人胎肝干细胞体外分离培养及脾内移植的初步研究  

Intrasplenic heterotransplantation of in vitro cultured human fetal hepatic stem cells for treatment of acute liver injury in mice with severe combined immunodeficiency

作　　者：周高速[1] 刘思德[1] 张振书[1] 张明军[1] 李旭[2] 王亚东[1] 何敬东[1] 

机构地区：[1]南方医科大学南方医院消化病研究所,广东广州510515 [2]南方医科大学南方医院急诊科,广东广州510515

出　　处：《南方医科大学学报》2007年第6期817-820,共4页Journal of Southern Medical University

基　　金：军队重大临床技术研究项目(02MA102)

摘　　要：目的将人胎肝干细胞脾内移植23肝切除的严重联合免疫缺陷(SCID)小鼠,观察其在受体小鼠内定植、迁移及分化的可行性。方法①以临床上自然流产废弃的孕期16～24周健康胎肝为供体,分离纯化胎肝干细胞,并行光镜、电镜观察及免疫细胞化学和流式细胞检测鉴定。②人胎肝干细胞经脾内移植到23肝切除的SCID小鼠。③免疫组化检测15、30、60、90d受体小鼠脾脏和肝脏的人Hepatocyte、α1-AT、AFP的定位和表达。PAS染色检测受体小鼠各时间点脾脏组织糖原表达。④RT-PCR检测30、60、90d受体小鼠脾脏组织AFP和白蛋白(Alb)基因的表达。结果①镜下观察分离细胞体积较小、约为肝细胞的16～13,核与浆比例大,内质网、线粒体和核糖体不发达。免疫细胞化学和流式细胞检测分别显示分离的人胎肝干细胞表达AFP、Thy-1、C-kit和CD34、CK19。②各时间点免疫组化均可检测到肝干细胞移植小鼠的肝脏和脾脏组织人Hepatocyte、α1-AT和AFP阳性表达。PAS染色显示各时间点受体小鼠脾脏组织不同程度的糖原表达。③人胎肝干细胞移植后30、60、90d受体小鼠脾脏组织表达人AFP和Alb基因。结论人胎肝干细胞经脾内移植可在异体内存活、定植并向受体受损肝脏迁移,在受体内增殖和定向分化为肝细胞。Objective To observe the in vivo colonization, migration, and differentiation of in vitro cultured human fetal hepatic stem cells （HSCs） following intrasplenic transplantation for treatment of acute liver injury in mice with severe combined immunodeficiency （SCID）. Methods Human fetal HSCs were isolated from the normal fetal liver （16-24 weeks） and purified, and the morphology of HSCs was observed under optical and transmission electron microscopes. The expressions of stem cell markers were examined in these HSCs by means of immunocytochemistry and flow cytometry. The passaged human fetal HSC suspension （0.2 ml） were injected into the spleen of SCID mice with acute liver injury induced by two-third partial hepatectomy, and 15, 30, 60, and 90 days after cell transplantation, immunohistochemistry was performed to examine the location and expressions of human hepatocytes, α-AT and AFP antigen in the spleen and liver of the recipient SCID mice. PAS staining was used to examine the expression of glycogen and RT-PCR employed for detection of the expressions of AFP and albumin mRNA in the spleen of the mice on the scheduled time points. Results Under optical microscope and transmission electron microscope, most of the HSCs were small, about 1/6 to 1/3 of the size of the hepatocyte, with relatively large nucleus-cytoplasm ratio and only small quantities of endocytoplasmic reticulum, chondriosome, and ribosome. Immunohistochemistry and flow cytometry identified positive expressions ofAFP, Thy-1, C-kit, CD34 and CK19 in the HSCs, and after cell transplantation, positive expressions of human hepatocyte, α1-AT, and AFP antigen occurred in the liver and spleen of the recipient SCID mice. PAS staining confirmed the presence ofglycogenosome in the spleen of the mice following cell transplantation. RT-PCR on days 30, 60, and 90 showed positive expressions of human AFP and albumin mRNA in the spleen of the mice. Conclusion Human fetal HSCs can survive and settle in the spleen and liver, and migrate to the damage

关 键 词：肝干细胞 分离 培养 移植 分化 

分 类 号：R657.6[医药卫生—外科学]