论文部分内容阅读
目的:探索人胎儿肝干细胞的分离及鉴定方法。方法:改进Seglen胶原酶(IV型)原位灌注结合Percoll密度梯度离心分离纯化4～6个月水囊引产男性胎儿的肝干细胞,CO2培养箱培养,光镜、电镜下观察细胞特点,进行肝干细胞细胞标志SABC免疫组化染色。结果:平均细胞产量(2.10±0.50)×107,细胞活性率为(92.30±1.23)%。光镜下呈游离状态、大小不等的单个细胞,约为正常肝细胞1/5～1/3大小;电镜下细胞表面可见少量短而小的微绒毛状突起,卵圆形细胞核,核/浆比例较大等特点;甲胎蛋白、细胞角蛋白8、19及α-1-抗胰蛋白酶免疫组化染色呈阳性,白蛋白及白细胞共同抗原染色呈阴性表现,培养3周可形成克隆样结构。结论:改进的Seglen胶原酶原位灌注结合Percoll密度梯度离心法可成功分离、纯化人胎儿肝干细胞。
Objective: To explore the method of isolation and identification of human fetal liver stem cells. Methods: Hepatic stem cells were isolated and purified from male fetal fetuses induced by hydatid cyst in 4 ~ 6 months after the in situ perfusion of Seglen collagenase (IV type) and Percoll density gradient centrifugation. The cells were cultured in CO2 incubator and observed under light microscope and electron microscope. Stem cell markers SABC immunohistochemistry. Results: The average cell yield (2.10 ± 0.50) × 107, cell viability was (92.30 ± 1.23)%. Under the light microscope, a single cell with different size was about 1/5 ~ 1/3 of that of normal hepatocytes. A small amount of short and small microvilli were seen on the cell surface under electron microscope. The oval nucleus and nucleus / The proportion of plasma albumin was higher. AFP, cytokeratin 8, 19 and α-1-antitrypsin were immunohistochemically stained. The albumin and leukocyte common antigen staining was negative. After cultured for 3 weeks, structure. Conclusion: The improved Seglen collagenase in situ perfusion combined with Percoll density gradient centrifugation can be successfully isolated and purified human fetal liver stem cells.