宫腔注射PBMC调节Th17/Treg细胞免疫平衡改善胚胎着床的机制研究

Repeated implantation failure (RIF) has a serious impact on the success rate of IVF, which brings high cost and heavy psychological burden to infertile couples. Our previous studies have found that intrauterine injection of autologous peripheral blood mononuclear cells (PBMC) could increase pregnancy rate and livebirth rate in RIF patients, this method was more simple, safe, easy and no allergy compared to the method of subcutaneous husband or other's lymphocyte, but its mechanism remains to be further explored. Recent studies have shown that Th17/Treg cells immune balance was closely related to the maternal fetal immune tolerance during pregnancy, and our results indicated that: intrauterine injection of PBMC could increase the number of Treg cells and the mRNA expression of Foxp3 and PD-L1 in placental tissue. Therefore, we hypothesized that: intrauterine injection of PBMC regulates Th17/Treg cells immune balance in the maternal-fetal interface to promote embryo implantation. To test this hypothesis, we implore the effect of intrauterine injection of PBMC on embryo implantation and its mechanism of Th17/Treg cell immune balance in mice of embryo implantation dysfunctional. It provides new ideas for the immune-related diseases’ prevention and treatment such as RIF and recurrent spontaneous abortion.

反复着床失败（RIF）严重影响着试管婴儿的成功率，给不孕夫妇带来高昂的治疗费用及沉重的心理负担。我们前期研究发现宫腔注射自体外周血单个核细胞（PBMC）可以提高RIF患者的妊娠率和活产率，此方法与传统皮下注射丈夫或第三者淋巴细胞的方法相比具有操作简单、安全简便、无变态反应等优点，但其内在机理尚待阐明。近年来研究显示Th17/Treg细胞分化及平衡与妊娠期母胎免疫耐受密切关联，我们的预实验结果提示：PBMC宫腔注射可以增加胎盘组织中Treg细胞数目及Foxp3，PD-L1mRNA的表达。为此，我们提出假说: 宫腔注射PBMC通过调节母胎界面的Th17/Treg细胞免疫平衡，利于胚胎着床。为了验证这一假说，我们通过胚胎着床障碍模型初步探讨宫腔注射PBMC对胚胎着床的影响及其Th17/Treg免疫平衡机制研究。为反复着床失败及复发性流产等免疫相关疾病的防治提供新的思路。

反复着床失败（RIF）严重影响着试管婴儿的成功率，给不孕夫妇带来高昂的治疗费用及沉重的心理负担。胚胎作为母体的半同种异体移植物，其是否被母体免疫系统识别，将直接影响妊娠结局。阐明反复着床失败的发生和免疫的内在机理从而制定有效的免疫治疗策略，意义重大。我们前期临床研究发现宫腔注射HCG诱导的自体外周血单个核细胞（PBMC）可以提高反复着床失败患者的临床妊娠率（39.58% vs 14.29%）和活产率（33.33% vs 9.58%），但其内在机理尚待阐明。.本项目首次从Th17/Treg细胞免疫平衡这个新视点对PBMC宫腔注射改善胚胎着床的机理进行阐述，研究内容分为三个部分：第一，体外共培养PBMC与HCG发现HCG有利于PBMC细胞分泌TGF-β等细胞因子，并选择出最佳培养条件为24小时和最佳HCG浓度为10U/L；第二，构建胚胎着床障碍动物模型，研究发现宫腔注射PBMC对着床期和妊娠早期外周血及脾脏组织中Th17细胞和Treg细胞的表达无显著影响，而子宫内膜免疫微环境中Th17细胞及其分泌的细胞因子减少，Treg细胞及其分泌的细胞因子增多，说明可能作用于子宫内膜的Th17/Treg细胞免疫平衡；第三，阻断剂PD-L1 Fc阻断PD-1/PD-L1通路后发现着床率降低，且Treg及其细胞因子的表达减少，说明宫腔注射PBMC可能通过PD-1/PD-L1通路调节子宫内膜局部Th17细胞和Treg细胞免疫平衡来改善胚胎着床。.本研究明确PBMC 体外培养的最佳条件，并从Th17/Treg细胞免疫平衡来探讨宫腔注射PBMC免疫治疗对胚胎着床和宫腔内免疫微环境的影响，以期验证反复着床失败的有效治疗方法，使纠正Th17/Treg细胞平衡体系为核心的治疗手段成为可能，为丰富反复着床失败的临床治疗方法奠定理论基础。