种感染小鼠后,可引起急性感染,造成宿主机体组织损伤,因此,小鼠常作为弓形虫急性感染的动物模型。由于小鼠具有饲养方便、繁殖快、易于实验操作、经济等特点,因此,建立弓形虫RH强毒株急性感染小鼠建立获得性弓形虫眼病动物模型具有重要的实际意义。弓形虫感染后,细胞免疫起着强有力的抗弓形虫效应,参与细胞免疫的细胞有T淋巴细胞、自然杀伤细胞、巨噬细胞等。T细胞按其功能可分为CD4+T细胞和CD8+T细胞等亚群,弓形虫在宿主细胞内寄生,其感染引起机体的免疫反应以CD4+T细胞亚群介导的免疫应答为主。CD4+T细胞多为Th细胞(辅助性T细胞)。根据产生细胞因子和生物功能的不同,传统上将CD4+T细胞分为Thl和Th2两类细胞亚群,它们在免疫过程中相互调节并相互制约。已有的研究表明：急性弓形虫眼部感染时,房水中Thl细胞分泌的γ-干扰素(interferon-γ,IFN-γ)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平明显升高,Th2细胞分泌的白细胞介素—10(interleukin-10, IL-10)的水平明显降低。IFN-γ和TNF-α在巨噬细胞活化、杀灭寄生虫方面十分重要,是机体对抗弓形虫侵害的主要活性分子。但过量的IFN-γ和TNF-α可能导致免疫应答过度,加重组织的炎症反应甚至导致组织坏死等。IL-10又称为细胞因子生成抑制因子(CSIF),是IL-10家族的首要成员。已有的研究表明IL-10通过下调IFN-γ和TNF-α从而在保护性免疫和免疫病理的平衡中起着重要作用。那么,在小鼠获得性弓形虫眼病模型中,通过静脉注射IL-10,对外周血和眼内房水中的IFN-γ和TNF-α水平如何影响?是否又能进一步减轻眼部的炎症反应?文献中未见到相关报道。CD4+CD25+调节性T细胞(regulatory T cells,Treg)是近年来发现的一类不同于Thl和Th2细胞的CD4+T细胞,作为一种调节性T细胞,此类细胞在抑制病原体感染免疫中起重要的作用。其发挥效应的一条重要途径为分泌IL-10。Th17是另一类新发现的不同于Thl和Th2细胞的CD4+T细胞亚群,以分泌IL-17为主,研究表明,Th17细胞在介导炎症反应和自身免疫病发病中发挥重要作用。正常情况下,T细胞及各亚群数目在周围组织中相对稳定,T细胞总数或其亚群的绝对数和比值发生改变,可视为免疫异常,并与某些疾病的发生和发展相关。Treg/Th17的分布在很多疾病的发生过程中出现了失衡现象,在小鼠获得性弓形虫眼病模型中,脾脏中Treg/Th17的平衡状态是否发生了变化,是否能进一步阐明弓形虫感染的发病机制?文献中未见报道。体外研究表明,IL-10可通过作用于Treg降低Th17的数量。那么,在获得性弓形虫眼病模型中,通过静脉注射IL-10,对脾脏中Treg/Th17的平衡状态如何影响?研究目的本研究通过建立获得性弓形虫眼病的C57BL/6小鼠动物模型,检测小鼠房水和血清中IFN-γ和TNF-α浓度以及小鼠脾脏Treg与Th17细胞数量的变化,并探讨IFN-γ和TNF-α浓度的变化以及Treg/Th17平衡状态与弓形虫眼部感染的关系。通过外源性细胞因子IL-10干预,探讨IL一10对获得性弓形虫眼病小鼠体内IFN-γ和TNF-α浓度、Treg/Th17的平衡以及对视网膜组织炎症的影响。进而为弓形虫眼病的发病机制及进一步治疗提供重要理论依据。研究方法将160只C57BL/6小鼠分为4组：①对照组40只；②实验A组(实验A组)40只；③0.01ug IL-10干预组(实验B组)40只；④O.1ug IL-10干预组(实验C组)40只。对实验组每只小鼠均通过腹腔注射给予102个／只(200ul无菌生理盐水)弓形虫强毒株(RH株)速殖子,对照组小鼠注射等体积的生理盐水,实验B组于感染虫体后第2d和第4d通过尾静脉注射0.0lug IL-10(溶于200ul无菌生理盐水中)；实验C组于感染虫体后第2d和第4d通过尾静脉注射O.1ug IL-10(溶于200ul无菌生理盐水中)；感染小鼠后第6天,分别处死小鼠,摘取眼球,收集外周血,取对照组和实验组小鼠脾脏。应用套式PCR技术对外周血做基因检测；对小鼠眼球做石蜡切片和HE染色,观察小鼠眼部的组织病理学切片改变；通过ELISA试剂盒检测对照组和实验组房水和血清中IFN-γ和TNF-α浓度,采用流式细胞术检测脾脏Treg和Th17细胞的数量变化,计算脾脏Treg/Th17的比值。通过小鼠尾静脉注射IL-10后,观察其对血清和房水中IFN-γ和’INF-α的浓度、Treg/Th17以及视网膜炎症的影响。研究结果1.PCR检测弓形虫感染第6天,在实验组小鼠外周血中可检测到小鼠P30基因。2.不同组视网膜组织学切片的变化弓形虫感染第6天,实验A组视网膜组织可见到弓形虫,且出现明显的炎症反应；0.01ugIL-10注射后,视网膜组织中炎症反应仍比较明显；0.1ugIL-10注射后,视网膜组织中仍可见到弓形虫,但炎症反应减轻。3.不同组房水中IFN-γ和TNF-α的浓度变化弓形虫感染第6天,实验A组房水中IFN-γ (4531.4pg/ml)和TNF-α (1572.3pg/ml)的浓度升高较对照组房水中IFN-γ (12.3pg/ml)和TNF-α的浓度(1165.2pg/m1)；0.0lugIL-10注射后,房水中IFN-γ (3838.7pg/ml)和TNF-α (1431.1pg/ml)的浓度变化甚微,0.1ugIL-10注射后,房水中IFN-γ (2482.7pg/ml)和TNF-α (1248.1pg/ml)的浓度较实验A组降低。4.不同组血清中IFN-γ和TNF-α的浓度变化弓形虫感染第6天,实验A组血清中IFN-γ (455.7±39pg/ml)较对照组血清中IFN-γ的浓度(16.4±3.1pg/m1)升高；实验A组血清中TNF-α (14.6±2.2pg/ml)较对照组血清中TNF-α的浓度(8.7±2.1pg/m1)升高；0.01ugIL-10注射后,实验B组血清中IFN-γ(389.8±42pg/m1)和TNF-α(13.1±1.7pg/ml)的浓度较实验A组降低,统计学分析有统计学意义,但炎症反应仍比较明显；0.1ugIL-10注射后,血清中IFN-γ(265.7±27pg/ml)和TNF-α(12.1±1.9pg/ml)的浓度较实验A组降低更为明显,统计学分析有统计学意义,视网膜组织中仍可见到弓形虫,但炎症反应减轻。5.