【作者】 朱姝；

【导师】 陆启滨；

【作者基本信息】 南京中医药大学 ， 中医妇科学， 2011， 博士

【摘要】 目的：1.临床试验：通过检测ACA阳性先兆流产患者治疗前后外周血CD4+CD25+FOXP3+Treg细胞的比例,观察安子合剂对ACA阳性先兆流产患者外周血CD4+CD25+FOXP3+Treg细胞的影响,从临床方面探讨ACA阳性先兆流产的免疫病理机制及安子合剂的免疫调节机理。2.动物实验：通过检测ACA导致妊娠丢失动物模型孕鼠外周血CD4+CD25+FOXP3+Treg细胞比例,观察安子合剂对ACA导致妊娠丢失动物模型孕鼠外周血CD4+CD25+FOXP3+Treg细胞的影响,从实验方面探讨ACA导致妊娠丢失的免疫病理机制及安子合剂的免疫调节机理。方法：1.临床试验：①选取27例ACA阳性先兆流产患者及15例正常孕妇为观察对象,用流式细胞仪检测两组外周血CD4+CD25+FOXP3+Treg细胞的比例,比较ACA阳性先兆流产患者治疗前与正常孕妇外周血CD4+CD25+FOXP3+Treg细胞比例的差异。②用流式细胞仪检测27例ACA阳性先兆流产患者经安子合剂治疗后外周血CD4+CD25+FOXP3+Treg细胞的比例,观察治疗前后外周血CD4+CD25+FOXP3+Treg-eg细胞比例的变化。③用ELISA法测定安子合剂治疗前后ACA定量和定性指标,观察ACA的变化。④根据中医证候分级量化及评分标准,记录安子合剂治疗前后腹痛、腰酸、阴道出血等先兆流产临床症状的积分变化,评价中医证候的改善情况；动态检测安子合剂治疗期间血清E2、P、β-HCG值及B超检查孕囊大小、形态及胎心搏动等的变化,评价胚胎发育情况。⑤根据临床综合疗效评定标准,评价安子合剂的临床综合疗效。2.动物实验：①将动物随机分为6组：空白对照组、模型组、安子合剂低剂量组、安子合剂中剂量组、安子合剂高剂量组、阿司匹林组。观察阴道脱落细胞涂片及阴栓,来确定妊娠日。②通过给孕鼠腹腔注射ACA-IgG/NH-IgG,建立动物模型。③用流式细胞仪检测各组孕鼠外周血CD4+CD25+FOXP3+Treg细胞的比例,比较各组细胞比例的差异。④用ELISA法测定各组孕鼠外周血ACA的滴度,观察各组ACA滴度的变化。⑤常规组织病理切片,观察各组子宫蜕膜、胎盘组织病理变化；免疫组化检测各组母胎界面上FOXP3.C3.TNF-α、IgG的表达,观察各组胚胎吸收率及胎鼠发育情况。结果：1.临床试验：1.1. ACA阳性先兆流产患者治疗前CD4+CD25+FOXP3+Treg细胞的比例显著低于正常妊娠对照组(P<0.01)。1.2.安子合剂治疗后,ACA阳性先兆流产患者外周血CD4+CD25+FOXP3+Treg细胞的比例显著高于治疗前(P<0.01)。1.3.安子合剂治疗后,27例患者中20例ACA定性指标—ACA阳性的转阴率达85.00%,7例ACA定量指标—ACA滴度较治疗前显著下降(P<0.01)。1.4.安子合剂治疗后,27例ACA阳性先兆流产患者,B超检查25例可见胎心搏动,21例胎儿成形,保胎的成功率为92.59%。1.5.安子合剂治疗后,先兆流产中医证候明显改善,治愈率33.33%,显效率33.33%,总有效率88.89%;临床综合疗效为痊愈率84.00%,好转率14.81%,总有效率92.59%。2.动物实验：2.1.成功复制了ACA导致妊娠丢失的动物模型。2.2.模型动物外周血CD4+CD25+FOXP3+Treg细胞的比例较空白对照组显著下降(P<0.01)；与模型组相比,安子合剂低、中、高剂量组、阿司匹林组均能显著升高CD4+CD25+FOXP3+Treg细胞的比例(P<0.01),其中安子合剂低剂量组疗效优于中、高剂量组和阿司匹林组(P<0.01)。2.3.与模型组相比,安子合剂低、中剂量组、阿司匹林组能显著降低模型动物外周血ACA的滴度(P<0.01),安子合剂低剂量组疗效优于中剂量组和阿司匹林组(P<0.01)。2.4.安子合剂中、低剂量组及阿司匹林组能明显改善模型动物母胎界面上免疫复合物的沉积、血栓病灶、坏死区域等病理变化。2.5.安子合剂低、中剂量组、阿司匹林组都能增加模型动物母胎界面上FOXP3的表达(P<0.01),安子合剂低剂量组优于中剂量组和阿司匹林组(P<0.01)。2.6.安子合剂低、中、高剂量组、阿司匹林组都能减少模型动物母胎界面上C3、IgG、TNF-α的沉积(P<0.05),安子合剂低剂量组疗效优于中、高剂量组和阿司匹林组(P<0.01)。2.7.安子合剂低剂量组、阿司匹林组能显著降低模型动物胚胎吸收率,显著增加模型动物胎鼠、胎盘重量(P<0.05)。结论：1.临床研究表明,ACA阳性先兆流产患者外周血CD4+CD25+FOXP3+Treg细胞的比例低于正常妊娠妇女,存在免疫调节异常；中药安子合剂治疗ACA阳性先兆流产的作用机理之一与增加CD4+CD25+FOXP3+Treg细胞的比例、调节妊娠期机体免疫功能有关。2.实验研究表明,ACA导致妊娠丢失的病理机制之一是机体免疫调节细胞-CD4+CD25+FOXP3+Treg细胞比例的减少,免疫调节功能紊乱,免疫复合物沉积于母胎界面,引起补体C3过度激活,诱导TNF-α等炎性细胞聚集并活化,引起母胎界面组织损伤,母体对胚胎排斥,导致妊娠丢失。安子合剂干预ACA导致妊娠丢失的免疫调节机制是通过增加CD4+CD25+FOXP3+Treg细胞的比例来实现的,其作用的强弱与药物的剂量密切相关,适当的药物剂量,可发挥最佳的免疫调节功效。更多还原

【Abstract】 Objective:1. Clinical trials:By detecting the percentage of peripheral blood CD4+CD25+FOXP3+Treg cells in ACA-positive patients with threatened abortion pre and post therapy, to observe the impact of Anziheji on the percentage of peripheral blood CD4+CD25+FOXP3+Treg cells in ACA-positive patients with threatened abortion, to explore the immune pathological mechanism of ACA-positive threatened abortion and the immune regulation mechanism.of Anziheji from the clinical aspects.2. Animal experiments:By detecting the pregnant mice’ percentage of peripheral blood CD4+CD25+FOXP3+Treg cells in animal model of ACA induced pregnancy loss, to observe the impact of Anziheji on the pregnant mice’ percentage of peripheral blood CD4+CD25+FOXP3+Treg cells in animal model of ACA induced pregnancy