弓形虫中国分离株毒力相关的发病机制：巨噬细胞极化引起ICR孕鼠不良妊娠结局

发布时间：2018-06-06 13:23

本文选题：刚地弓形虫 + 胎盘　；参考：《安徽医科大学》2012年硕士论文

【摘要】：目的弓形虫是一种专性细胞内寄生原虫，可以寄生于所有温血动物包括人类。孕期感染弓形虫可导致流产、早产、死胎和胎儿畸形，弓形虫可经胎盘垂直传播导致新生儿先天性弓形虫病，影响其出生后的生长发育和智力水平[1]。欧洲和北美的弓形虫主要有三个克隆系谱（Ⅰ型、Ⅱ型和Ⅲ型）。最近研究显示，小鼠巨噬细胞感染Ⅰ型和Ⅲ型虫株后宿主的巨噬细胞亚型向M2极化；而感染了Ⅱ型虫株的巨噬细胞则向类似于M1的方向极化。虫体所分泌的毒力相关因子等可以不依赖于模式识别受体来诱导M1或M2的极化。宿主在孕期主要是Th2应答占优势的免疫环境，对于本室分离的两个分离株，TgCtwh3和TgCtwh6，它们有着相同的基因型，却有着不同的毒力。本研究探讨了孕期感染上述两个虫株后，小鼠体内M1/M2和Th1/Th2应答的偏移及其差异，巨噬细胞极化方向，相同基因型不同毒力虫株诱导不良妊娠结局及其相关机制。本文旨在为基因型相关的先天性弓形虫病的防治提供理论和实验基础。方法体内试验：将ICR小鼠以雌雄2:1的比例合笼，得到90只孕鼠，随机分为三组：TgCtwh3组、TgCtwh6组和对照组。于孕第8天（G8）TgCtwh3组和TgCtwh6组分别腹腔注射0.1ml生理盐水含200个速殖子；对照组只注射等量生理盐水。分别于妊娠第10、12、14、16、18天麻醉下颈椎脱臼处死小鼠，取腹腔巨噬细胞和胎盘。同一组的部分胎盘4%甲醛固定，石蜡包埋，HE染色和TUNEL检测；部分胎盘用荧光定量PCR做虫荷定量。巨噬细胞上清检测尿素和NO；用Western-blotting检测巨噬细胞内iNOs和Arg-1的蛋白水平；流式检测巨噬细胞表面CD206、MHCⅡ、PDL-2、CD80、CD86、PDL-1的水平。取G14天的三组动物脾脏，流式检测CD4+IFN-γ和CD4+IL-4的百分比。体外实验：孕鼠于孕12~14天无菌操作取胎盘滋养层细胞和巨噬细胞，Transwell共培养，速殖子与巨噬细胞按照1：1比例分别感染TgCtwh3和TgCtwh6虫株，另设一组无感染对照。各组分别于2h、6h、12h、24h收集上清和巨噬细胞，滋养层细胞固定做TUNEL检测。巨噬细胞上清检测IL-10、IL-23、IL-12的水平；巨噬细胞内的蛋白检测同体内实验。结果1、三组小鼠的胎盘随着妊娠天数的增加凋亡率上升，依次为TgCtwh3组。 TgCtwh6组大于对照组。 2、HE染色结果显示，与TgCtwh3组和对照组相比，TgCtwh6感染组胎盘局部在感染早期出现大量淋巴细胞浸润；TgCtwh6虫株感染的小鼠腹腔巨噬细胞在孕14天开始出现高水平的NO，而其他两组未能检测到NO分泌；脾脏淋巴细胞流式细胞术检测结果提示，TgCtwh6感染组PDL-1、CD80、CD86表达明显增高。 3、 TgCtwh3虫株感染组Arg-1水平高于TgCtwh6组和对照组；孕14天的小鼠TgCtwh3感染后腹腔巨噬细胞CD206、PDL-2和MHC-Ⅱ表达明显增高；脾脏细胞流式检测结果显示，与对照组和TgCtwh6组相比，TgCtwh3组更趋向Th2应答的偏移。 4、体外实验中， Transwell共培养2h、6h、12h、24h的巨噬细胞上清中，TgCtwh3感染组的各时间点上清均出现高水平的尿素（urea），提示精氨酸酶（arginase）的活性较高；而TgCtwh6感染小鼠的上清液略比对照组稍有增高；TUNEL检测结果显示，胎盘滋养细胞凋亡率TgCtwh6组＞TgCtwh3组＞对照组；TgCtwh6组巨噬细胞的上清中M1型细胞因子的水平高于其他两组。结论采用相同基因型Chinese1的不同毒力虫株，毒力较强的TgCtwh3虫株和毒力较弱的TgCtwh6虫株感染孕鼠都导致了不良妊娠结局。TgCtwh3虫株感染的孕鼠巨噬细胞向M2型偏移，致体内免疫应答类型更偏向于Th2型，结果可能使得弓形虫速殖子大量繁殖，破坏宿主细胞，直接或间接导致诱导滋养层细胞凋亡率增加；而弱毒TgCtwh6组虫株感染后，孕鼠巨噬细胞向M1型偏移，，分泌的M1型细胞因子可能改变孕鼠Th2型生理状态下的优势应答的环境，产生不良的妊娠结局。以上结果提示，同一基因型的不同毒力虫株感染可以诱导不同的巨噬细胞亚群（M1，M2）的活化；或者Chinese1基因型（11个位点分型）内尚存在着其他不同的基因型。上述不同虫株导致的不同免疫结局是否与ROPs和GRAs的多态性有关，尚待深入研究。
[Abstract]:Toxoplasma gondii is a special type of parasitic protozoa that can parasitism in all warm blooded animals including humans. Toxoplasma gondii infection during pregnancy can lead to abortion, preterm birth, stillbirth and fetal malformation. Toxoplasma can be transmitted vertically by the placenta to cause congenital toxoplasmosis of the newborn, and influence the growth and intelligence of [1]. in Europe and north of its birth. There are three main clones (type I, type II and type III) of the Toxoplasma gondii (type I, type II and type III). Recent studies show that the macrophage subtypes of the host infected with type I and type III of the mouse macrophages are polarized to M2, while the macrophages infected with the type II strain are polarized to the direction similar to that of M1. The virulence related factors secreted by the body can not be dependent on the strain. The polarization of M1 or M2 is induced by the pattern recognition receptor. The host is mainly the immune environment with the dominant Th2 response during pregnancy. For the two isolates of this room, TgCtwh3 and TgCtwh6, they have the same genotypes but have different virulence. This study explored the M1/M2 and Th1/Th2 in mice infected by pregnancy. The migration and difference of the response, the direction of macrophage polarization, the undesirable pregnancy outcome and the related mechanism of the same genotypic different strains of virulence. This article aims to provide the theoretical and experimental basis for the prevention and control of the genotype of congenital toxoplasmosis. Methods in vivo, 90 pregnant rats were obtained by the proportion of male and female 2:1 in ICR rats. Randomly divided into three groups: TgCtwh3 group, TgCtwh6 group and control group. Eighth days (G8) TgCtwh3 group and TgCtwh6 group were intraperitoneally injected with 200 tachyonite respectively, and the control group was only injected with equal amount of physiological saline. The peritoneal macrophages and placenta were taken in the same group. Partial placenta was fixed with 4% formaldehyde, paraffin embedding, HE staining and TUNEL detection; partial placenta was quantified by fluorescent quantitative PCR. Urea and NO were detected by macrophage supernatant; the protein levels of iNOs and Arg-1 in macrophages were detected by Western-blotting; flow cytometry was used to detect the surface CD206 of macrophages, MHC II, PDL-2, CD80, CD86, and CD86. The percentage of CD4+IFN- gamma and CD4+IL-4 in spleen of three groups of G14 days was detected by flow cytometry.