各组Treg占CD4+细胞的百分比实验A组(18.41±5.32)Treg占CD4-细胞的百分比较对照组(39.16±12.53)降低,统计学分析差异有统计意义(P＜0.05)；实验B组Treg占CD4+细胞的百分比(20.57±5.61)和实验A组相比统计学分析差异无统计意义(P>0.05)；实验C组(31.27±8.47)Treg占CD4+细胞的百分比较实验A组升高,两者相比差异有统计意义(P<0.05)。6.各组Thl7占CD4+细胞的百分比实验A组Thl7占CD4+细胞的百分比(33.15±8.37)较对照组(9.41±7.54)升高,统计学分析差异有统计意义(P<0.05)；实验B组(29.27±7.54)Thl7占CD4+细胞的百分比和实验A组相比统计学分析差异无统计意义(P>0.05)；实验C组Th17占CD4‘细胞的百分比(14.57±9.32)较实验A组降低,两者相比差异有统计意义(P<0.05)。7.脾脏中Treg/Th17的比值的变化。实验A组(0.34±0.17)脾脏中Treg/Th17的比值低于对照组(3.76±1.23),两者比较差异有统计意义,实验B组(0.51±0.31)脾脏中Treg/Th17的比值和实验A组相比差异无统计意义(P>0.05)；实验C组(2.33±1.16)脾脏中Treg/Th17的比值和实验A组相比升高,差异有统计意义(P<0.05)。研究结论经腹腔注射弓形虫强毒株(RH株)感染C57BL/6小鼠可建立获得性弓形虫眼病模型,病变主要集中在视网膜,套式PCR检测具有高度的特异性和敏感性。弓形虫感染后,血清和房水中的IFN-γ和TNF-α的浓度升高,脾脏局部Treg细胞数量下降,Thl7细胞数量升高,脾脏中Treg/Th17的比值明显降低,打破了正常的免疫平衡。Treg/Thl7的比值降低以及IFN-γ和TNF-α的浓度升高可能与弓形虫眼部感染炎症反应有关。通过尾静脉注射0.01ug IL-10后,房水和血清中的IFN-γ和TNF-α的浓度降低,Treg/Th17变化不明显,增加IL-10(0.1ugIL-10)注射后,脾脏中Treg/Thl7比值升高,房水和血清中的IFN-γ和TNF-α的浓度继续降低,统计学分析有统计学意义,视网膜炎症反应减轻,从而推测外源性IL-10可能通过降低体内的IFN-γ和TNF-α的浓度以及改变体内的Treg/Th17的平衡状态减轻弓形虫感染小鼠的眼部炎症反应。研究意义通过腹腔注射弓形虫RH株感染C57BL/6小鼠,建立了获得性弓形虫眼病的动物模型,为获得性弓形虫眼病发病机制、治疗方法等相关研究奠定了坚实的基础。造模成功后,应用组织化学方法(HE染色)观察弓形虫眼病的眼部病理改变特点,并对眼部组织和血液标本行套式PCR检测弓形虫P30基因,建立了敏感特异的弓形虫检测方法,PCR检测方法不仅能弥补血清学方法不能检测的免疫缺陷患者或动物的弓形虫病,而且PCR检测方法更为快速和敏感,可作为临床上检测弓形虫感染的检验方法很好的选择。本研究首次从Treg/Thl7平衡的角度探讨弓形虫感染的发生机理,结果发现,弓形虫感染可以导致鼠脾脏Treg细胞水平降低,而Th17细胞水平升高,出现了Treg/Th17的失衡。结果提示,感染引起的Treg/Th17失衡在弓形虫感染的发病机制中可能起到重要的作用。然后,通过小鼠尾静脉注射IL-10,应用ELISA试剂盒检测小鼠外周血和眼内房水中炎症因子IFN-γ和TNF-α水平的改变,并检测脾脏中Treg/Th17平衡的变化。据实验结果推测在小鼠获得性弓形虫眼病模型中,眼部的炎症反应可能与体内的IFN-γ和TNF-α浓度明显升高及Treg/Th17的降低有关,外源性IL-10可能通过降低血清和房水中IFN-γ和TNF-α的浓度及改变体内Treg/Th17的平衡及减轻了小鼠弓形虫感染的眼部炎症反应,IL-10注射有可能成为治疗弓形虫眼病的一种新的途径。更多还原

【Abstract】 BackgroundToxoplasma gondii is an intracellular parasitic Protozoa that infects a wide variety of hosts. Nearly one billion people worldwide have been infected with Toxoplasma gondii at present. According to the routes of infection, there are two kinds of infection which is acquired infection and congenital infection. Acquired infection occurs due to ingesting ainimal meats with cysts of the parasite which are not cooked, raw milk, raw eggs or eating food with Toxoplasma gondii oocysts in cat feces.In recent years, researches have shown that acquired infections of Toxoplasma gondii are increasing, which can cause full pigment film inflammatory or necrosis retinal inflammatory. Heavy disorder of pigment film inflammatory can make retinal detachment, glaucoma and endophthalmitis leading to blindness. As Toxoplasma gondii infection rates constantly increase, the pathogenesis and the effective treatment of Toxoplasmosis ophthalmopathy has cause widely concerning. After infected by Toxoplasma gondii, cells of our bodies play a strong resistance of immune effects to Toxoplasma, such as T lymphocytes, natural killer cells, and macrophages. According to the different function, T cells can be divided into