从诱导调节性T细胞分化探讨补肾活血中药治疗免疫性卵巢早衰的机制

In recent years, studies have shown that abnormal autoimmunity is one of the major cause of premature ovarian failure(POF). Multiple regulations of Bushenhuoxue medicine in the neural - immune - endocrine network have be founded in the long-term clinical and scientific research. The pre-experiment found Bushenhuoxue tang containing serum induced mouse Treg generation，put forward the speculation: Bushenhuoxue method may interfere with autoimmune premature ovarian failure through the induction of Treg differentiation. In order to further reveal the occurrence and development of molecular immunological mechanisms to clarify the role and mechanism of Bushenhuoxue for the treatment of this disease,our topic assumes that Bushenhuoxue exert immunomodulatory effects through the induction of regulatory T,to interfere with the occurrence and development of this disease. We design the following research：1. Detecting Bushenhuoxue prescription for drug-containing serum in vitro induction of Treg conversion and its biological properties by induced Treg phenotype and function detection; 2. Mouse model of infusion of Bushenhuoxue prescription for induction of regulatory T cells, observation of ovarian function and functional changes, the assessment of Bushenhuoxue prescription treatment of autoimmune premature ovarian failure mice; 3. Proven Bushenhuoxue prescription for induction of regulatory T cells play a regulatory role of sites and mechanisms by fluorescent labeling Treg and peripheral blood cytokine detection. New scientific theoretical basis for Chinese medicine to better treatment of premature ovarian failure has important clinical implications and the value of drug development.

近年研究表明自身免疫异常是卵巢早衰（POF）主要病因之一。本研究团队既往多项研究发现补肾活血中药可在多个环节调控神经-免疫-内分泌网络功能。预实验发现补肾活血中药可诱导小鼠调节性T细胞（Treg）生成，推测Treg可能在本病的转归中发挥重要作用。为了深入探讨补肾活血中药治疗POF的免疫学机制，拟从Treg分化角度进行以下研究内容：1.通过对诱生的Treg细胞表型及功能检测，分析含药血清体外诱导Treg的生物学性能；2.建立小鼠免疫性POF模型,通过输注经补肾活血中药扩增的Treg，观察其对卵巢功能和形态的影响，阐明其治疗作用；3.采用荧光标记Treg，观察其在体内定位，并通过外周血细胞因子检测，探讨补肾活血中药诱导的Treg发挥调节作用的场所及机制。从而为中医药治疗卵巢早衰提供新的科学依据。

本研究从Treg角度探讨研究补肾活血中药治疗卵巢早衰的免疫学机制。本项目总体按计划进行，研究结果如下：1、在补肾活血中药及含药血清质谱研究中发现血清中药物的有效成分浓度具有时间量效关系，0.5h取血组血清中药物浓度最高，且血清中检出知母皂苷、盐酸小檗碱药物原型成分。2、成功建立了免疫性卵巢早衰小鼠模型。3、从小鼠脾脏提取Treg细胞纯度达到94%，补肾活血中药血清培养增殖发现中剂量组中药血清组增殖较为明显。中药血清组诱生的Treg细胞中Foxp3mRNA的表达上调，CD4+CD25+Treg细胞培养上清液中IL-10水平升高，MLR法检测增殖后CD4+CD25+Treg对CD4+ CD25-T细胞增殖的抑制率升高，提示补肾活血中药能够增强Treg细胞抑制功能。4、采用小鼠活体成像观察回输入小鼠体内荧光标记Treg细胞，发现模型组小鼠子宫卵巢部位部分可见荧光表达。Treg回输组卵巢HE染色可见卵泡数目较模型组稍增多，可见较成熟卵泡，形态尚规则。子宫HE染色较模型组变厚，间质增厚，褶皱稍增多，腺腔增大。5、通过对小鼠体内Treg、Foxp3、Th17、ROR-γt、ER、PR、CD4+淋巴细胞及相关细胞因子IL-10和IFN-γ的检测，发现补肾活血中药可使卵巢免疫荧光抗透明带抗体减弱或消失，促进了雌激素受体和孕激素受体的表达，能够降低免疫性卵巢早衰小鼠模型中Th17细胞水平及ROR-γt表达，有效抑制了CD4+T淋巴细胞的活化，增加了Treg的比例，促进了Foxp3 mRNA的表达，使IL-10 and IFN-γ水平显著下降，提高了卵巢功能。考虑补肾活血中药可能通过调节Treg细胞，对卵巢早衰起到了有效的免疫保护作用。.在本项目的资助下，课题组完成设计内容，已发表国内期刊论文10篇，定刊待发表国内论文2篇，SCI已录取待发表论文2篇，课题组成员参加国内会议3次，课题完成期间培养硕士研究生3名。