人羊膜间充质干细胞分离培养:胰蛋白酶及胶原酶消化时间及浓度的选择  

Isolation and culture of human amniotic mesenchymal stem cells: proper digestion time and concentrations of trypsin and collagenase

作　　者：张惠娟[1] 丛姗[1] 梁美萍[1] 刘俊平[1] 黄利刚[1] 宋瑾[1] 曹贵方[1] 

机构地区：[1]内蒙古农业大学动物组织胚胎与发育生物学实验室,内蒙古自治区呼和浩特市010018

出　　处：《中国组织工程研究》2014年第6期944-949,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家高技术研究发展863项目(2008AA101005);课题名称:家畜胚胎干细胞技术研究~~

摘　　要：背景:文献报道人羊膜间充质干细胞提取方法各不一致,得到的细胞数量各不相同。目的:探索人羊膜间充质干细胞在体外分离培养的最适方法。方法:无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,分别通过4个实验7种方法体外培养人羊膜间充质干细胞。①实验一:分别采用3种方法:0.05 g/L胰蛋白酶消化10 min后,再加0.75 g/L胶原酶Ⅰ消化60 min;0.75 g/L胶原酶Ⅰ直接消化120 min;0.05 g/L胰蛋白酶与0.75 g/L胶原酶Ⅰ同时消化60 min。②实验二:采用0.05 g/L的胰蛋白酶消化30 min后,再加0.75 g/L胶原酶Ⅰ消化30 min。③实验三:分别采用2种方法:0.05 g/L的胰蛋白酶连续2次消化30 min后,再加入0.75 g/L的Ⅰ型胶原酶消化60 min;0.05 g/L的胰蛋白酶连续2次消化40 min后,再加0.75 g/L胶原酶Ⅰ消化60 min。④实验四:采用0.05 g/L的胰蛋白酶连续2次消化30 min后,再加1 g/L的Ⅰ型胶原酶消化60 min。显微镜下观察其形态,研究人羊膜间充质干细胞在体外分离培养的最适方法。结果与结论:用0.05 g/L的胰蛋白酶连续消化2次每次消化30 min,然后用1 g/L的胶原酶消化60 min是最合适的体外分离培养条件。细胞成细长梭形或星形,胞质丰富,细胞核呈圆形,1-3个核仁。说明实验四采用的胰酶和胶原酶消化的时间合适,而且胶原酶的浓度合适,得到的细胞数量最多。BACKGROUND：Extraction methods of human amniotic mesenchymal stem cells are inconsistent in the number of cells. OBJECTIVE：To explore the optimal method to in vitro isolate and culture human amniotic mesenchymal stem cells.METHODS：Under sterile conditions, ful-term cesarean fetal amniotic membrane was cut into pieces, then to isolate human amniotic mesenchymal stem cells by seven methods in four experiments. In experiment 1, human amniotic mesenchymal stem cells were isolated by the fol owing three methods：（1） 0.05 g/L trypsin digestion for 10 minutes fol owed by 0.75 g/L col agenase digestion for 60 minutes;（2） 0.75 g/L col agenase I for 120 minutes;（3） co-digestion with 0.05 g/L trypsin and 0.75 g/L col agenase for 60 minutes. In experiment 2, the samples were digested with 0.05 g/L trypsin digestion for 30 minutes fol owed by 0.75 g/L col agenase digestion for 30 minutes. In experiment 3, the samples were digested by two methods：（1） 0.05 g/L trypsin digestion for 30 minutes×2, fol owed by 0.75 g/L col agenase digestion for 60 minutes;（2） 0.05 g/L trypsin digestion for 40 minutes×2, fol owed by 0.75 g/L col agenase digestion for 60 minutes. In experiment 4, the samples were digested with 0.05 g/L trypsin digestion for 30 minute×2, fol owed by 1 g/L col agenase digestion for 60 minutes. Fol owing morphology observation under a microscope, we studied the most suitable method for isolating human amniotic mesenchymal stem cells. RESULTS AND CONCLUSION：Digestion with 0.05 g/L trypsin for 30 minutes twice fol owed by 1 g/L of col agenase digestion of 60 minutes was the most suitable isolation and culture condition in vitro. cells became elongated fusiform or star-shaped with rich cytoplasm, and nuclei were round with 1-3 nuts. We can harvest the most number of human amniotic mesenchymal stem cells using the method described in experiment 4.

关 键 词：干细胞 培养 间充质干细胞 羊膜间充质干细胞 细胞培养 胰蛋白酶 胶原酶 863项目 

分 类 号：R394.2[医药卫生—医学遗传学]