subgroups of CD4+T cells and CD8+T cells. Most CD4+T cells are Th cells (helper T cells). According to different cytokine production and biological function, traditional CD4+T cells are divided into subgroups of Thl and Th2type cells, they control and restraint in the immune process. Researches have shown that when acute Toxoplasma infection occurs in eyes, the concentration of y-interferon (IFN-y) and tumor necrosis factor-alpha (TNF-a) secreted by Thl cells in aqueous humor significantly increased, and the concentration of interleukin-10(IL-10) secreted by Th2cells significantly decreased. IFN-y and TNF-a plays very important roles in macrophage activation and killing parasites, which are the main active elements of the body fighting against Toxoplasma gondii. But excess IFN-y and TNF-a can cause excessive immune response and increase inflammatory response of the organization or even cause tissue necrosis. IL-10can reduce the secretion of IFN-y and TNF-a. Researches showed that IL-10played an important role in the balance of protective immunity and immune pathology. Then, early in the acute infection of Toxoplasma gondii Whether intravenous injection of IL-10can affect the concentration of IFN-y or TNF-a in peripheral blood and aqueous humor and further reduce the inflammation of the eyes or not?CD4+CD25+T cells (Treg) were discovered different from Thl and Th2cells in recent years. As a kind of regulatory T cells, they play an important role in the regulation of immune function of pathogen infecting disease by secreting IL-10and other cytokines. Thl7is another type of CD4+T cell subsets different from Thl and Th2cells, secreting IL-17, studies have shown that Th17cells play an important role in mediating inflammatory responses and pathogenesis of autoimmune disease. Under normal circumstances, the number of T cells and subsets in the surrounding tissue is relatively stable, the change of absolute number and ratio of the total number of T cells or subsets can be regarded as immune abnormalities and the occurrence and development of certain diseases. Distribution of Treg/Th17imbalance occurred in many disease processes. In acute Toxoplasma infection in a mouse model, Treg/Th17balance in the spleen is broken. Whether the Treg/Th17imbalance can further elaborate in pathogenesis of Toxoplasma gondii infection or not? There are no reports in the literature. Studies in vitro have shown that IL-10can reduce number of Th17through Treg. Then, Whether IL-10by intravenous injection affect on the splenic Treg/Th17balance or not in acquired toxoplasmosis ophthalmopathy model?ObjectiveTo understand Treg/Th17imbalance in mice spleen and effects of IL-10on murine ocular toxoplasmosis, this research established acquired toxoplasmosis ophthalmopathy C57BL/6mouse model, detected