人胎儿肝干细胞的分离及鉴定  

Isolation and Identification of Human Fetal Hepatic Stem Cell

作　　者：张金卷[1,2] 杜智[1,2] 李涛[1,2] 朱争艳[1,2] 王毅军[1,2] 聂福华 宋继昌[1,2] 

机构地区：[1]天津市第三中心医院肝胆外科,300170 [2]卫生部人工细胞工程技术研究中心

出　　处：《天津医药》2005年第5期268-270,共3页Tianjin Medical Journal

基　　金：国家863课题基金资助项目(项目编号:2001AA216051)

摘　　要：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 study the isolation, purification and identification of human fetal hepatic stem cells (HSCs). Methods: Human fetal HSCs were obtained from liver in abortion fetus in the second trimester of pregnancy. Liver cells were isolated by collagenase (type IV) digestion method, and purified by Percoll gradient centrifugation. The makers of HSCs were identified by immunohistochemical staining. Results: The purified human fetal HSC had a yield of (2.10 ± 0.50)×107, and the viability was(92.30 ± 1.23)%. The diameter of cells was less than 1/3 of the normal hepatocytes under phase contrast microscope. The electron microscopic examination showed that there were protruding microvilli on the surface, oval shaped nuclei and high nuclear/cytoplasm ratio. There were cytokeratin (CK)8,CK19, alpha-fetoprotein (AFP) and alpha-1 -antitrypsin(AAT) positive in stem cell. The Albumin and leucocyte common antigen (LCA) were not stained in HSCs. The isolated cell could clonally proliferate and form colonies in primary culture within 3 weeks. Conclusion: Liver stem cells could be easily isolated from human fetus by collagenase perfusion technique, and purified successfully by Percoll gradient centrifugation.

关 键 词：胎儿肝干细胞 Percoll密度梯度离心 免疫组化染色 白细胞共同抗原 密度梯度离心法 CO2培养箱 原位灌注 抗胰蛋白酶 鉴定方法 男性胎儿 水囊引产 分离纯化 细胞特点 镜下观察 SABC 细胞产量 细胞活性 单个细胞 细胞表面 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R321-33[医药卫生—基础医学]