分娩发动前后孕妇蜕膜及外周血中NK、NKT细胞生物学特性的研究

发布时间：2018-01-13 02:13

本文关键词：分娩发动前后孕妇蜕膜及外周血中NK、NKT细胞生物学特性的研究　出处：《安徽医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 研究背景: 正常的妊娠,是母体免疫系统对带有父源抗原的胎儿产生的一种免疫耐受现象,这种耐受是由多种因素之间相互制约、相互调节产生的。近年生殖免疫学者对自然杀伤细胞(NK cells)和自然杀伤T细胞(NKT cells)在其中的作用给予更多关注。NK细胞、NKT细胞在妊娠期产生Th2的免疫平衡,当这种平衡被打破,即产生各种病理妊娠,并可能诱导产程的发动。人类分娩发动的机理至今仍是个迷,目前人们转向组织水平、免疫水平找寻分娩动因。母-胎界面免疫微环境由蜕膜中的免疫活性细胞及其分泌的细胞因子组成,母体的免疫调节系统包括抗原特异T细胞、调节性T细胞、NK细胞、γδT细胞、NKT细胞和细CTL细胞等,它们共同参与调节Th1/Th2平衡构成妊娠期Th2偏倚的免疫微环境,使母体对胎儿产生特异性免疫耐受以维持妊娠。已有的研究证实,NK细胞大量存在于妊娠早期蜕膜并与胎盘绒毛滋养细胞密切接触,在妊娠中发挥重要作用(其中70%左右为CD56brightCD16+NK细胞),而在妊娠晚期则明显减少。最近大量的研究发现,NKT细胞也参与了母胎耐受的形成。在人妊娠早期的蜕膜和外周血中存在NKT细胞的表达,人蜕膜组织富集TCRαβ+CDl61+非恒定型NKT细胞(也称为II型NKT细胞,non-iNKT cell)并表现了显著的Th2型偏移(产生IL-4),为机体提供一个Th2型的微环境,并发现该群NKT细胞具有免疫抑制作用。提示蜕膜组织中的非恒定型NKT细胞可能通过提供一个Th2型的微环境从而抑制胎儿排斥和维持正常的妊娠。本研究采用流式细胞荧光测定技术检测孕足月分娩发动前后外周血和蜕膜组织单个核细胞中NK细胞、NKT细胞构成及其表面NKG2D受体和NKG2A受体表达情况的变化,并进一步检测外周血单个核细胞中Th1/Th2平衡的变化及NK细胞、NKT细胞分泌Th1、Th2型细胞因子的情况,探讨其与分娩发动的关系,揭示NK细胞及NKT细胞在分娩发动中的作用。并进一步研究母胎界面趋化因子CXCR4的表达情况,试图探讨其与分娩发动前后NK细胞、NKT细胞重新分布之间的关系。研究外周血及蜕膜组织中NK细胞、NKT细胞的功能状态与分娩发动的关系,无疑从一个全新的角度诠释分娩这一生理现象,也为揭示妊娠时限异常疾病的发生机制及最终有效防治提供新思路,并为生殖免疫的发展提供新的理论依据。目的: 检测分娩发动前后蜕膜及外周血中NK细胞及NKT细胞的含量、表型、Th1/Th2型细胞因子表达方面的变化,探讨NK细胞、NKT细胞独特的表型、功能与分娩发动之间的重要联系。检测母胎界面趋化因子CXCR4表达的变化,以发现其与NK细胞、NKT细胞在产程发动后在母胎界面重新分布及功能变化的联系。探讨母胎免疫平衡在分娩发动中的作用。方法: 1)收取正常晚孕分娩组(实验组,32例)和正常晚孕剖宫产组(对照组,34例)壁蜕膜组织及同一人外周血,机械研磨法联合密度梯度离心法分离出蜕膜单个核细胞,单纯密度梯度离心法分离出外周血单个核细胞,流式细胞术检测蜕膜及外周血中NK细胞、NKT细胞的含量及表型。 2)流式细胞术检测正常晚孕分娩组和剖宫产组外周血中Th细胞平衡的变化及NK细胞、NKT细胞内Th1/Th2型细胞因子IFN-γ和IL-4的表达。 3)收取正常晚孕自然分娩及剖宫产孕妇壁蜕膜及胎盘组织(各6例),RT-PCR方法检测胎盘及壁蜕膜中CXCR4的表达情况。结果: 1)与剖宫产组相比,分娩组蜕膜中NK细胞及NKT细胞的构成低于剖宫产组,外周血中NK细胞及NKT细胞构成高于剖宫产组(P0.01),两组蜕膜中NK细胞构成均高于外周血(P0.01)。 2)蜕膜单个核细胞中,分娩组CD56+NKG2A+细胞、CD56+ NKG2D+细胞、NK细胞表面NKG2A、NKG2D受体表达均低于剖宫产组(P0.05)。两组中NKT细胞表面NKG2A、NKG2D受体表达无显著差异(P0.05)。 3)外周血单个核细胞中,分娩组CD56+NKG2A+细胞构成、NK细胞表面NKG2A受体表达低于剖宫产组,(P0.05)。CD56+NKG2D+细胞构成、NK细胞表面NKG2D的表达及NKT细胞表面NKG2A/NKG2D受体的表达高于剖宫产组,(P0.01)。 4)外周血单个核细胞中,分娩组外周血CD3+T细胞中Th1细胞含量高于剖宫产组(P0.01),Th2细胞含量与剖宫产组无差异(P0.05)。分娩组NKT细胞、NK细胞分泌IFN-γ的能力高于剖宫产组。分娩组NKT细胞分泌IL-4的能力高于剖宫产组,NK细胞分泌IL-4的能力与剖宫产组无显著差异。 5)两组胎盘、胎膜均有CXCR4表达,剖宫产组胎盘、胎膜上CXCR4表达相对高于分娩组。结论: 分娩发动后外周血及蜕膜中NK细胞、NKT细胞的构成、表型及功能有变化。分娩组蜕膜中NK细胞、NKT细胞含量降低,外周血中含量增加且倾向于Th1偏倚。外周血中NK细胞、NKT细胞中NKG2D受体表达增高,提示其功能活化与分娩发动有一定联系,并且这种变化与趋化因子存在一定关联。在分娩发动过程中免疫系统存在变化,不仅表现在全身,在母胎界面也有明显变化,免疫平衡的改变可能在分娩发动中起着重要作用。
