【摘要】：目的：探索Treg細(xì)胞相關(guān)功能分子在早孕期弓形蟲(chóng)感染致不良妊娠結(jié)局中的作用機(jī)制以及過(guò)繼轉(zhuǎn)輸調(diào)節(jié)性T細(xì)胞對(duì)弓形蟲(chóng)感染致不良妊娠結(jié)局影響的分子機(jī)制。方法：為探索Treg細(xì)胞相關(guān)功能分子在早孕期弓形蟲(chóng)感染致不良妊娠結(jié)局中的作用機(jī)制,建立剛地弓形蟲(chóng)感染孕鼠模型,設(shè)感染組和正常對(duì)照組,感染組于孕8天腹腔感染剛地弓形蟲(chóng)(Toxoplasma gondii, T. gondii)速殖子,孕14天頸椎脫臼處死孕鼠,記錄妊娠結(jié)局。制備母胎界面(子宮、胎盤)和脾淋巴細(xì)胞懸液,流式檢測(cè)Treg細(xì)胞凋亡率及Treg細(xì)胞表面抑制性分子CTLA-4和PD-1的表達(dá)水平；同時(shí)制備胎盤上清,ELISA檢測(cè)TGF-β、IL-10和IFN-γ的水平。為了進(jìn)一步研究過(guò)繼轉(zhuǎn)輸調(diào)節(jié)性T細(xì)胞對(duì)弓形蟲(chóng)感染致不良妊娠結(jié)局影響的分子機(jī)制,分別提取正常孕鼠母胎界面和脾中Treg細(xì)胞并用CFSE標(biāo)記,然后分別過(guò)繼轉(zhuǎn)輸給弓形蟲(chóng)感染的孕鼠,追蹤過(guò)繼轉(zhuǎn)輸?shù)腡reg細(xì)胞在受體孕鼠體內(nèi)的分布情況。實(shí)驗(yàn)對(duì)照組感染孕鼠注射相同劑量PBS。在孕第14天觀察并記錄妊娠結(jié)局,采用流式檢測(cè)母胎界面和脾中CTLA-4+Treg和PD-1+Treg細(xì)胞的表達(dá)水平,同時(shí)觀察來(lái)自母胎界面和脾的CFSE+Treg細(xì)胞在受體孕鼠體內(nèi)的分布情況;并用ELISA檢測(cè)過(guò)繼轉(zhuǎn)輸組與對(duì)照組感染孕鼠胎盤中TGF-β、IL-10和IFN-γ的水平。結(jié)果：正常對(duì)照組孕鼠精神狀態(tài)良好,胎鼠、胎盤血供正常。感染組孕鼠精神萎靡不振,聳毛、拱背,活動(dòng)明顯減少；胎鼠血供不足,胎鼠、胎盤形狀小,重量輕,胎盤有出血壞死。感染組Treg細(xì)胞CTLA-4和PD-1表達(dá)較正常組顯著降低,并且弓形蟲(chóng)感染可以使Treg細(xì)胞凋亡明顯增加。與正常對(duì)照組比,感染組胎盤上清TGF-β/IFN-γ和IL-10/IFN-γ的比值顯著下降。另外,研究發(fā)現(xiàn)正常孕鼠母胎界面的Treg細(xì)胞CTLA-4和PD-1的表達(dá)量比脾臟中的高。實(shí)驗(yàn)發(fā)現(xiàn),只有當(dāng)轉(zhuǎn)輸來(lái)自于母胎界面的Treg細(xì)胞時(shí),妊娠結(jié)局才得到改善。過(guò)繼轉(zhuǎn)輸母胎界面Treg細(xì)胞的感染組孕鼠相比較未轉(zhuǎn)輸Treg細(xì)胞的感染組孕鼠精神狀態(tài)好,吸收胎率降低,胎鼠血供改善、重量增加,胎盤出血壞死情況減輕；而過(guò)繼轉(zhuǎn)輸脾Treg細(xì)胞的感染組孕鼠的精神狀態(tài)與未過(guò)繼轉(zhuǎn)輸Treg細(xì)胞的感染組孕鼠相似,其胎鼠、胎盤的外觀、重量、吸收胎率也無(wú)明顯改善。此外發(fā)現(xiàn),無(wú)論是來(lái)自母胎界面的Treg細(xì)胞還是來(lái)自脾的Treg細(xì)胞到達(dá)受體孕鼠相同部位(母胎界面或脾)的數(shù)量無(wú)明顯差異。轉(zhuǎn)輸母胎界面Treg細(xì)胞的感染組孕鼠CTLA-4和PD-1增加的幅度顯著高于轉(zhuǎn)輸脾臟Treg細(xì)胞的感染孕鼠。母胎界面TGF-β/IFN-γ和IL-10/IFN-γ的比值在轉(zhuǎn)輸母胎界面Treg細(xì)胞的感染組比未轉(zhuǎn)輸Treg的感染組顯著升高,而在轉(zhuǎn)輸脾Treg細(xì)胞的感染組相對(duì)于未轉(zhuǎn)輸Treg細(xì)胞的感染組則無(wú)明顯差異。結(jié)論：Treg細(xì)胞相關(guān)功能分子CTLA-4和PD-1在弓形蟲(chóng)感染致不良妊娠結(jié)局中發(fā)揮重要作用；過(guò)繼轉(zhuǎn)輸母胎界面Treg細(xì)胞能顯著改善弓形蟲(chóng)感染導(dǎo)致的不良妊娠結(jié)局。
[Abstract]:Aim: to investigate the role of Treg cell-related functional molecules in poor pregnancy outcome induced by Toxoplasma gondii infection in early pregnancy and the molecular mechanism of the effect of adoptive transfer of regulatory T cells on poor pregnancy outcome induced by Toxoplasma gondii infection. Methods: in order to explore the role of Treg cell-related functional molecules in the poor pregnancy outcome induced by Toxoplasma gondii infection in early pregnancy, a pregnant rat model of Toxoplasma gondii infection was established, and the infected group and the normal control group were set up. In the infected group, Toxoplasma gondii (Toxoplasma gondii, T. gondii) Tachyzoites were infected intraperitoneally on the 8th day of gestation, and the pregnant rats were killed on the 14th day of gestation with cervical dislocation to record the pregnancy outcome. The maternal-fetal interface (uterus, placenta) and splenic lymphocyte suspension were prepared. The apoptosis rate of Treg cells and the expression of CTLA-4 and PD-1 on the surface of Treg cells were detected by flow cytometry. At the same time, the placental supernatant was prepared and the levels of TGF- 尾, IL-10 and IFN- 緯 were detected by ELISA. In order to study the molecular mechanism of Toxoplasma gondii (Toxoplasma gondii) infection, Treg cells were extracted from maternal fetal interface and spleen of normal pregnant rats and labeled with CFSE, respectively, in order to study the molecular mechanism of the effect of adoptive transfer regulatory T cells on poor