胎儿骨髓源性胚胎后亚全能干细胞分离培养及向肝细胞的诱导分化(英文)  

Isolation, culture, induction and differentiation of fetal bone marrow-derived postembryonic pluripotent stem cells

作　　者：朱争艳[1] 张金卷[1] 李涛[1] 杜智[1] 

机构地区：[1]天津市第三中心医院、卫生部人工细胞工程技术研究中心、天津市人工细胞重点实验室,天津市300170

出　　处：《中国组织工程研究与临床康复》2009年第1期192-196,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：funded by the National “863”Fund Project “Liver and Blood Stem Cell Culture, Amplification and Transplantation” (2001AA216051)

摘　　要：背景:目前研究认为在胚胎发育后的多种组织中存在一类干细胞群体,可分化为不同胚层的组织细胞;但又不同于胚胎干细胞,随着妊娠期间胚胎的发育胚胎干细胞会逐渐失去部分分化潜能,会出现一些特殊表型或分子标记,如 CD105,称其为胚胎后亚全能干细胞。目的:根据胚胎后亚全能干细胞表达 CD105的特性分离胎儿骨髓源性胚胎后亚全能干细胞,经体内外诱导使其分化为肝细胞样细胞并检测其功能。设计、时间及地点:动物随机分组,细胞分子生物学实验,于 2003-03/2005-03 在天津市第三中心医院卫生部人工细胞工程技术研究中心完成。材料:取 22 周龄左右胎儿股骨和胫骨骨髓分离出胚胎后亚全能干细胞。以雌性成年SCID 鼠作为干细胞移植的受体。CD105 免疫磁珠为德国 Miltenyi Biotec 产品。鼠抗人白蛋白抗体为美国 Sigma 产品。碱性成纤维细胞生长因子、肝细胞生长因子为英国PEPROTECH 产品。方法:利用密度梯度离心结合免疫磁珠方法分离胎儿骨髓 CD105(+)胚胎后亚全能干细胞,体外培养,用含 30 μg/L 肝细胞生长因 20 μg/L 碱性成纤维细胞生长因子的诱导培养基将其向肝细胞样细胞诱导分化。将24 只 SCID 鼠随机分为干细胞治疗组和对照组,每组 12 只,均按 800 mg/kg 的剂量向腹腔内注射 D-氨基半乳糖制备肝损伤模型。造模次日,干细胞移植组鼠在肝原位输注106 左右 CD105(+)细胞,对照组分别输注106左右的CD105(-)细胞或同等体积的培养液。主要观察指标:于干细胞移植后 2,7 d,1,3 个月对肝脏组织行免疫组化检测人源白蛋白表达。结果:免疫磁珠筛选后的细胞免疫细胞化学检测 CD105 呈弱阳性表达;细胞在对数生长期的倍增时间为 30 h 左右;约传 10 代后进入衰退期。SCID 鼠移植胚胎后亚全能干细胞 3 个月后在小鼠肝脏中可见有点状或小灶状的人白蛋白表达,对照组未见表达。结�BACKGROUND： At present, studies show that a kind of stem cell community which in many kinds of organizations can differentiate into tissue cells of different embryonic layers; but those are different from embryonic stem cells, embryonic stem cell will lose the part differentiation potential gradually during the development of pregnancy, and will present some special phenotypes or the molecular markers, as CD105 and so on, will call it postembryonic pluripotent stem cells. OBJECTIVE： To study the isolation of postembryonic pluripotent stem cells from fetal bone marrow, proliferative culture in vitro, induction and differentiation; transplantation to the liver of SCID mice with hepatic failure, and detect therapy effects. DESIGN, TIME AND SETTING： Cell observation and animal randomization expedment which was completed in the Ministry of Health of Cell Engineering Technology Research Center, Tianjin Third Central Hospital from March 2003 to March 2005. MATERIALS： The postembryonic pluripotent stem cells were extracted from thighbone and shinbone of 22-week old fetuses under sterile circumstance. Adult female SCID mice were regarded as the recipients. CD105 immunomagnetic beads were provided by Miltenyi Biotec, Germany; mouse-anti-human albumin by Sigma, USA; basic fibroblast growth factor （bFGF） and hepatocyte growth factor （HGF） by PEPROTECH, UK. METHODS： Postembryonic pluripotent stem cells obtained from fetal bone marrow were isolated using density gradient centrifugation and micromagnetic beads technique. The hepatocyte-like cells were induced and differentiated with culture media containing HGF （30 ng/mL） and bFGF （20 ng/mL）. Twenty-four SCID mice were randomly divided into experimental group and control group with 12 mice in each group. Hepatic injury models were established with intraperitoneal injection of D-galactosamine. On the next day, about 10^6 CD105（＋） cells were perfused into liver in situ in the experimental group, and about 10^6 CD105（-） cells or isovolumic cultu

关 键 词：胚胎后亚全能干细胞 骨髓 肝细胞样细胞 

分 类 号：R394.2[医药卫生—医学遗传学]