【摘要】 目的:观察脐带中含有的间充质干细胞体外分离、纯化及培养条件,并对其进行生物学特性、分化能力和表面抗原的鉴定。方法:实验于2004-10/2005-07在江苏省人民医院中心实验室完成。①无菌条件下取正常足月剖腹产的脐带近胎儿段,剔除动静脉,取出其中的间质组织,胶原酶消化法原代培养细胞进行培养和纯化获得贴壁细胞。②用流式细胞仪检测间充质干细胞表面抗原的表达情况。③对间充质干细胞多向分化的能力进行初步鉴定。结果:①来源于脐带的单个核细胞经体外培养贴壁后出现间充质样的细胞,间充质细胞为成纤维样的细胞形态。②第1,3,5,7代细胞生长曲线基本相同。其生长共同特性:传代培养潜伏期约为24～36h;传代培养对数增殖期约为4～6d;对数增殖期结束后至接种后八九天,生长进入平台期。③表达间充质干细胞相关的抗原CD105,CD44,但不表达造血细胞抗原CD34,CD45,这与源于骨髓的间充质干细胞一致。④加入诱导剂5h后表现出典型的神经元样细胞形态,伴少量细胞开始死亡。24h后死亡细胞明显增多;诱导后的神经元样细胞NSE表达明显增强。结论:人脐带来源的间充质干细胞能在体外培养、扩增,并且具有和骨髓源间充质干细胞类似的生物形态和抗原表型。更多还原

【Abstract】 AIM: To observe the conditions of in vitro isolation, purification and culture of mesenchymal stem cells of human umbilical cord, and to identify its biological characters, differentiative ability and surface antigen.METHODS: This experiment was conducted at the Central Laboratory of People’s Hospital of Jiangsu Province from October 2004 to July 2005. ①Under aseptic condition, the umbilical cord was collected from full term deliveries scheduled for cesarean section. Artery and vein were removed and mesenchymal tissue was taken out. The primary cells were performed culture and purification with collagenase digestion method to obtain the adherent cells. ② Flow cytometry was used to detect the expression of surface antigen of mesenchymal stem cells. ③ To primarily identify the multiple differentiation potential of mesenchymal stem cells.RESULTS: ①Mesenchyme-like cells appeared after the umbilical cord-derived mononuclear cells were cultured in vitro and attached to the wall. Mesenchymal stem cells presented fibroblast-like form. ②The growth curve of the 1st, 3rd, 5th and 7th generation cells were the same basically. The common characters of the growth: the latency of passage culture was about 24 to 36 hours; The logarithmic increasing phase of passage culture was about 4 to 6 days; From the end of logarithmic increasing phase to 8 or 9 days after inoculation, the growth entered the platform phase. ③ Express mesenchymal stem cell-related antigen CD105 and CD44, but not express hematopoietic cell antigen CD34 and CD45 , which was identical to human bone marrow-derived mesenchymal stem cells. ④ 5 hours after inducer was added, typical neuron-like cellular morphology appeared and a few cells began to die. 24 hours later, the dead cells increased obviously; neuron-like cell NSE expression was increased obviously after induction. CONCLUSION: Human umbilical cord-derived mesenchymal stem cells can be cultured and proliferated in vitro, and also have the similar biological morphology and antigen phenotype to bone marrow-mesenchymal stem cells.更多还原