【作者】 赖毛华；

【导师】 尤昭玲；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的：通过研究小鼠母胎界面SOCS 1,SOCS2,SOCS3mRNA的表达及Th1/Th2型细胞因子与反复自然流产的关系及寿胎丸对自然流产模型母-胎界面SOCS1,SOCS3mRNA的表达和Th 1/Th2型细胞因子表达及平衡的调控,并通过用体外培养的方法,进一步了解寿胎丸的调控不同SOCS基因表达及影响Th1/Th2的偏移的免疫调节作用,探讨寿胎丸在诱导母一胎免疫耐受中的作用机制。方法：1.CBA/J×BALB/c建立正常妊娠模型,CBA/J×DBA/2建立反复自然流产模型,将自然流产模型CBA/J×DBA/2孕鼠按怀孕先后顺序随机分为四组,分别为模型组,寿胎丸低、中、高剂量组,从妊娠第一天开始给药,孕14天后处死孕鼠,计算各组胚胎吸收率,RT-PCR测定各组母-胎界面SOCS1和SOCS3的转录水平,ELISA检测蜕膜及胎盘组织共培养上清细胞因子IL-4,IL-10,IFN-y,TNF-a的含量,以IL-4／IFN-γ、IL-10／TNF-α衡量免疫平衡的指标。2.以非孕期BALB/c雌鼠脾细胞来源CD4+T细胞为反应细胞,以OVA为,抗原灭活处理的DBA/2雄鼠脾细胞APC为抗原呈递细胞,加入IL-2和IL-12细胞因子共培养,诱导制备CD4+T细胞Th1偏移模型。用寿胎丸含药血清在低中高剂量和最有效剂量在Oh,1h,2h,4h,8h分别对CD4+T细胞Th1偏移模型进行体外诱导其分化,分别采RT-PCR测定细胞模型的SOCS1和SOCS3的转录水平;ELISA测定细胞培养上清液IL-4,IL-10,IFN-γ,TNF-α的分泌水平,以IL-4／IFN-γ、IL-10／TNF-α衡量免疫平衡的指标.结果：模型组的胚胎吸收率模型高于正常妊娠组,与模型组比较,寿胎丸低剂量未能显著改善自然流产模型孕鼠的胚胎吸收率(P>0.05)寿胎丸中高剂量组孕鼠的胚胎吸收率均显著下降,与正常妊娠组比较无明显差异(P>0.05)。模型组母胎界面IFN-γ、TNF-α表达显著高于正常妊娠组(P<0.05),IL-4、IL-10表达、IL-4／IFN-γ、IL-10／TNF-α比值显著低于正常妊娠组(P<0.05)。与模型组相比,寿胎九低中高剂量组均能使孕鼠母胎界面TNF-a和IFN-γ的表达显著下降(P<0.05),寿胎丸中高剂量组能使母胎界面IL-4、IL-10的表达显著增加(P<0.05)；与正常妊娠组比较,孕鼠母-胎界面IL-4,IL-10,IFN-γ,TNF-a.的表达水平无显著性差异(P>0.05)；寿胎丸低中高剂量组使Th1/Th2平衡的平衡朝Th2偏移,其中中高剂量组与正常组无明显差异(P>0.05). SOCS1、SOCS2、SOCS3mRNA在模型组及正常妊娠组蜕膜和胎盘组织均有表达,但与正常妊娠组比较,SOCS1mRNA在模型组升高,而SOCS3mRNA表达降低,SOCS2mRNA表达无明显差异,寿胎丸低中高剂量组能够下调模型组孕鼠母胎界面的SOCS1mRNA的表达,寿胎丸中高剂量组升调模型组孕鼠SOCS3mRNA的表达(P<0.05)。寿胎丸低中高剂量组SOCS1mRNA的表达与正常妊娠组无明显差异,寿胎丸中高剂量组的SOCS3mRNA的表达与正常妊娠组无明显差异(P>0.05)。寿胎丸中剂量和高剂量药物血清可显著降低Thl偏移模型的CD4+T细胞SOCS1mRNA,TNF-α、IFN-γ的表达(P<0.05),同时高剂量药物血清还可显著提高其SOCS3mRNA, IL-4,IL-10表达水平(P<0.05)；寿胎丸高剂量药物血清分别作用2、4、8h均可显著降低SOCS1mRNA,TNF-α、IFN-γ的表达(P<0.05),作用4h时达到最小值,高剂量药物血清分别作用2、4h可显著提高SOCS3mRNA, IL-4,IL-10表达水平(P<0.05),作用4h时达到最大值。结论：正常妊娠组母胎界面局部均呈现明显的Th2型免疫偏倚。而模型组呈现明显的Th1型免疫偏倚。母胎界面Thl/Th2型细胞因子表达调节失衡是导致流产的重要原因之一。SOCS1、SOCS3可能在调节Th1/Th2型平衡中发挥重要作用。寿胎丸可能通过降低母胎界面SOCS1的表达水平,升高母胎界面SOCS3的表达水平,恢复母—胎界面局部Th1型／Th2型细胞因子表达的生理平衡,形成维持正常妊娠所需的Th2型免疫偏离,诱导母—胎免疫耐受,从而降低胚胎吸收率,维持妊娠。更多还原

【Abstract】 Objective:To study the relationship between abortion and the expression of mouse SOCS1,2,3 and Th1/Th2 type cytokines at the maternal-fetal interface. To evaluate the role that Shoutaiwan regulate SOCS1, SOCS3mRNA expression and Thl/Th2 type cytokine expression and balance of control in maternal-fetal interface and By cultured in vitro,to study shoutaiwan can regulate gene expression of different SOCS and the impact of Thl/Th2 immune deviation and the adjustment mechanism. In order to shoutaiwn can induce the maternal-fetal immune tolerance Of mechanism.Methods:1.The abortion-prone CBA/JxDBA/2 matings were established as the model of spontaneous abortion and the nonabortion-prone CBAxBALB/c matings were used as the model of normal pregnancy.The model of spontaneous abortion CBA/JxDBA/2 of mice pregnant were randomly divided into four groups according to the sequence of pregnancy, From the 1st day of pregnant,pregnant rats of every group are oral administration in different doses (Og/kg,3g/kg,6g/kg, 12 mg/kg).on the 14th days of pregnancy,the mice are killed. The embryo resorption rate was counted,RT-PCR was applied to analyze the SOCS1, SOCS2,SOCS3 transcription of decidua and placenta, ELISA Was applied to determinate the IL-4, IL-10, IFN-y, TNF-a concentration in culture supemates of decidual and placenta, The ratio of IL-4/IFN-y, IL-10/TNF-a was used as the index to evaluate the immune regulation balance.2.the CD4+T cells coming from the non-pregnant BALB/c female spleen cells are think as respond cell, the inactivation of DBA/2 male spleen cells are think as APC t, add OVA antigen, IL-2 and IL-12 Co-culture of cytokines, induce CD4+T cells Thl diviated model. shoutaiwan serum containing low middle high-dose and the most effective dose of Oh, 1h,2h,4h,8h, respectively, induce CD4+T cells of Thl deviation differentiation in vitro model, RT-PCR was applied to analyze the SOCS1, SOCS2,SOCS3 transcription of decidua and placenta, ELISA Was applied to determinate the IL-4, IL-10, IFN-y, TNF-a concentration in culture supemates of decidual