pregnancy outcome. Then the Treg cells were transferred to the pregnant mice infected with Toxoplasma gondii respectively and the distribution of Toxoplasma gondii cells in the recipient pregnant mice was tracked. The infected pregnant rats in the experimental control group were injected with the same dose of PBS.. The pregnancy outcome was observed and recorded on the 14th day of gestation. The expression levels of CTLA-4 Treg and PD-1 Treg cells in the maternal-fetal interface and spleen were detected by flow cytometry, and the distribution of CFSE Treg cells from the maternal-fetal interface and spleen in the recipient pregnant rats was observed at the same time. The levels of TGF- 尾, IL-10 and IFN- 緯 in placenta of pregnant rats were measured by ELISA. Results: pregnant rats in normal control group were in good mental state, fetal rats and placental blood supply were normal. The pregnant rats in the infected group showed mental malaise, shrug, arched back and decreased activity, and the blood supply of fetal rats was insufficient, the placentas were small in shape and light in weight, and the placenta was bleeding and necrotic. The expression of CTLA-4 and PD-1 in Treg cells in infection group was significantly lower than that in normal group, and Toxoplasma gondii infection could significantly increase the apoptosis of Treg cells. Compared with the normal control group, the ratio of TGF- 尾 / IFN- 緯 and IL-10/IFN- 緯 in the supernatant of infected group decreased significantly. In addition, the expression levels of CTLA-4 and PD-1 in Treg cells at the maternal-fetal interface of normal pregnant rats were higher than those in spleen. It was found that pregnancy outcomes improved only when Treg cells from the maternal-fetal interface were transferred. The pregnant rats with Treg cells transferred to the mother fetal interface were better in mental state, lower fetal absorption rate, better blood supply, higher weight and lower placental hemorrhage and necrosis than those without Treg cells transfusions in pregnant rats, and the placental hemorrhage and necrosis were alleviated in the pregnant rats after being transferred to the mother's fetal interface (P < 0. 05). However, the mental state of pregnant rats transferred to spleen Treg cells was similar to that of pregnant rats without transfer of Treg cells. The appearance, weight and absorption rate of fetal rats and placentas were not significantly improved. It was also found that there was no significant difference in the number of Treg cells from the maternal-fetal interface or the Treg cells from the spleen reaching the same site (maternal-fetal interface or spleen) of the pregnant mice. The increase of CTLA-4 and PD-1 in pregnant mice was significantly higher than that in infected pregnant mice with Treg cells transferred to spleen. The ratio of TGF- 尾 / IFN- 緯 and IL-10/IFN- 緯 at the mother-fetal interface was significantly higher in the infected group than that in the non-Treg infected group. However, there was no significant difference between the infection group with Treg cells transferred to spleen and those infected with non-transfused Treg cells. Conclusion: Treg cell-related functional molecules CTLA-4 and PD-1 play an important role in poor pregnancy outcome caused by Toxoplasma gondii infection, and Treg cells can significantly improve the adverse pregnancy outcome caused by Toxoplasma gondii infection.
【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.251
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本文編號(hào)：2444373