[Abstract]:Research background:
Normal pregnancy is a phenomenon of immune tolerance to maternal immune system with paternal antigens of the fetus, the tolerance is restrict each other by a variety of factors, the mutual regulation of reproductive immunology. In recent years, scholars on natural killer cells (NK cells) and natural killer T cells (NKT cells) to give more attention to.NK cells on the function of the immune balance of NKT cells to produce Th2 during pregnancy, when this balance is broken, which produce a variety of pathological pregnancy, and may start induction of labor. The mechanism of human parturition is still a mystery, the people turned to the organization level, the immune level of motivation. For delivery of maternal fetal immune micro the environment consists of immunocompetent cells in the decidua and the secretion of cytokines, including maternal immune system regulation of antigen specific T cells, regulatory T cells, NK cells, gamma delta T cells, NKT cells and small CTL Cells, etc., they participate in regulating Th1/Th2 balance to form immune microenvironment of Th2 bias during pregnancy, so that the mother has specific immune tolerance to the fetus to maintain pregnancy.
Research has confirmed that NK cells exist in early pregnancy decidua and trophoblast and placenta cells in close contact, play an important role in pregnancy (of which about 70% of CD56brightCD16+NK cells, and decreased) in late pregnancy. Many studies have found that NKT cells are involved in the formation of maternal fetal tolerance. Expression of NKT in cells during early pregnancy decidua and peripheral blood in human decidual tissue enriched TCR alpha beta +CDl61+ non constant type NKT cells (also known as II NKT cells, non-iNKT cell) and the performance of the Th2 type offset significantly (IL-4), provide a microenvironment for the Th2 body, and found the group of NKT cells with immunosuppression. Tips in decidua of non constant NKT cells may provide a microenvironment by Th2 to inhibit the fetal rejection and normal pregnancy.
