过继转输调节性T细胞对弓形虫感染致不良妊娠结局影响的分子机制研究
发布时间：2019-03-20 16:08
【摘要】：目的：探索Treg细胞相关功能分子在早孕期弓形虫感染致不良妊娠结局中的作用机制以及过继转输调节性T细胞对弓形虫感染致不良妊娠结局影响的分子机制。方法：为探索Treg细胞相关功能分子在早孕期弓形虫感染致不良妊娠结局中的作用机制,建立刚地弓形虫感染孕鼠模型,设感染组和正常对照组,感染组于孕8天腹腔感染刚地弓形虫(Toxoplasma gondii, T. gondii)速殖子,孕14天颈椎脱臼处死孕鼠,记录妊娠结局。制备母胎界面(子宫、胎盘)和脾淋巴细胞悬液,流式检测Treg细胞凋亡率及Treg细胞表面抑制性分子CTLA-4和PD-1的表达水平；同时制备胎盘上清,ELISA检测TGF-β、IL-10和IFN-γ的水平。为了进一步研究过继转输调节性T细胞对弓形虫感染致不良妊娠结局影响的分子机制,分别提取正常孕鼠母胎界面和脾中Treg细胞并用CFSE标记,然后分别过继转输给弓形虫感染的孕鼠,追踪过继转输的Treg细胞在受体孕鼠体内的分布情况。实验对照组感染孕鼠注射相同剂量PBS。在孕第14天观察并记录妊娠结局,采用流式检测母胎界面和脾中CTLA-4+Treg和PD-1+Treg细胞的表达水平,同时观察来自母胎界面和脾的CFSE+Treg细胞在受体孕鼠体内的分布情况;并用ELISA检测过继转输组与对照组感染孕鼠胎盘中TGF-β、IL-10和IFN-γ的水平。结果：正常对照组孕鼠精神状态良好,胎鼠、胎盘血供正常。感染组孕鼠精神萎靡不振,耸毛、拱背,活动明显减少；胎鼠血供不足,胎鼠、胎盘形状小,重量轻,胎盘有出血坏死。感染组Treg细胞CTLA-4和PD-1表达较正常组显著降低,并且弓形虫感染可以使Treg细胞凋亡明显增加。与正常对照组比,感染组胎盘上清TGF-β/IFN-γ和IL-10/IFN-γ的比值显著下降。另外,研究发现正常孕鼠母胎界面的Treg细胞CTLA-4和PD-1的表达量比脾脏中的高。实验发现,只有当转输来自于母胎界面的Treg细胞时,妊娠结局才得到改善。过继转输母胎界面Treg细胞的感染组孕鼠相比较未转输Treg细胞的感染组孕鼠精神状态好,吸收胎率降低,胎鼠血供改善、重量增加,胎盘出血坏死情况减轻；而过继转输脾Treg细胞的感染组孕鼠的精神状态与未过继转输Treg细胞的感染组孕鼠相似,其胎鼠、胎盘的外观、重量、吸收胎率也无明显改善。此外发现,无论是来自母胎界面的Treg细胞还是来自脾的Treg细胞到达受体孕鼠相同部位(母胎界面或脾)的数量无明显差异。转输母胎界面Treg细胞的感染组孕鼠CTLA-4和PD-1增加的幅度显著高于转输脾脏Treg细胞的感染孕鼠。母胎界面TGF-β/IFN-γ和IL-10/IFN-γ的比值在转输母胎界面Treg细胞的感染组比未转输Treg的感染组显著升高,而在转输脾Treg细胞的感染组相对于未转输Treg细胞的感染组则无明显差异。结论：Treg细胞相关功能分子CTLA-4和PD-1在弓形虫感染致不良妊娠结局中发挥重要作用；过继转输母胎界面Treg细胞能显著改善弓形虫感染导致的不良妊娠结局。
[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- 纬 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- 纬 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- 纬 and IL-10/IFN- 纬 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- 纬 and IL-10/IFN- 纬 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.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.251
，
本文编号：2444373
[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- 纬 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- 纬 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- 纬 and IL-10/IFN- 纬 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- 纬 and IL-10/IFN- 纬 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.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.251
，
本文编号：2444373
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