loss, to explore the immune pathological mechanism of ACA induced pregnancy loss and the immune regulation mechanism.of Anziheji from the experiment aspects.Methods:1. Clinical trials:①Select the 27 cases of ACA-positive patients with threatened abortion and 15 cases of normal pregnant women as the observed object, detect the percentage of peripheral blood CD4+CD25+FOXP3+Treg cells in the two group by flow cytometry, compare the difference of peripheral blood CD4+CD25+FOXP3+Treg cells’percentage between ACA-positive patients with threatened abortion before treatment and normal pregnant women.②Detect the percentage of peripheral blood CD4+CD25+FOXP3+Treg cells in 27 cases of ACA-positive patients with threatened abortion after treatment of Anziheji by flow cytometry, compare the difference of peripheral blood CD4+CD25+FOXP3+Treg cells’percentage between pre and post therapy.③Detect the quantitative and qualitative indicators of ACA by ELISA before and after treatment by Anziheji to evaluate the changes of ACA.④According to traditional chinese syndrome quantitative grading and scoring criteria, recording abdominal pain, backache, vaginal bleeding and other symptoms of the integral change before and after treatment by Anziheji, to evaluate the improvement in traditional chinese syndrome;dynamic monitoring of serum E2, P,β-HCG levels and B-monitoring of gestational sac size, shape and fetal heart beat and so on, evaluation of embryo development⑤According to comprehensive clinical evaluation standard, to evaluate the comprehensive effect of Anziheji. 2. Animal experiments:①Animals were randomly divided into 6 groups:control group model group、low dose group of Anziheji、middle dose group of Anziheji、high dose group of Anzihej、aspirin group. See vaginal smears of keratinocytes and the vaginal plug to make the day of pregnancy.②Inject ACA-IgG/NH- IgG to pregnant mice by intraperitoneal injection to establish animal model.③Detected the peripheral blood CD4+CD25+FOXP3+ Treg cells ratio of pregnancy mice by flow cytometry, compare the difference in each group.④Detected ACA level of mice pregnancy by ELISA, compare the difference in each group.⑤Observe the decidual and placental pathological changes by routine pathological section, detecte the expression of FOXP3, C3, TNF-α, IgG on maternal-fetal interface by immunohistochemical, observe the embryo resorption and fetal development situation.Results:1.Clinical trials:1.1.The proportion of CD4+CD25+FOXP3+Treg cells in ACA-positive patients with threatened abortion before treatment of Anziheji was significantly lower than the control group (P<0.01).1.2.The proportion of CD4+CD25+FOXP3+Treg cells in ACA-positive patients with threatened abortion after treatment of Anziheji was significantly higher than before treatment (P<0.01).1.3.After treatment of Anziheji,20 cases of 27 patients in the ACA negative rate of the qualitative indicators of 85.00%,7 cases of quantitative indicators of ACA titers were significantly decreased compared with before treatment (P<0.01).1.4. After treatment of Anziheji,27 cases of abortion patients, B-ultrasound fetal heart beat