本文選題：剛地弓形蟲 + 胎盤�。� 參考：《安徽醫(yī)科大學》2012年碩士論文

【摘要】：目的弓形蟲是一種專性細胞內寄生原蟲，可以寄生于所有溫血動物包括人類。孕期感染弓形蟲可導致流產(chǎn)、早產(chǎn)、死胎和胎兒畸形，弓形蟲可經(jīng)胎盤垂直傳播導致新生兒先天性弓形蟲病，影響其出生后的生長發(fā)育和智力水平[1]。歐洲和北美的弓形蟲主要有三個克隆系譜（Ⅰ型、Ⅱ型和Ⅲ型）。最近研究顯示，小鼠巨噬細胞感染Ⅰ型和Ⅲ型蟲株后宿主的巨噬細胞亞型向M2極化；而感染了Ⅱ型蟲株的巨噬細胞則向類似于M1的方向極化。蟲體所分泌的毒力相關因子等可以不依賴于模式識別受體來誘導M1或M2的極化。宿主在孕期主要是Th2應答占優(yōu)勢的免疫環(huán)境，對于本室分離的兩個分離株，TgCtwh3和TgCtwh6，它們有著相同的基因型，卻有著不同的毒力。本研究探討了孕期感染上述兩個蟲株后，小鼠體內M1/M2和Th1/Th2應答的偏移及其差異，巨噬細胞極化方向，相同基因型不同毒力蟲株誘導不良妊娠結局及其相關機制。本文旨在為基因型相關的先天性弓形蟲病的防治提供理論和實驗基礎。方法體內試驗：將ICR小鼠以雌雄2:1的比例合籠，得到90只孕鼠，隨機分為三組：TgCtwh3組、TgCtwh6組和對照組。于孕第8天（G8）TgCtwh3組和TgCtwh6組分別腹腔注射0.1ml生理鹽水含200個速殖子；對照組只注射等量生理鹽水。分別于妊娠第10、12、14、16、18天麻醉下頸椎脫臼處死小鼠，取腹腔巨噬細胞和胎盤。同一組的部分胎盤4%甲醛固定，石蠟包埋，HE染色和TUNEL檢測；部分胎盤用熒光定量PCR做蟲荷定量。巨噬細胞上清檢測尿素和NO；用Western-blotting檢測巨噬細胞內iNOs和Arg-1的蛋白水平；流式檢測巨噬細胞表面CD206、MHCⅡ、PDL-2、CD80、CD86、PDL-1的水平。取G14天的三組動物脾臟，流式檢測CD4+IFN-γ和CD4+IL-4的百分比。 體外實驗：孕鼠于孕12~14天無菌操作取胎盤滋養(yǎng)層細胞和巨噬細胞，Transwell共培養(yǎng)，速殖子與巨噬細胞按照1：1比例分別感染TgCtwh3和TgCtwh6蟲株，另設一組無感染對照。各組分別于2h、6h、12h、24h收集上清和巨噬細胞，滋養(yǎng)層細胞固定做TUNEL檢測。巨噬細胞上清檢測IL-10、IL-23、IL-12的水平；巨噬細胞內的蛋白檢測同體內實驗。結果1、三組小鼠的胎盤隨著妊娠天數(shù)的增加凋亡率上升，依次為TgCtwh3組。 TgCtwh6組大于對照組。 2、HE染色結果顯示，與TgCtwh3組和對照組相比，TgCtwh6感染組胎盤局部在感染早期出現(xiàn)大量淋巴細胞浸潤；TgCtwh6蟲株感染的小鼠腹腔巨噬細胞在孕14天開始出現(xiàn)高水平的NO，而其他兩組未能檢測到NO分泌；脾臟淋巴細胞流式細胞術檢測結果提示，TgCtwh6感染組PDL-1、CD80、CD86表達明顯增高。 3、 TgCtwh3蟲株感染組Arg-1水平高于TgCtwh6組和對照組；孕14天的小鼠TgCtwh3感染后腹腔巨噬細胞CD206、PDL-2和MHC-Ⅱ表達明顯增高；脾臟細胞流式檢測結果顯示，與對照組和TgCtwh6組相比，TgCtwh3組更趨向Th2應答的偏移。 4、體外實驗中， Transwell共培養(yǎng)2h、6h、12h、24h的巨噬細胞上清中，TgCtwh3感染組的各時間點上清均出現(xiàn)高水平的尿素（urea），提示精氨酸酶（arginase）的活性較高；而TgCtwh6感染小鼠的上清液略比對照組稍有增高；TUNEL檢測結果顯示，胎盤滋養(yǎng)細胞凋亡率TgCtwh6組＞TgCtwh3組＞對照組；TgCtwh6組巨噬細胞的上清中M1型細胞因子的水平高于其他兩組。 結論采用相同基因型Chinese1的不同毒力蟲株，毒力較強的TgCtwh3蟲株和毒力較弱的TgCtwh6蟲株感染孕鼠都導致了不良妊娠結局。TgCtwh3蟲株感染的孕鼠巨噬細胞向M2型偏移，致體內免疫應答類型更偏向于Th2型，結果可能使得弓形蟲速殖子大量繁殖，破壞宿主細胞，直接或間接導致誘導滋養(yǎng)層細胞凋亡率增加；而弱毒TgCtwh6組蟲株感染后，孕鼠巨噬細胞向M1型偏移，，分泌的M1型細胞因子可能改變孕鼠Th2型生理狀態(tài)下的優(yōu)勢應答的環(huán)境，產(chǎn)生不良的妊娠結局。 以上結果提示，同一基因型的不同毒力蟲株感染可以誘導不同的巨噬細胞亞群（M1，M2）的活化；或者Chinese1基因型（11個位點分型）內尚存在著其他不同的基因型。上述不同蟲株導致的不同免疫結局是否與ROPs和GRAs的多態(tài)性有關，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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R382.5

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