【摘要】 背景:早期肝的发育与横隔间充质和原始心脏的发育在时间和空间上存在密切关系,来自原始心脏和横隔间充质的信号分子可诱导前肠内胚层细胞向肝细胞的特化,横隔间充质为肝芽的形成提供了一个适宜的环境,并可促进其生长和分化。但这一诱导过程的分子机制尚不清楚。目的:利用发育第3～5周人胚标本,选择甲胎蛋白、细胞角蛋白19、c-Met作为肝干细胞的标记物,观察早期人胚胎的发育及肝干细胞的形态特征,以明确肝干细胞的特征和影响其增殖、分化的因素,为肝干细胞的基础研究和临床应用提供实验依据。设计:开放性实验。单位:潍坊医学院人体解剖学教研室。材料:实验于2004-09/2005-01在成都医学院科研中心完成。选择2个月内流产的新鲜人胚胎标本20例,20min内用40g/L多聚甲醛固定,常规石蜡包埋、连续切片,苏木精-伊红染色,显微镜下观察,参照Jirasek的人胚发育分期标准,根据胚胎的长度、体节的数目及器官发育状况确定胚龄。方法:选择胚龄第3～5周的标本,SABC法进行免疫组织化学染色。多克隆抗肝细胞生长因子、c-Met、胰岛素样生长因子I及其受体、转化生长因子β1、转化生长因子β受体1、转化生长因子β受体2抗体,单克隆抗增殖细胞核抗原、甲胎蛋白和细胞角蛋白19抗体,4℃孵育过夜,羊抗兔或羊抗鼠IgG及SABC液室温下分别孵育2h,二氨基联苯胺显色。以磷酸盐缓冲液代替一抗作阴性对照。光学显微镜下观察、拍照。主要观察指标:第3～5周人胚肝干细胞的形态特征及标记物的免疫组织化学染色,肝细胞生长因子、转化生长因子β1、胰岛素样生长因子I及其受体在第3～5周人胚肝、原始心脏及横隔间充质细胞中的表达。结果:①第3～5周人胚肝干细胞的形态特征及标记物的免疫组织化学染色观察:第3周末肝芽形成,第4周肝芽的细胞伸入横隔间充质内形成肝索,构成肝索的细胞具有与第3周末相同的形态特点。第5周时这些细胞数量明显增加,细胞体积有所增大,胞核嗜碱性稍减弱,胞质的嗜酸性增强,但细胞的形态仍均匀一致。第3～4周时肝索的细胞呈增殖细胞核抗原的反应阴性,5周时开始出现阳性反应,主要分布在细胞核,少数细胞的胞质为弱阳性。第3～5周时多数肝索细胞都为甲胎蛋白和c-Met阳性,甲胎蛋白阳性细胞的免疫反应沉淀物在胞核、胞质及细胞膜中均存在;c-Met阳性反应主要分布于细胞核,胞质亦可见较弱的阳性反应。而这些细胞为细胞角蛋白19阴性。②肝细胞生长因子、转化生长因子β1、胰岛素样生长因子I及其受体在第3～5周人胚肝、原始心脏及横隔间充质细胞中的表达:发育第4周组成肝索的细胞表达c-Met,而不表达其他的因子或受体。第5周时,除了肝细胞生长因子以外的其他因子均在肝索细胞表达。转化生长因子β1及其受体转化生长因子β受体1、转化生长因子β受体2的免疫反应沉淀物主要存在于细胞质和细胞膜,胰岛素样生长因子I及其受体的阳性反应在细胞核、细胞质以及细细胞膜均可见。第3～5周时,肝芽或肝索周围的心肌细胞和间充质细胞呈肝细胞生长因子、转化生长因子β1、胰岛素样生长因子I免疫反应阳性,阳性信号主要聚集在细胞质,少数胞核亦有阳性。结论:①人胚胎发育的第3周末,前肠腹侧的部分内胚层细胞特化为肝干细胞。②发育第3～5周人胚肝的细胞为未分化的肝干细胞,若拟研究人胚肝干细胞,此时取材、并利用肝干细胞特异性的标记物,可得到具有双向分化潜能的肝干细胞。对于鼠胚肝干细胞的研究,也可利用人、鼠胚龄的对应关系,推算出其肝干细胞存在的时间、从而决定取材的时间点。③肝细胞生长因子、胰岛素样生长因子I、转化生长因子β1等生长因子促进早期人胚肝的发育。更多还原

【Abstract】 BACKGROUND: There is an intimate temporal and spatial relationship between growth of primitive cardiac cells,septum transversum mesenchyme and liver development. The signal from primitive cardiac cells and septum transversum mesenchyme induces the ventral foregut endoderm cells specialize toward hepatocytes. While the septum transversum mesenchyme provides a suitable environment for forming the liver bud and promoting the growth and differentiation. However,the molecular mechanism of this induction is not yet delineated. OBJECTIVE: Using alpha-fetal protein (AFP),c-Met and cytokeratin (CK) 19 as markers of hepatic stem cells,the growth of early human embryo of 3-5 weeks and morphologic characteristic of hepatic stem cells were observed to demonstrate the characteristic and factors that affected the proliferation and differentiation of hepatic stem cell,which provided experimental evidence for basic research and clinical application of hepatic stem cells. DESIGN: An opening experiment was designed. SETTING: Department of Anatomy,Weifang Medical College. MATERIALS: The experiment was carried out at the Scientific Research Center of Chengdu Medical College between September 2004 and January 2005. Twenty cases fresh human embryos aged less than 2 months were collected with signed agreements of the pregnant women suffering from pregnancy termination with mifepristone. The samples were fixed with 40 g/L polymerisatum in 20 minutes and embedded routinely in paraffin,and then 5 μm thick series sections were continuously made. After hematoxylin-eosin staining,the embryonic age was determined