can be seen in 25 cases,21 cases of fetal shape, success rate of miscarriage is 92.59%.1.5. After treatment of Anziheji, the patient improved traditional chinese syndrome,33.33% cure rate, markedly effective rate 33.33%, total effective rate was 88.89%; the clinical comprehensive efficacy cure rate was 84.00%, improvement rate was 14.81%, total efficiency was 92.59%.2. Animal experiments:2.1. Successful established the animal model of ACA induced fetal loss.2.2.Model animal, the peripheral blood CD4+CD25+FOXP3+Treg cells ratio was significantly lower than the control group(P<0.01); The low、high、middle dose group of Anziheji and the aspirin group can increase CD4+CD25+FOXP3+Treg cells ratio(P<0.01), compared with model group, The low dose group of Anziheji was better than the high、 middle dose group of Anziheji and the aspirin group (P<0.01).2.3. Compared with model group, The low、middle dose group of Anziheji and the aspirin group can significantly reduce the titer of ACA in the peripheral blood of animal models (P <0.01), the low dose group of Anziheji was better than the middle dose group of Anziheji and the aspirin group (P<0.01).2.4.The low、middle dose group of Anziheji and the aspirin group can reduce deposition of immune complexes, necrotic regions, thrombosis lesions on maternal-fetal interface of model animal.2.5.Compared with model group, the low、middle dose group of Anziheji and the aspirin group can increased FOXP3 expression of maternal-fetal interface (P<0.01), the low dose group of Anziheji was better than the middle dose group of Anziheji and the aspirin group (P<0.01).2.6. Compared with model group, the low、high、middle dose group of Anziheji and the aspirin group can decreased model animal. maternal-fetal interface C3, TNF-a, IgG deposition (P< 0.05), the low dose group of Anziheji was better than the high、middle dose group of Anziheji and the aspirin group (P<0.01).2.7.Compared with model group, the low dose group of Anziheji and the aspirin group can significantly reduce the resorption rate of model animal(P<0.05), increased fetal weight and placental weight of model animal(P<0.05).Conclusions:1. Clinical studies have shown that, ACA-positive patients with threatened abortion peripheral blood CD4+CD25+FOXP3+Treg cells percentage were lower than normal pregnant women, there is abnormal immune regulation, one of the mechanism of Anziheji is to increase the CD4+CD25+FOXP3+Treg cells percentage, regulate the immune function of pregnancy2. Animal experiments have shown that, ACA induced pregnancy loss was the pathological mechanism of decreased immune cells-CD4+CD25+FOXP3+Treg cells percentage, immune regulation disorders, immune complex deposition in the maternal-fetal interface, complement activation, recruitment of inflammatory mediators, tissue damage on maternal-fetal interface, the mother of the embryo rejection. The immune regulatory mechanism of Anziheji on ACA induced pregnancy loss was achieved by increasing the CD4+CD25+FOXP3+Treg cells ratio, strength and its role is closely related to the dose of drug, appropriate dosage, can play the best immune function.更多还原