【作者】 施晓波；

【导师】 林秋华；

【作者基本信息】 中南大学 ， 妇产科学， 2009， 博士

【摘要】 目的探讨自身免疫性POF的免疫学诊断方法;建立自身免疫性POF动物模型,采用糖皮质激素和雄激素治疗自身免疫性POF模型小鼠为临床治疗提供理论依据。方法分三个部分进行。第一部分自身免疫性卵巢早衰的诊断的研究:52例卵巢早衰并不孕症患者为研究组,20例其他原因不孕症患者为对照组,分别检查血清抗核抗体,抗卵巢组织抗体,抗心磷脂抗体,抗甲状腺微粒体抗体,抗双链DNA抗体,免疫球蛋白IgG,IgA,IgM,IgE,补体C₃,C₄含量及抗透明带抗体浓度,并进行统计学分析。第二部分实验性自身免疫性卵巢早衰小鼠模型的建立:取近交系雌性小鼠（C57BL/6J）,鼠龄7-9周130只,分别为模型组120只、空白对照组10只,各组小鼠均于双后脚掌掌心处进行皮下注射。模型组注射CFA—pZP3混合液0.1毫升,其中包含CFA:0.05毫升,pZP3:72.225ug;空白对照组注射三蒸水0.1毫升。建模观察指标为①各组小鼠阴道脱落细胞涂片观察性周期的改变;②采用ELISA检测小鼠外周血清中抗透明带抗体的浓度;③光学显微镜检查各组小鼠卵巢组织学改变:以生长卵泡所占比例表示卵巢萎缩受损程度;④免疫组化法检测卵巢组织中CD45阳性细胞的浸润程度判断其自身免疫性卵巢炎严重程度。模型组随机选择10只小鼠与空白组进行统计学比较和分析,评估建模是否成功。小鼠的研究:以出现明显性周期紊乱为标准,选择建模成功的自身免疫性POF小鼠80只,随机分为4组:每组20只,模型对照组同期每日腹腔注射三蒸水0.05ml,其余三组分别给以糖皮质激素、雄激素、糖皮质激素加雄激素联合治疗三个性周期,以上述建模指标进行检测作为疗效观察指标,判断其治疗效果,并进行统计学分析。结果第一部分:研究组ANA,AOAb,ds-DNA,A-TG阳性率及抗透明带抗体浓度分别为38.5%,40.4%,35.5%,44.2%和110.42±10.38pg/ml,均显著性高于对照组,P＜0.05;研究组ACA、免疫球蛋白IgM、补体C₃异常率较高,分别28.8%、23.1%和25%,但与对照组无显著性差别,P＞0.05;研究组免疫球蛋白IgG,IgA,IgE,补体C₄异常率较低分别为5.8%,1.9%,1.9%,3.8%,与对照组无显著性差别,P＞0.05。第二部分:模型组小鼠80%表现为性周期紊乱,血清抗透明带抗体平均浓度高达17.55±3.52（ng/ml）,生长卵泡比例低达22.44±5.88（%）,出现自身免疫性卵巢炎,即CD45阳性细胞浸润的小鼠比例为90%,上述指标均较空白组有显著性差异,P＜0.01。第三部分:①性周期正常者糖皮质激素组60%,雄激素组70%,联合治疗组65%,模型对照组仅10%。各治疗组与模型对照组均存在显著差异,P＜0.01。但三个治疗组进行两组间比较,均无显著差异,P＞0.05。②皮质激素组、雄激素组及联合治疗组小鼠血清抗透明带抗体平均浓度分别为（ng/ml）21.18±6.72,16.12±4.64和18.32±5.28,与模型对照组（32.61±8.77）比较均存在显著差异,P＜0.01。糖皮质激素组明显高于与雄激素组,P＜0.01。糖皮质激素组与联合治疗组、雄激素组与联合治疗组之间均不存在显著差异,P＞0.05。③糖皮质激素组、雄激素组及联合治疗组各组小鼠组织学检查生长卵泡比例分别为（%）36.8±8.00,37.5±6.06和30.71±4.36,与模型对照组（25.72±5.31）比较均存在显著差异,P＜0.01。糖皮质激素组与雄激素组无显著差异,P＞0.05;糖皮质激素组与联合组、雄激素组与联合治疗组之间均存在显著差异,P＜0.01。④各组出现自身免疫性卵巢炎,即CD45阳性细胞浸润的小鼠比例分别为糖皮质激素组25%,雄激素组30%,联合治疗组50%,均与模型对照组90%存在显著差异,P＜0.01。三个治疗组进行两组间比较,均无显著差异,P＞0.05。结论1.卵巢早衰患者存在多种自身免疫性抗体,ANA,AOAb,ds-DNA,A-TG,抗透明带抗体等检测对诊断自身免疫性卵巢早衰有临床实际意义。2.通过给小鼠皮下免疫注射小鼠透明带多肽片段,成功地建立了小鼠的自身免疫性卵巢早衰的实验动物模型,为进行卵巢早衰的诊断学及治疗学方面的研究提供了前提条件。3.糖皮质激素和雄激素均能显著改善实验性自身免疫性卵巢早衰小鼠的临床症状及免疫反应指标。且二者疗效无显著性差异。4.糖皮质激素和雄激素联合治疗实验性自身免疫性卵巢早衰小鼠的效果并不优于上述两者的单独治疗效果。更多还原

