【作者】 张旸；

【导师】 曹颖平；

【作者基本信息】 福建医科大学 ， 临床检验诊断学， 2008， 硕士

【摘要】 目的对比研究NKT细胞在正常妊娠组小鼠与原因不明复发性自发性流产模型小鼠子宫滋养层细胞中的数量,细胞因子分泌情况、成熟度以及滋养层淋巴细胞T-bet mRNA的表达水平。方法将CBA/J雌性与BABL/C雄性各一只同笼饲养,建立正常妊娠模型,将CBA/J雌性与DBA/2J雄性各一只同笼饲养,建立原因不明复发性自发性流产模型,每日观察小鼠阴道有无栓塞,在小鼠妊娠第9、11、13、15天,终止小鼠妊娠。并取子宫滋养层,用200目钢丝筛网,进行研磨,获得单个细胞悬液。裂解红细胞,取1.5×106的细胞用CD3、α-Galcer加载的CD1d四聚体、CD69抗体,流式细胞检测。另取5×106的细胞接种于RPMI1640+15%FBS培养液中,两天后收集培养液上清,与悬浮细胞并提取总RNA,逆转录成cDNA后,进行荧光定量PCR分析,检测T-bet相对于HPRT的表达水平。结果1.正常妊娠组与原因不明自发性复发性流产组,在妊娠过程中,CD3+T细胞数量无显著性变化。且两组间无显著性差异。2.正常妊娠组,在妊娠过程中,滋养层细胞分泌IFN-γ的量逐渐下降,伴随有NKT细胞数量、成熟型频率逐渐下降。3.原因不明自发性复发性流产组,在妊娠过程中,滋养层细胞分泌IFN-γ的量逐渐上升,伴随有NKT细胞数量、成熟型频率逐渐上升。4.无论是正常妊娠组还是原因不明自发性复发性流产组,滋养层分泌IFN-γ的量都与NKT细胞成熟型频率正相关。5.原因不明自发性复发性流产组与正常妊娠组相比,T-bet mRNA存在表达异常。T-bet mRNA的表达水平与NKT细胞成熟型频率,滋养层分泌IFN-γ的量正相关。结论1.妊娠过程中,NKT细胞可能是滋养层分泌细胞因子的主要细胞。2.原因不明自发性复发性流产的发生,可能与NKT细胞失调相关。妊娠早期与NKT细胞成熟型频率偏低,分泌IFN-γ不足有关;妊娠中后期则与NKT细胞成熟型频率偏高,分泌IFN-γ过量有关。3. T-bet mRNA的表达异常是造成NKT细胞失调的因素之一。更多还原

【Abstract】 ObjectiveComparative Study of the NKT cells numbers、cytokine secretion、maturity and expression levels of T-bet in mice trophoblast with normal pregnancy and unexplained recurrent spontaneous abortion.MethodsEstablish normal pregnancy model by feeding CBA / J and BABL/C in a cage, unexplained recurrent spontaneous abortions model by feeding CBA / J and DBA/2J in a cage. Observe the vagina embolization in mice daily. In the 9、11、13、15 days,terminate pregnancy. Grind the trophoblast wire on the mesh 200 to get cells suspension. Crack erythrocytes. Stain 1.5×106 cells with the CD3,α-loaded CD1d Galcer tetramer, CD69 antibody, then detected by flow cytometry. Culture 5×106 cells in RPMI1640 +15% FBS medium, two days after the collection medium supernatant, and the suspension cells and from the total RNA, reverse transcriptase into cDNA, fluorescence quantitative PCR analysis, testing T - bet in relation to the expression of HPRT.Results1. Normal pregnancy and unexplained recurrent spontaneous abortion group, in the course of pregnancy, classical CD3 + T cells without significant changes. Moreover, there is no significant difference between the two groups.2.Normal pregnancy group, in pregnancy process, the trophoblast cells secreting IFN-γdecreased gradually;the number and mature frequency gradually declining.3.Unexplained recurrent spontaneous abortion group, in pregnancy process, trophoblastic cells secreting IFN-γincreased gradually, the number and mature frequency of NKT cells also increased gradually.4. No matter the normal pregnancy group or the unexplained recurrent spontaneous abortion group, trophoblast cells IFN-γsecretion is related to NKT cells mature frequency.5.There is a difference of T-bet mRNA expression between Unexplained recurrent spontaneous abortion group compared with normal pregnancy,. T-bet mRNA expression levels is related to the frequency of mature NKT cells and trophoblastic cells IFN-γsecretion.Conclusions1. In the course of pregnancy, NKT cells may be the major secreting cells in the trophoblast.2.Unexplained recurrent spontaneous abortion occurred, may be related to NKT cells disorders. NKT cells have low- mature frequency and inadequate secretion of IFN-γduring early pregnancy; whereas have high- mature frequency and excessive secretion of IFN-γduring latter pregnancy.3.The anomaly of T-bet mRNA expression may be one of the factors lead to NKT cells disorder.更多还原