| 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. |