【Abstract】 Objective:To establish the immunologic diagnostic methods of autoimmune premature ovary failure(POF)and to induce the animal model of autoimmune POF and provide theoretical evidence for treatment of autoimmune POF by glucocorticoids and androgen.Methods:Part 1 Study of diagnosis of autoimmune POF:52 patients with POF complicating infertility were chosen for test group,and 20 patients with infertility caused by others factors were chosen for control We detect and analyze the serum levels of ANA,AoAb,AcA,A-TG,ds-DNA, immunoglobulins IgG、IgA、IgM、IgE,complement C3、C4 and AzpAb.Part 2 Induction of animal model of autoimmune POF:We bought 130 seven-to nine-week old female inbred line mice(C57BL/6J)from the SLACCAS animal centre,and divided them into two groups:10 mice in the control group and 120 in the model group.Each mouse accepted subcutaneous injections in both posterior sole of feet.The model group was injected with CFA—pZP3 mixed solution,which contained CFA: 0.05ml,pZP3:72.225ug.And the control group was injected with 0.1ml of distilled water.Observation indices included:①Smear of the vaginal cast-off cell of the mice for sexual cycle observation;②Detection of AzpAb serum level by ELISA;③Observation of the histological changes of the mice by HE slicing under optical microscope; ④Evaluation of the severity of autoimmune oophoritis via examining infiltration of cells expressing CD45 in the ovary by immunohistochemisty to.Part 3 Study of therapy methods of glucocorticoids or androgen for autoimmune POF:We choose 80 mice of autoimmune POF,and randomly distributed them into 4 groups,20 mice per group.Three groups were treated by glucocorticoids,androgen,or therapeutic alliance of glucocorticoids and androgen repsectively.We evaluated and analyzed the treatment effectiveness of these three therapeutic methods according to the index above.Results:Part 1 The serum level of AzpAb and positive rates of ANA, AoAb,ds-DNA and A-TG in the test group were 110.42±10.38pg/ml, 38.5%,40.4%,35.5%,44.2%respectively,which were all higher than the control group(P＜0.05).Abnormality rates of ACA,immunogloblin IgM,complement C3 in the test group were 28.8%、23.1%and 25% respectively,which were found to be higher than the control group. However,the difference was not significant(P＞0.05).Abnormality rates of immunogloblin IgG,IgA,IgE and complement C4 were 5.8%,1.9%, 1.9%,3.8%respectively,which was found to be lower than the control group,but not significantly so(P＞0.05).Part 2 Eighty percent of mice in the model group were found to have disordered sexual cycles.The mean serum level of AzpAb was at 17.55±3.52ng/ml,whereas the proportion of growing follicles was only 22.44±5.88%.In addition,ninety percent of mice suffered from ovary infiltration of cells positively expressing CD45,which supported autoimmune oophoritis.Thus,all theindices in the model group were significantly different from the control group(P＜0.01).suggesting that the animal model of autoimmune POF was successful.Part 3①Comparison of sexual cycles of each group after treatment: Sexual cycle of 60%of mice in the glucocorticoids group was normal, while 70%in the androgen group,65%in the therapeutic alliance group, but 10%model control group were normal.Each therapeutic group was significantly different from the control group(P＜0.01)However,the differences among the three therapeutics groups were not significant(P＞0.05)②Comparison of serum levels of AzpAb of each group after treatment:The serum levels of AzpAb in glucocorticoids group,androgen group and therapeutic alliance group was 21.18±6.72ng/ml,16.12±4.64 ng/ml and18.32±5.28ng/ml respectively.All of the above values were significantly different from the model control group(32.61±8.77 ng/ml)(P＜0.01).The difference between the glucocorticoids group and androgen group was significant(P＜0.01).But the difference between the glucocorticoids group and the therapeutic alliance group,or androgen group and the therapeutic alliance group were not significant(P＞ 0.05).③Comparison of the proportion of growing follicles in each group after treatment:The proportion of growing follicles in glucocorticoids group,androgen group and therapeutic alliance group was36.8±8.00%, 37.5±6.06%and30.71±4.36%respectively,all of which was significantly different from the model control group(25.72±5.31%,P＜0.01).The difference between the glucocorticoids group and androgen group was not significant,(P＞0.05).But the difference between the glucocorticoids group and the therapeutic alliance group,or androgen group and the therapeutic alliance group were significant,(P＜0.01).④Comparison of infiltration of lymphocytes in the ovaries:Twenty five percent of mice in the glucocorticoids group suffered from oophoritis,that was,the infiltration of cells expressing CD45 in the ovaries,while 30%in the androgen group,50%in the therapeutic alliance group,90%in the model group suffered from oophoritis.Each therapeutic group was significantly different from the control group(P＜0.01).But the differences among the three therapeutics groups were not significant(P＞0.05).Conclusion:1.We found many kinds of autoimmune antibodies in patients with POF.Detection of ANA,AOAb,ds-DNA,A-TG and AzpAb was of clinical significance to diagnosing autoimmune premature ovary failure.2.We successfully developed animal model of autoimmune premature ovary failure by active immunization of adult mouse with a murine ZP3 peptide(pZP3)in complete Freund’s adjuvant(CFA),and provided conditions for further research of POF in diagnostics and therapeutics.3.We found that both glucocortcoids and androgen could significantly redress the clinical symptons and immunical indicators of autoimmune POF.But the differences between them were not significant (P＞0.05).4.We found that the therapeutic alliance of glucocorticoids and androgen was not better than either glucocorticoids or androgen alone.更多还原