This study used flow cytometry fluorescence detection of full-term delivery technology before and after the onset of NK cells in peripheral blood mononuclear cells and decidual tissues, changes of NKT cell structure and surface NKG2D receptor and NKG2A receptor expression, and further detected in peripheral blood mononuclear cells Th1 and NK cells balance changes of /Th2, NKT cells the secretion of Th1, Th2 type cytokines, to explore its relationship with the onset of labor, to reveal the role of NK cells and NKT cells in the initiation of labor. And further study on maternal fetal interface expression of chemokines CXCR4, to explore its and NK cells before and after parturition, the relationship between NKT cell NK cell redistribution. Study on peripheral blood and decidual tissues, the relationship between functional status and delivery of NKT cells to launch, no doubt the interpretation from a new perspective of delivery this physiological phenomenon, also to reveal the abnormal pregnancy time. The pathogenesis and the final effective prevention and control of disease provide new ideas, and provide new theoretical basis for the development of reproductive immunity.
Objective:
The phenotype of content detection before and after the onset of delivery in decidual and peripheral blood NK cells and NKT cells, the expression of Th1/Th2 type cytokines of NK cells, NKT cell phenotype unique and important link between the function and the onset of labor. The detection of maternal fetal interface of chemokines in the expression of CXCR4 and NK, to find the cells, the changes of NKT cells started in labor after redistribution in the maternal fetal interface and function. To investigate the role of maternal fetal immune balance in laboronset.
Method:
1) charged the normal fetal group (32 cases in experimental group, normal pregnant) and cesarean section group (control group, 34 cases) wall of decidua and peripheral blood of the same person, mechanical grinding method combined with density gradient centrifugation of decidual mononuclear cells isolated by density gradient centrifugation separation out peripheral blood mononuclear cells, NK cells were detected by flow cytometry in decidua and peripheral blood during the operation, the content and the phenotype of NKT cells.
2) flow cytometry was used to detect the changes of Th cell balance in peripheral blood of normal late pregnancy delivery group and cesarean section group, and the expression of Th1/Th2 cytokines IFN- gamma and IL-4 in NK cells and NKT cells.
3) the expression of CXCR4 in the placenta and the decidua of the placenta and wall decidua was detected by RT-PCR method for the normal delivery of late pregnancy and the caesarean section of the decidua and placenta (6 cases each).
Result:
1) compared with the cesarean section group, the NK cells and NKT cells in the decidua group were lower than those in the cesarean section group, and the NK cells and NKT cells in the peripheral blood were higher than those in the cesarean section group (P0.01). The NK cells in the two groups were higher than those in the peripheral blood (P0.01).
2) in decidual mononuclear cells, the expression of NKG2A and NKG2D receptors on CD56+NKG2A+ cells, CD56+ NKG2D+ cells and NK cells in the decidua mononuclear cells were lower than those in the cesarean section group (P0.05). There was no significant difference in the expression of NKG2A and NKG2D receptors on NKT cells in the two groups (P0.05).
3) in peripheral blood mononuclear cells, the CD56+NKG2A+ cells in the delivery group were made up. The expression of NKG2A receptors on the surface of NK cells was lower than that in the cesarean section group. (P0.05).CD56+NKG2D+ cells were formed, the expression of NKG2D on NK cells and the expression of NKG2A/NKG2D receptors on NKT cells were higher than those in caesarean section group (P0.01).
4) in peripheral blood mononuclear cells, Th1 cells in CD3+T cells in the peripheral blood of childbirth group is higher than that of cesarean section group (P0.01), Th2 cell content and cesarean section was no difference (P0.05). Delivery group NKT cells, NK cells secreted IFN- ability is higher than that of cesarean section group. Delivery capacity group NKT cells secreting IL-4 higher than that in the cesarean section group, NK cell secretory capacity of IL-4 and cesarean section group had no significant difference.
5) the expressions of placenta and placenta in the two groups were CXCR4, and the placenta in the caesarean section and the expression of CXCR4 on the membranes were higher than those in the delivery group.
Conclusion:
Parturition after NK cells in the peripheral blood and decidua, NKT cells, the phenotype and function change. NK cell delivery group in decidua of NKT cells decreased the content of content in peripheral blood increased and tended to bias Th1. NK cells in peripheral blood, increased expression of NKG2D receptor in NKT cells, suggesting that the the activation function and the onset of labor has certain relation, and this change and there is a certain correlation. Chemokines in laboronset immune system in the process of change, not only in the body, at the maternal fetal interface also has obvious changes, the change of the balance of immunity may play an important role in the onset of labor.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】