percentage of Treg and Thl7cells to total CD4+cells in mice spleen infected by Toxoplasma gondii and analysis the relationship between Treg/Th17balance and Toxoplasma gondii infection of the eye. By exogenous cytokine IL-10intervention, we discussed the effects of IL-10on Treg/Th17balance, the concentration of IFN-y and TNF-a in vivo and effects on retinal inflammation of murine ocular toxoplasmosis.MethodsOne hundred and sixty C57BL/6mice were divided into four groups:forty mice in the control group, forty mice in the infected group (experiment A),forty mice in the injecting0.01ug IL-10group (experiment B),and forty mice in the injecting0.1ug IL-10group (experiment C).102(0.2ml) tachyzoites of Toxoplasma gondii virulent strain (RH strains) were given by intraperitoneal injection of each mouse in experiment group, while each mouse in control group was injected with saline. On2d and4d after infection parasites, each mouse was given0.01μg I1-10by tail vein injection (dissolved in200ul sterile saline) in experimental B group, and each mouse was given0.1μg I1-10by tail vein injection (dissolved in200ul sterile saline) in experimental C group. After six days of infection, all mice were sacrificed and removed the eyes. Histological sections of the four groups were detected. Peripheral blood was collected. The DNA of Toxoplasma gondii in eyes and serum was detected.The mouse spleens were removed in control group and experimental groups to detect percentage of Treg and Th17cells to total CD4+cells by flow cytometry. The concentration of IFN-γ and TNF-α in eyes and serum were detected in experimental groups.Results1. PCR detection. P30gene could be detected in experiment groups on the sixth day.2. Retinal change in histological slices of differerent groups. Toxoplasma and apparent inflammation were watched in retinal slices in experiment A and B group.Toxoplasma and light inflammation were watched in retinal slices in experiment C group.3. Concentration of IFN-γ and TNF-α in aqueous humor. Compared with the concentration of IFN-γ (12.3pg/ml))and TNF-α (1165.2pg/ml)of aqueous humor in the control group, the concentration of IFN-γ (4531.4pg/ml)and TNF-α (1572.3pg/ml) in aqueous humor was higher in experiment A group; Compared with experiment A group, the concentration of IFN-γ (3838.7pg/ml) and TNF-α (1431.1pg/ml) in aqueous humor was lower in experiment B group; Compared with experiment A group, the concentration of IFN-γ (2482.7pg/ml) and TNF-α (1248.1pg/ml) in aqueous humor was lower in experiment C group.4. Concentration of IFN-y and TNF-α in serum. Compared with the concentration of IFN-γ (16.4±3.1pg/ml) and TNF-a (8.7±2.1pg/ml) in serum of the control group, the concentration of IFN-y (455.7±39pg/ml) and TNF-a (14.6±2.2pg/ml) in serum was higher in experiment A