卵巢癌微环境中树突状细胞前体异常分化的体内研究及其与Notch1信号传导的相关性

Immune deficiency is confirmed to exit in ovarian cancer microenvironment, whereas the mechanisms of DC differentiation which has close relationship with it is no clearly. Based on our previous researches that epithelial ovarian cancer cells can induce abnormal differentiation of precursors of myeloid dendritic cells with function deficiency and the relationship with Notch signal transduction way, the project is established to study the differentiation pathway and outcome of precursors of mDC in mice burdened with human ovarian cancer cell. The study also focuses on the function of Notch signal pathway. We plan to establish a transplantation model of mDC precursors in NOD/SCID mice burdened with human ovarian cancer cells, which will offer a good platform for in vivo experiments. The next step is to silence the receptor of Notch1 by transfection of shRNA by lentivirus vector. Another way is blocking the Notch1 signal way by y-secretion inhibitor. We will observe the differentiation outcomes of precursors in vitro and in vivo,the expression of surface markers, the subtypes and the maturation. The results will disclose the new mechanisms that how ovarian cancer cell can escape from human immunity system, and disclose a new target for tumor immunity therapy.

上皮性卵巢癌存在着免疫微环境的缺陷，与之密切相关的DC分化异常的机制并不明确。基于我们既往的研究基础上皮性卵巢癌细胞培养上清能诱导mDC前体细胞分化异常和功能异常，且Notch信号与DC分化相关，本课题拟从体内和体外实验研究人mDC前体细胞在上皮性卵巢癌荷瘤鼠体内的分化途径和结局以及Notch1信号途径在其中起到的关键作用。首先建立人mDC前体荷瘤鼠体内移植模型，更为真实地模拟其人体内分化过程，为DC的体内研究提供良好的平台。在基因水平采用慢病毒载体转染shRNA干扰技术沉默Notch1受体的表达，采用r-分泌酶抑制剂阻断Notch1信号通路，观察处理后的mDC前体在体内和体外分化的结局，细胞表面分子，分群以及成熟度，从而为卵巢癌的免疫逃逸机制增添新的认识，为肿瘤的免疫治疗提供新的靶点。

本课题从体外和体内实验两方面阐明mDC 前体在EOC 免疫微环境中的分化异常与Notch1 信号途径具有密切相关性，Notch1 信号在调控其分化中发挥重要作用。本课题从足月分娩产妇的脐血中提取CD34+造血干细胞，并分离扩增和诱导成熟Lin-CD45RA-DC前体细胞；采用RT-PCR检测Lin-CD45RA-DC前体细胞中Notch1受体及配体的表达；采用慢病毒载体转染shRNA干扰技术及r-内分泌酶抑制剂下调 Notch1 mRNA的表达；观察Lin-CD45RA-DC前体细胞在体外卵巢癌微环境中的分化结局。构建NOD/SCID鼠卵巢癌皮下移植瘤模型，将Lin-CD45RA-DC前体细胞及慢病毒转染后的DC前体细胞分别注入小鼠尾静脉，观察DC前体细胞的体内分化结局。研究结果发现在Lin-CD45RA-DC前体细胞中检测到Notch1受体及配体的表达。ShRNA- Notch1慢病毒转染及r-分泌酶抑制剂均可以成功下调Lin-CD45RA-DC细胞中 Notch1 mRNA的表达。下调Notch1 mRNA的表达后，Lin-CD45RA-DC前体细胞向HLA-DR+CD11C-CD123+浆细胞样树突状细胞(pDCs)的分化减少，向HLA-DR+CD11C+CD123-髓样树突状细胞（mDCs）的分化增多或不变。在SKOV3培养上清环境下，下调Notch1的表达后，向pDC的分化仍减少，向mDC的分化增多或不变。 在荷瘤鼠体内同样发现Lin-CD45RA-DC前体细胞分化为mDC减少，pDC增多。但Notch1mRNA表达抑制后的前体细胞在荷瘤鼠体内的分化未收到影响。研究结论为Lin-CD45RA-DC前体细胞分化中， pDC的分化有赖于Notch1信号通路的激活。在肿瘤微环境中，阻断Notch1后， pDC减少，提示Notch1信号通路对于pDC的分化不受肿瘤微环境的影响。本研究结果提示抑制Notch1途径有可能成为重要的作用靶点，这对于卵巢癌的精准治疗，免疫治疗，抗肿瘤的瘤苗制备，攻克复发性难治性卵巢癌等具有重要意义。