In vitro experiment: pregnant rats were given placental trophoblast cells and macrophages on 12~14 days of pregnancy. Transwell co culture, tachyonus and macrophages were infected with TgCtwh3 and TgCtwh6 respectively in proportion to 1:1, and another group without infection control. Each group was collected in 2H, 6h, 12h, 24h to collect supernatant and macrophage, and the trophoblast cells were fixed for TUNEL. Test. The level of IL-10, IL-23 and IL-12 was detected by macrophage supernatant; the protein inside the macrophage was detected in the androgyny experiment. Results 1, the rate of apoptosis in the three groups of mice increased with the increase of the number of days of pregnancy, followed by group TgCtwh3. The TgCtwh6 group was larger than the control group.
2, the results of HE staining showed that compared with the TgCtwh3 group and the control group, the placenta in the TgCtwh6 infection group had a large number of lymphocytic infiltration in the early stage of infection; the peritoneal macrophages of the mice infected by the TgCtwh6 strain began to appear high level of NO at the 14 day of pregnancy, while the other two groups failed to detect the secretion of NO, and the splenic lymphocyte flow cytometry was detected. The results showed that the expression of PDL-1, CD80 and CD86 increased significantly in TgCtwh6 infection group.
3, the level of Arg-1 in the TgCtwh3 infection group was higher than that in the TgCtwh6 group and the control group. The expression of CD206, PDL-2 and MHC- II in the peritoneal macrophages of the mice after the 14 day pregnancy was significantly higher, and the splenic cell flow cytometry showed that the TgCtwh3 group was more likely to shift to the Th2 response compared with the control group and the TgCtwh6 group.
4, in the in vitro experiment, Transwell co culture 2h, 6h, 12h, 24h of macrophage supernatant, TgCtwh3 infection group had high level of urea (urea) at all time points, suggesting that the activity of arginine enzyme (arginase) was higher, and the supernatant of TgCtwh6 infected mice was slightly higher than that of the control group; TUNEL detection results showed that placental trophoblast cells were detected by TUNEL. The apoptotic rate in group TgCtwh6 > TgCtwh3 > control group; in group TgCtwh6, the level of M1 cytokines in supernatant of macrophages was higher than that of other two groups.
Conclusion using the different virulent strains of the same genotypic Chinese1, the virulent TgCtwh3 strain and the weak TgCtwh6 strain infected pregnant mice resulted in the migration of macrophage to the M2 type of the pregnant mice infected by the bad pregnancy outcome, and the type of immune response in the body was more biased to the Th2 type. The result may make the tachyonus of the Toxoplasma gondii big. To reproduce, destroy the host cells directly or indirectly lead to the increase of the apoptosis rate of the trophoblast cells, and after the infection of the weakly toxic TgCtwh6 group, the macrophages of the pregnant mice shift to the M1 type, and the secreted M1 cytokine may change the environment of the dominant response under the Th2 physiological state of the pregnant mice and produce a bad pregnancy outcome.
The above results suggest that different strains of the same genotype can induce the activation of different macrophage subpopulations (M1, M2), or there are other different genotypes in the Chinese1 genotype (11 loci typing). Research.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R382.5

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