体外特异微环境诱导人胎盘间充质干细胞分化成子宫平滑肌细胞的研究

Simulated uterus microenvironment induced human placental mesenchymal stem cells differentiation to uterus smooth muscle cells in vitro

目的体外建立人胎盘间充质干细胞（pMSC）的分离扩增方法，研究其在模拟的特异性微环境中向子宫平滑肌细胞（uSMC）的分化。方法取足月健康新生儿胎盘组织，纯化并扩增pMSC，检测表面标志和并鉴定多向分化潜能。同时原代培养uSMC，改良Trans well培养体系，建立模拟特异性微环境，共培养诱导pMSC向uSMC定向分化。并通过标志性蛋白及细胞功能进行鉴定。结果pMSC同骨髓间充质干细胞一样，具有活跃增殖的能力，表达于细胞标志，具有间充质细胞的特征及多向分化能力。pMSC与uSMC共培养后，其形态逐渐向uSMC形态过渡，并表达uSMC最具特异性的分化晚期标志蛋白肌球蛋白重链（MHC）。诱导获得的细胞表达雌激素受体，并对雌激素的刺激具有反应性的功能变化。结论利用胎盘组织可获取大量pMSC，为组织工程的种子细胞和基因工程的载体细胞提供新的、可行的来源。pMSC的分化具有环境依赖性，在体外模拟的特异性微环境中pMSC可分化为有功能的子宫平滑肌细胞。

Objective To develop a new method to promote the differentiation of mesenchymal stem cells derived from human placenta （pMSC） to uterus smooth muscle cells （uSMC） in simulated uterus microenvironment. Methods MSCs were isolated from human placenta, cultivated, and analyzed for their phenotype by flow cytometry. The muhipotential differentiation of the pMSC was examined by chondrogenic, adipogenic, and osteogenetic induction, uSMC were isolated from uteri resected during operation and cocultivated with the pMSC in a Transwell chamber simulating Two, 4, and 8 days later RT-PCR and Western blotting were used to detect the mRNA and protein expression of α-actin, calmodulin, and myosin heavy chains （ MHC）, the markers of smooth muscle differentiation at the early, middle, and late stages. On day 8 RT-PCR was used to detect the expression of estrogen receptor in these 2 groups of cells, then estrogen was used to stimulate these cells and the protein kinase C （PKC） activity was examined. Results The pMSC could be induced into adipocytes, osteocytes, and chondrocytes respectively. After co-culture with uSMC, the morphology of the pMSC changed closely into that of the uSMC, and MHC was expressed in the pMSC. Estrogen receptor was positive in both groups of cells. The PKC activity increased, especially in the cell membrane, after stimulation of estrogen. Conclusion The postpartum human placenta can be used as an important and novel source of muhipotent stem cells for tissue engineering and genetic engineering. Placental MSC have the potential to differentiate into smooth muscle cells under the simulated uterus microenvironment in vitro.