大鼠胚胎来源肝干细胞分离纯化方法的比较

目的 观察比较机械分离法和酶消化法分离纯化孕鼠胚胎来源肝干细胞的优劣.方法 取孕13.5 d SD大鼠胚胎肝后分别用机械分离法和酶消化法分离纯化肝干细胞,比较两种方法所得细胞总量和存活率的差异,用免疫荧光染色法鉴定细胞,并比较原代培养前后肝干细胞存活率的变化.结果两种方法分离培养出的细胞形似卵圆形,经免疫荧光染色法检测表达干细胞标志物CD34和胆管细胞标志物CK19,机械分离法分离的肝干细胞数量(57.49±14.25)×106与酶消化法(63.36±12.67)× 106相当,两者比较差异无统计学意义(P>0.05),但机械分离法所得肝干细胞的存活率(96.37±0.82)%高于酶消化法(93.53±0.63)%(P<0.05),并且培养前后细胞存活率也明显高于酶消化法,两两比较差异有统计学意义(P<0.05).结论 机械分离法是一种简单、经济、实用的胚胎来源肝干细胞分离纯化方法.

Comparison of mechanical separation and enzymatic digestion methods in isolating fetal hepatic stem cells

Objective To compare the number and viability of fetal hepatic stem cells isolated by mechanical separation and enzymatic digestion methods.Methods Two kinds of cell isolation methods,namely,mechanical separation procedure and enzymatic digestion method,were used to obtain hepatic stem cells from fetal liver tissues.The total number and viability of cells were compared.The isolated cells were identified by immunofluorescence.Results There was no significance in the number of cells obtained by combination of enzymatic digestion (63.36 ± 12.67) × 106 and mechanical procedure (57.49 ± 14.25)× 106 ( P ＞ 0.05 ).But the survival rate by mechanical procedure ( 96.37 ± 0.82 ) % was significantly higher than that by combination of enzymatic digestion (93.53 ± 0.63 )%(P＜0.05).Conclusion The fetal hepatic stem cells are of high viability by mechanical procedure.Mechanical procedure is a simple,economic and effective method to isolate fetal hepatic stem cells.