中文摘要：

目的：三维可降解生物材料是组织工程的重要载体,观察胚胎干细胞在聚合三维支架上的生长、增殖和分化状态,为胚胎干细胞分化形成任何组织修复替代治疗机体组织器官的损伤和功能衰竭提供实验学依据。方法：实验于2007-05/09在解放军第三军医大学西南医院感染病研究所人工肝实验室完成。①材料：鼠胚胎干细胞CCE株为加拿大Stem Cell Technologies公司产品,由聚乳酸和聚乙醇酸聚合而成的非编织型三维支架为美国Synthecon公司产品,细胞外基质Matrigel TM由BD Biosciences公司提供。②实验方法：将鼠胚胎干细胞株接种于被覆明胶的培养瓶内,加入含0.1mmol/L非必需氨基酸、2mmol/LL-谷酰胺、0.1mmol/L二羟基丙硫醇、15%胎牛血清、1000U/mL白血病抑制因子的IMDM培养液。3d后采用＂悬滴法＂使细胞在不含白血病抑制因子的上述IMDM培养液中形成胚胎体,6d后胰酶消化得到胚胎干细胞,浓度调整为（3-4）×109L-1,与Matrigel TM按1∶1混合并接种于剪成5mm×4mm×1mm小片的三维支架内,加入含1%二甲基亚酚、10-7mol/L地塞米松、5mg/LITS、15%胎牛血清的IMDM培养液进行诱导分化。③实验评估：激光共聚焦显微镜观察胚胎干细胞在三维支架上的生长与增殖情况;应用荧光染色检测细胞活性;苏木精-伊红染色光镜下观察胚胎干细胞的组织分化情况。结果：①细胞生长与增殖：接种培养1d多数胚胎干细胞粘连在一起形成聚集体,状似球形,附着于三维支架的纤维上。7d时聚集的胚胎干细胞开始沿纤维支架生长,互相粘连,呈片状和网状。细胞数随培养时间延长而不断扩大,至14d细胞交织在一起呈现多层面立体景象,布满整个三维支架。②活细胞观察：接种培养1,7,14d的胚胎干细胞绝大多数发出绿色荧光,始终保持良好的细胞活性。③胚胎干细胞的组织分化：在非特异诱导培养液作用下,胚胎干细胞可向?

英文摘要：

AIM： Three-dimensional （3D） biodegradable polymer scaffold is the important carrier in tissue engineering. We observed the growth, proliferation, and differentiation of embryonic stem cells （ES cells） on this 3D scaffold. It can provide experimental evidences for ES cells differentiation and for repairing tissue and organ injure and failure. METHODS： Experiments were performed at the Artificial Liver Laboratory, Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University of Chinese PLA from May to September 2007. （1）Mouse ES cells （CCE strain） were purchased from Stem Cell Technologies Inc. （Canada）. The non-woven 3D scaffold was the copolymer of poly-L-lactic acid （PLLA） and poly-glycolic acid （PGA） （Synthecon, USA）. Extracellular matrix, MatrigelTM was provided by BD Biosciences. （2）ES cells were maintained on gelatin-coated dishes in Iscove＇ s modified Dulbecco medium （IMDM） cofltaining 0.1 mmol/L of nonessential amino acids, 2 mmol/L of L-glutamine, 0.1 mmol/L of monothioglycerol, 15% fetal bovine serum （FBS）, and 1000 U/mL leukemia inhibitory factor （LIF）. Embryonic bodies （EBs） were firstly formed by ES cells with hang drip method in IMDM without LIF. Six-day-old EBs were trypsinized and the cells were resuspended at a concentration of （3-4） × 10^9 L^-1 cells per 1 mL of a 1：1 mixture of culture medium and MatrigelTM, and then seeded onto scaffold （5 mm×4 mm× 1 mm） and cultured in differentiation medium supplemented with 1% DMSO, 10^-7 mol/L of dexamethasone, 5 mg/L ITS （insulin, transferrin, selenium）, and 15% FBS for cell differentiation. （3）The growth, proliferation, activity, and differentiation of ES cells were respectively examined by laser confocal microscope, calcein-AM staining, and hematoxylin/eosin （HE） staining. RESULTS： （4）Cell growth and proliferation： ES cells were attached to the copolymer scaffolds as globular-aggregates at 1 day after cell seeding. Cells grew along fabric