羊水间充质干细胞作为转运载体介导干扰素-γ靶向治疗卵巢癌的研究

The traditional method of treatment of ovarian cancer has poor efficacy. For the treatment of ovarian cancer interferon-γ (IFN-γ) has exact effect, but Its efficacy is dose-dependent. Its toxicity increases with increasing dose. So enhancing the targeting of drug therapy is undoubtedly an effective way to improve its efficacy. Studies have shown that mesenchymal stem cell (AF-MSC) has immunogenicity and unique tumor tropism and so on characteristics. As a transporter-mediated drug targeted therapy has potential advantages. Studies have found that AF-MSC from amniotic fluid also has not tumorigenicity. And it can be obtained through amniocentesis which is a safe operation and has a negligible effect on the fetus and the mother..This project intends to separate AF-MSC from the amniotic fluid in the second trimester and then culture it. In vitro and in animal experiments, by using genetic engineering, cell transfection, flow cytometry, immunohistochemistry, molecular biology and cell biology, study the immunogenicity, tumorigenicity, and ovarian cancer tropism of AF-MSC from the second trimester, and explore its feasibility as a transporter-mediated drug targeting the treatment of ovarian cancer, and the effectiveness of AF-MSC as a transporter-mediated IFN-γ for the targeting treatment of ovarian cancer, and lay the theoretical and practical basis for the use of the AF-MSC-mediated IFN-γ targeted therapy of ovarian cancer.

传统的方法治疗卵巢癌疗效欠佳。干扰素-γ对卵巢癌的治疗效果确切，但其疗效呈剂量依赖性，随着剂量的增加，毒副反应也增加，提高药物治疗的靶向性无疑是改进其疗效的一个有效途径。研究显示间充质干细胞具有免疫原性低和独特的肿瘤亲嗜性等特点，作为转运载体介导药物的靶向治疗具有潜在的优势。有研究发现羊水来源的间充质干细胞（AF-MSC）还具有不成瘤性，而且可以通过羊膜腔穿刺获得，操作安全，对胎儿、母体的影响轻微。.本课题拟从妊娠中期的羊水中分离、培养AF-MSC，利用基因工程、细胞转染、流式细胞术、免疫组化等分子生物学和细胞生物学方法，通过体外及动物实验，研究妊娠中期AF-MSC的免疫原性、成瘤性和卵巢癌亲嗜性，探讨其用作转运载体介导药物靶向治疗卵巢癌的的可行性；并且对AF-MSC作为干扰素-γ的转运载体靶向治疗卵巢癌的有效性进行检测，为利用AF-MSC介导干扰素-γ靶向治疗卵巢癌奠定理论与实践基础。

研究羊水间充质干细胞（AF-MSCs）的生物学特性及其作为转运载体介导干扰素-γ药物靶向治疗卵巢癌的可能性分析。.贴壁分离法分离提取妊娠中期羊水间充质干细胞。流式细胞术、RT-PCR、免疫组织化学等技术用于鉴定羊水间充质干细胞的生物学特性。茜素红钙染色、油红O染色用于评价AF-MSCs的体外分化潜能。分子克隆、慢病毒质粒的构建、ELISA等方法用于构建IFN-γ-AF-MSCs细胞系，并评估IFN-γ的表达情况。鼠肿瘤模型、AF-MSCs鼠尾注射、免疫组织化学技术研究AF-MSCs的体内肿瘤趋向性。Tranwell共培养技术评估IFN-γ-AF-MSCs细胞系对卵巢癌细胞系skov3的作用。.成功分离出具有高度增殖力的妊娠中期羊水间充质干细胞。流式细胞术分析显示高表达CD166、CD90、CD73、CD117、HLA-ABC（MHC-I），弱表达CD105 ，不表达CD45、CD14、CD34、HLA-DR（MHCII）。成功构建表达IFN-γ的慢病毒载体，并构建稳定表达IFN-γ的IFN-γ-AF-MSCs细胞系。建立鼠卵巢癌组织模型，证明AF-MSCs具有体内的宫颈癌组织亲嗜性。AF-MSCs鼠皮下注射证明其不成瘤性。IFN-γ-AF-MSCs有促进卵巢癌细胞系SKOV3凋亡的作用。结论：AF-MSCs是人羊水中存在的间充质干细胞，并且具有能够作为转运载体运载IFN-γ靶向治疗卵巢癌的可能性。