【摘要】 背景:多系分化应激耐受(multilineage-differentiating stress-enduring,Muse)细胞是一种具有自我更新和多向分化潜能、可向3个胚层分化的细胞,并且具有非致瘤性和低端粒酶活性,是组织工程、细胞移植及基因治疗领域的理想种子细胞,为临床上多种疾病的治疗提供了广阔的前景。目的:探讨脐带间充质干细胞与脂肪间充质干细胞分选获得的Muse细胞的生物学特性,为临床研究获得足量的Muse细胞提供实验数据。方法:胰酶长时间消化处理脐带间充质干细胞、脂肪间充质干细胞,锥虫蓝染色及流式细胞仪检测比较不同消化时间下两种细胞的存活率与SSEA-3表达阳性率;通过悬浮贴壁法获取Muse细胞,碱性磷酸酯酶染色检测获取Muse细胞的多能干性,Muse细胞在不同诱导分化液作用下进行多系分化,油红O、茜素红、Von Kossa染色检测比较成脂、成骨诱导分化能力,RT-PCR检测多能干细胞标志基因Oct-3/4,Nanog,SSEA-3的表达及Muse细胞诱导分化后双皮质素、神经元特异性烯醇化酶、脂蛋白脂肪酶、骨桥蛋白、甲胎蛋白的表达变化。结果与结论:(1)随着胰酶消化时间的延长,脐带间充质干细胞、脂肪间充质干细胞存活率下降,SSEA-3阳性率上升;(2)悬浮贴壁获得的Muse细胞碱性磷酸酯酶染色呈阳性反应,间充质干细胞碱性磷酸酯酶染色阴性;(3)Muse细胞SSEA-3、Nanog、Oct-3/4基因表达水平明显高于间充质干细胞,与脐带来源Muse细胞相比,脂肪来源Muse细胞Nanog、Oct-3/4基因表达增加;油红O、茜素红、Von Kossa染色结果显示Muse细胞成脂、成骨能力高于间充质干细胞;(4)间充质干细胞及Muse细胞在成脂、成骨诱导培养液、神经元细胞诱导液、肝细胞诱导液作用下分化效率不一样,但均高表达脂蛋白脂肪酶、骨桥蛋白、神经元特异性烯醇化酶、双皮质素及甲胎蛋白,Muse细胞相关基因表达高于间充质干细胞;(5)实验结果证明,脐带间充质干细胞、脂肪间充质干细胞在胰酶消化4 h时,SSEA-3阳性率与存活率达到最佳,脂肪间充质干细胞SSEA-3阳性率高于脐带间充质干细胞。脐带间充质干细胞、脂肪间充质干细胞分选获得的Muse细胞高表达多能干细胞基因,具有三胚层分化潜能。更多还原

【Abstract】 BACKGROUND:M ultilineage-differentiating stress-enduring(M use) cells possess self-renewal and multiple diffe rentiation potential.M use cells are able to differentiate into all three germ layers,and exhibit non-tumorigenicity and low telomerase activity.It is a seed cell in the fields of tissue engineering;cell transplantation and gene therapy,providing a broad prospect for the treatment of various clinical diseases.OBJECTIVE:To study the biological characteristics of Muse cells from umbilical cord derived and adipose derived mesenchymal stem cells in vitro,and provide experimental data for obtaining sufficient M use cells for clinical research.METHODS:Umbilical cord derived and adipose derived mesenchymal stem cells were incubated with trypsin for different time.The cell survival rates and SSEA-3 expression were detected by Trypan Blue staining and Flow cytomertry.Muse cells were obtained by suspension adherence method.Alkaline phosphatase staining was used to obtain the pluripotency of M use cells.Muse cells underwent multi-lineage differentiation under different induction conditions.Oil red O staining,Alizarin Red S staining,and Von Kossa staining were used to compare the adipogenic and osteogenic diffe rentiation.RT-PCR was used to detect the changes in pluripotent stem cell marker genes Oct-3/4,Nanog,SSEA-3 and Muse cells induced differentiation related genes doubleco rtin,neuro n-specific enolase,lipoprotein lipase,osteopontin,and alpha-fetoprotein expression.RESULTS AND CONCLUSION:(1) Under trypsin incubation extension,survival rates of umbilical cord derived mesenchymal stem cells and adipose derived mesenchymal stem cells were decreased,accompanied with SSEA-3 expression increased.(2) Alkaline phosphatase staining was positive in Muse cells and negative in mesenchymal stem cells.(3) SSEA-3,Nanog,and Oct-3/4 were highly expressed in Muse cells compared with mesenchymal stem cells.Nanog and Oct-3/4 were highly expressed in adipose-Muse cells compared with umbilical cord-Muse cells.Oil red O,Alizarin Red S,and Von Kossa staining results showed that Muse cells had stronger adipogenetic and osteogenetic abilities than mesenchymal stem cells.(4) Mesenchymal stem cells and Muse cells had different differentiation efficiencies under the action of adipogenesis,osteogenic induction culture medium,neuronal cell induction liquid,and hepatocyte induction liquid,but they all highly expressed lipoprotein lipase,osteopontin,neuron-specific enolase,doublecortin,and alpha-fetoprotein.The above genes were highly expressed in Muse cells compared with mesenchymal stem cells.(5) To conclude,umbilical co rd derived and adipose derived mesenchymal stem cells showed the best SSEA-3 positive rate and survival rate after trypsin digestion for 4 hours.The SSEA-3 positive rate of adipose derived mesenchymal stem cells was higher than that of umbilical cord derived mesenchymal stem cells.Muse cells obtained from umbilical cord derived mesenchymal stem cells and adipose derived mesenchymal stem cells highly express pluripotent stem cell genes and are able to differentiate into three germ layers.更多还原