group, The difference was statistically significant (P<0.05); Compared with experiment A group, the concentration of IFN-y (389.8±42pg/ml) and TNF-a (13.1±1.7pg/ml) in serum was lower in experiment B group, The difference was statistically significant (P<0.05); Compared with experiment A group, the concentration of IFN-y (265.7±27pg/ml) and TNF-a (12.1±1.9pg/ml) in serum was lower in experiment C group. The difference was statistically significant (P＜0.05).5. The percentage of Treg cells to total CD4+cells in spleen. Compared with control group (39.16±12.53) in the spleen, the percentage of Treg cells to total CD4+cells in the experiment A group (18.41±5.32) was lower. The difference was statistically significant (P＜0.05). Compared with experiment A group, the percentage of Treg cells to total CD4+cells in the experiment C group (31.27±8.47) was higher. The difference was statistically significant (P<0.05).6. The percentage of Th17cells to total CD4+cells in spleen. Compared with control group (9.41±7.54) in the spleen, the percentage of Th17cells to total CD4+cells in the experiment A group (33.15±8.37) was higher. The difference was statistically significant (P<0.05). Compared with experiment A group, the percentage of Th17cells to total CD4+cells in the experiment C group (14.57±9.32) was lower. The difference was statistically significant (P<0.05).7. Ratio of Treg/Th17in spleen. Compared with control group (4.26±1.23) in the spleen, the ratio of Treg/Th17in experiment A group (0.54±0.17) was lower. The difference was statistically significant (P＜0.05). Compared with experiment A group, the ratio of Treg/Th17in experiment C group (2.33±1.16) was higher. The difference was statistically significant (P<0.05).ConclusionAcquired toxoplasmosis ophthalmopathy model was established by intraperitoneal injection of virulent strain of Toxoplasma gondii (RH strain) infecting C57BL6mice. Lesions were mainly concentrated in the retina. Nested-PCR test had high specificity and sensitivity. After infection with Toxoplasma gondii, the concentrations of IFN-y and TNF-a in serum and aqueous humor increased, which might be associated with inflammation of the eye infected by Toxoplasma gondii. Percentage of Treg cells to total CD4+cells decline in spleen, Percentage of Th17cells to total CD4+cells increased, the ratio of splenic Treg/Th17significantly decrease and broke the normal immune response. By tail vein injection of IL-10, The concentration of IFN-γ and TNF-a in the aqueous humor and serum were lower, the ratio of Treg/Th17became higher, and retinal inflammation decreased. Thus we speculated that by changing Treg/Th17balance and making the concentration of IFN-γ and TNF-a lower in body, exogenous IL-10could reduce inflammation of eye infected by Toxoplasma gondii in mice.更多还原