HO-1基因修饰的胎盘间充质干细胞修复胎盘血管发育不良的机制研究

Placental vascular dysfunction is one of the most important reasons of preeclampsia. Promoting neovascularization to reverse of abnormal placental vascular development is of great value to improve the pregnancy outcome. Placental mesenchymal stem cells （PMSCs） can be recruited to ischemia, inflammatory sites, and exert their vasculogenesis and angiogenesis function. However, the effect of PMSCs in rescuing of impaired placental vascular is still unclear. We hypothesized that preeclampsia placental pathological environment induces PMSCs homing, PMSCs repair placental vascular dysplasia through angiogenesis and uterine vascular remodeling. In our previous research, Heme Oxygenase-1（HO-1）gene which has cytoprotective, anti-apoptotic, and proangiogenic properties was successfully transfected into PMSCs. Under ischemia condition, the HO-PMSCs apoptosis was decreased, angiogenesis ability was enhanced significantly. A series of in vitro and animal experiments will be done to explore the effect of HO-1 gene modified PMSCs in repairing placental vascular abnormal development. The superparamagnetic iron oxide will be used to label PMSCs, the stem cells homing and effect can be traced by MRI in vivo. It may throw a new light on the clinical treatment of the pregnancy complicated disease associated with placental vascular abnormalities.

胎盘血管发育不良是子痫前期的重要原因，修复其发育异常对增加胎盘灌流，改善妊娠结局具有重要价值。胎盘间充质干细胞（PMSCs）可归巢至缺血炎症部位，发挥促血管形成等修复作用。我们设想，PMSCs能够通过修复胎盘血管发育不良有效治疗子痫前期。子痫前期胎盘环境可诱发PMSCs归巢，PMSCs通过改善胎盘血管新生和螺旋动脉血管重塑完成修复胎盘血管发育不良过程。我们已成功分离鉴定PMSCs，并使用血红素加氧合酶-1（HO-1）进行基因修饰，促进干细胞活力及血管形成能力。我们将进行细胞及动物水平研究，深入探讨HO-1-PMSCs修复胎盘血管发育异常的具体机制。利用SPIO标记技术，实现干细胞的示踪，客观评价治疗后胎盘灌流。本项目的完成有望为相关病理妊娠治疗提供新思路。

背景：胎盘血管发育异常引起的胎盘低灌注可诱发子痫前期（PE）。基于间充质干细胞（MSCs）的疗法是一种很有前景的组织修复和促血管生成方法。血红素加氧酶-1(HO-1)是一种应激蛋白，对怀孕期间的血管生成具有有益作用。本课题组探讨了PMSCs/HO-1-PMSCs对胎盘血管生成的影响以及在硝基左旋精氨酸甲酯（L-NAME）诱导的PE大鼠模型中的治疗作用。方法：利用流式细胞术评估PMSCs表面标志物的表达，成脂和成骨分化实验验证PMSCs的分化能力。将携带HO-1基因的慢病毒载体转染PMSCs，分别通过CCK8、Transwell和流式细胞术检测各组细胞增殖、迁移和凋亡能力；通过qRT-PCR和ELISA实验检测促血管生成相关因子VEGF和PlGF的表达，并测定sFlt-1分子的表达量；利用各组细胞培养上清处理HUVEC和绒毛-蜕膜共培养体系，研究PMSCs/HO-1-PMSCs对血管形成能力和绒毛血管重塑的影响。成功构建子痫前期孕鼠模型，体内移植PMSCs/HO-1-PMSCs，检测孕鼠血压、尿蛋白；通过多普勒超声了解胎盘血流灌注、ELISA实验检测孕鼠血清中VEGF、PlGF、sFlt-1、Seng的水平；称量胎盘和胎鼠重量；分别通过HE染色、肾脏病理组织学检测胎盘组织微血管密度和肾脏微血管密度；在荧光显微镜下观察GFP标记的PMSCs在胎盘中的分布。结果：HO-1显着改善PMSCs的增殖和迁移并减少了PMSCs的凋亡。HO-1-PMSCs中VEGF水平升高、sFlt-1水平降低。管形成试验和绒毛-蜕膜共培养系统证实HO-1-PMSCs有利于胎盘血管生成和血管重塑。经PMSCs和HO-1-PMSCs处理的PE大鼠的血压和蛋白尿明显降低、肾损害明显改善、胎儿和胎盘质量增加；孕鼠血清中VEGF和PlGF表达增加、sFlt-1和sEng表达显著降低，多普勒超声结果显示胎盘血流灌注得到显著改善；更重要的是，HO-1-PMSCs治疗组的效果略优于PMSCs。绿色荧光示踪实验证实细胞已成功移植到胎盘并分布于血管内，提示PMSCs及HO-1-PMSCs可能参与了血管生成过程。随后课题组进一步研究发现低氧预处理可增强PMSCs的存活和迁移能力，并进一步探索了其机制；PMSCs来源外泌体可促进胎盘血管生成；血小板反应蛋白-4（TSP-4）在胎盘血管发育进程中起到重要作用。