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目的建立免疫性卵巢早衰(POF)动物模型,探讨模型小鼠POF的免疫调节机制,为临床研究POF的免疫机制提供实验依据。方法选取B6AF1雌鼠40只,分为空白组(20只)与模型组(20只)。模型组小鼠用透明带3(ZP3)的多肽片段(pZP3)为免疫原免疫小鼠建立自身免疫性POF小鼠模型。比较两组小鼠一般情况,以判断pZP3对模型小鼠状态的影响。14 d后两组小鼠均取脏器称重,计算脏器指数。两侧卵巢组织病理切片分别进行HE染色和免疫荧光染色,检测卵巢组织形态、淋巴细胞浸润和透明带抗体分布的情况。采用实时定量PCR检测叉状/翼状螺旋转录因子(Foxp3)及维甲酸相关孤核受体γt(ROR-γt)mRNA表达情况。结果经pZP3免疫的B6AF1小鼠一般情况较差,容易出现烦躁和愤怒等现象。与空白组相比,模型组小鼠脾脏指数升高(P<0.01),胸腺指数和卵巢指数均下降(P均<0.05),子宫指数有所下降,但无统计学差异(P>0.05);空白组卵巢组织中未见炎性细胞浸润及透明带抗体荧光,模型组小鼠卵巢组织中出现了不同程度的炎症细胞浸润,且有强度不等的透明带抗体荧光的显现。实时定量PCR结果显示,与空白组相比,模型组小鼠脾脏中Foxp3 mRNA相对表达水平明显下降(P<0.01),ROR-γt mRNA相对表达水平明显上升(P<0.05)。结论采用pZP3免疫B6AF1雌性小鼠能够成功建立免疫性POF模型,其中调节性T细胞(Treg细胞)和辅助性T细胞(Th)17中的特异性转录因子Foxp3和ROR-γt表达水平出现失衡,这有助于研究卵巢早衰的免疫调节机制。
OBJECTIVE: To establish an animal model of premature ovarian failure (POF) and to investigate the immunoregulatory mechanism of POF in model mice to provide an experimental basis for the clinical study of POF immune mechanism. Methods Forty female B6AF1 mice were divided into blank group (n = 20) and model group (n = 20). Model mice were immunized with the polypeptide fragment of zona pellucida 3 (ZP3) as immunogen to establish an autoimmune POF mouse model. The general situation of the two groups of mice was compared to determine the effect of pZP3 on the state of the model mice. After 14 days, the mice in both groups were weighed and the organ index was calculated. Histopathological sections of ovarian tissue on both sides were stained with HE and immunofluorescent staining, respectively. The ovarian tissue morphology, lymphocyte infiltration and antibody distribution of zona pellucida were detected. The expression of Foxp3 and ROR-γt mRNA in nucleus accumbens was detected by real-time PCR. Results The B6AF1 mice immunized with pZP3 were generally poor, prone to irritability and anger. Compared with the blank group, the spleen index (P <0.01), thymus index and ovarian index (P <0.05) and the uterus index decreased in the model group (P> 0.05) No inflammatory cell infiltration and fluorescence of zona pellucida were found in the ovarian tissue of the blank group. Inflammatory cell infiltration was observed in the ovarian tissue in the model group, and the fluorescence of the zona pellucida antibody with different intensities appeared. The results of real-time PCR showed that compared with the blank group, the relative expression level of Foxp3 mRNA in the spleen of the model group was significantly decreased (P <0.01) and the relative expression level of ROR-γt mRNA was significantly increased (P <0.05). CONCLUSION: Immunization of B6AF1 female mice with pZP3 can successfully establish an immune POF model in which the expression levels of specific transcription factors Foxp3 and ROR-γt in regulatory T cells (Tregs) and T-helper T cells (17) This helps to study the mechanism of immune regulation of premature ovarian failure.