and placenta, The ratio of IL-4/IFN-γ, IL-10/TNF-a was used as the index to evaluate the immune regulation balance.Results:The models of embryo resorption rate are higher than the normal pregnancy group, Compared with model group, low-dose shoutaiwan can not significantly improve the model of spontaneous abortion in pregnant mice embryo resorption rate (P> 0.05); shoutaiwan high dose group of pregnant rats absorption rate of the embryos were significantly decreased, IFN-y, TNF-a expression in model group are significantly higher than the normal pregnancy group (P<0.05), IL-4, IL-10 expression, IL-4/IFN-y, IL-10/TNF-a ratio are significantly lower than the normal pregnancy group at Maternal-fetal interface (P<0.05), Compared with model group, shoutaiwan low middle high-dose group can decreased significantly TNF-a and IFN-y expression of maternal-fetal interface of pregnant rats (P<0.05), shoutaiwan middle high dose group make IL-4, IL-10 expression in maternal-fetal interface increased significantly (P<0.05). Compared With the normal pregnancy group,the expression of IL-4, IL-10, IFN-y, TNF-a of maternal-fetal interface in pregnant rats show no significant change (P> 0.05); shoutaiwan low middle high-dose group shift Thl/Th2 balance towards Th2, the middle high-dose group show no significant difference,compared with the normal group (P> 0.05).when compared with normal pregnancy,no significant difference between groups (P> 0.05).SOCS1, SOCS2, SOCS3mRNA in the model group and normal pregnancy decidual and placental tissue were expressed, compared with the normal pregnancy group, SOCS1mRNA increase in the model group, SOCS3mRNA expression and, SOCS2mRNA show no significant differences (P> 0.05). shoutaiwan low middle high dose group model can reduce SOCS1 gene expression of maternal-fetal interface of pregnant rats, shoutaiwan middle high dose group increase model group SOCS3 gene expression (P<0.05). shoutaiwan low middle high dose group in pregnancy was no significant difference from the normal expression of SOCS1, shoutaiwan middle hign (?)ose group of SOCS3 gene expression are no significant difference in normal pregnancy with (P> 0.05). The models of embryo resorption rate are higher than the normal pregnancy group, SOCS1, SOCS2, SOCS3mRNA in the model group and normal pregnancy decidual and placental tissue were expressed, compared with the normal pregnancy group, SOCS1mRNA increase in the model group, SOCS3mRNA expression and, SOCS2mRNA show no significant differences (P> 0.05). Maternal-fetal interface IFN-y, TNF-a expression in model group are significantly higher than the normal pregnancy group (P<0.05), IL-4, IL-10 expression, IL-4/IFN-y, IL-10/TNF-a ratio are significantly lower than the normal pregnancy group (P<0.05). Middle and high dose Shoutaiwan containing serum could significantly decrease the expression of SOCS1, IFN-y, TNF-a(P<0.05).High dose Shoutaiwan-containing serum could significantly increase the expression of SOCS3, IL-4, IL-10 (P<0.05).In addition,high dose Shoutaiwan-containing serum could significantly decrease the expression of SOCS1, IFN-y, TNF-arespectivly at 2h,4h and 8h after treating (P<0.05),the peak was 4h.High dose Shoutaiwan-containing serum also significantly increase the expression of SOCS3,IL-4, IL-10 respectivly at 2h and 4h (P＜0.05),the peak was 4h.Conclusion:The maternal-fetal interface in normal pregnancy group showed a significant local Th2-type immune bias. And the model group showed a significant bias toward Thl-type immunity. Maternal-fetal interface Th1/Th2 type cytokine imbalance are an important cause one of the reasons for abortion. SOCS1, SOCS3 play an important role in regulate the balance of Thl/Th2 type. shoutaiwan can reduce the expression of SOCS1 and SOCS3 at maternal-fetal interface,and recover maternal fetal interface Thl-type/Th2-type cytokine expression of the physiological balance, induce the immune tolerance to the maternal-fetal, thereby reduce the rate of embryo resorption.更多还原