under the microscope according to the length of embryos,the number of somites and the development of organs,which was referring to the Jirasek’s staging standard of human embryo. METHODS: The immunohistochemical staining was conducted with SABC method on one of every ten sections,which were incubated overnight at 4 ℃ with polyclonal antibodies against hepatocyte growth factor (HGF),c-Met,insulin-like growth factor (IGF-I),IGF-I receptor (IGF-IR),transforming growth factor (TGFβ1),TGFβR1,TGFβR2 or monoclonal antibodies against proliferating cell nuclear antigen (PCNA),AFP and CK19. The following day,the sections were incubated for 2 hours at room temperature with biotinylated anti-mouse or anti-rabbit IgG and SABC liquid respectively,and then diaminobenzidine (DAB) was used to color them. The negative control was conducted with the phosphate buffer,then the sections were observed and photographed under light microscope. MAIN OUTCOME MEASUERS: ①the morphologic characteristic of human hepatic stem cells and immunohistochemical staining of markers②the expression of HGF,IGF-I,TGFβ1 and their receptors on human embryonic livers of 3-5 weeks,primitive cardiac cells and septum transversum mesenchyme. RESULTS: ①The morphologic characteristic of human hepatic stem cells and immunohistochemical staining of markers: The hepatic bud formed at the end of 3rd week and migrated into the septum transversum mesenchyme to form the hepatic cords at the 4th week. The cells structuring the hepatic cords displayed the typical characteristic of immature cells. At the 5th week,the number of cells within the hepatic cords,the size of cell body,the cytoplasmic acidophilia all increased,whereas the basophilia of nuclei decreased. However the cellular forms were still homogeneous and displayed the typical characteristic of immature cells. The cells of hepatic cords were negative for PCNA response during 3rd-4th week but began to express positive at the 5th week,mainly in the nucleus and minority cellular cytoplasm showed weak positive. Most hepatic cells during 3rd-5th weeks were positive for AFP,c-Met and negative for CK19. The immunologic reaction depositors of AFP positive cells were located in the nuclei,cytoplasm and membrane of the hepatocytes,and c-Met presented mainly in the nuclei and the positive signal was weak in the cytoplasm. ②Expressions of HGF,IGF-I,TGFβ1 and their receptors in the embryonic human liver,primitive heart and septum transversum mesenchyme: At the 4th week,the c-Met expressed only in all hepatocytes,whereas the other growth factors and their receptors were undetectable. At the 5th week,all the growth factors,except HGF,were expressed in the hepatocytes. The immunologic reaction depositors of TGFβ1,TGFβ1R1 and TGFβ1R2 were located in the cytoplasm and cell membrane. The positive response of IGF-I and IGF-IR were present at nuclei,cytoplasm and cell membrane. At the 3rd-5th week,myocardial cells surrounding liver bud or hepatic cord and the septum transversum mesenchyme were positive for HGF,TGFβ1 and IGF-I,with the signals were aggregated mainly in cytoplasm and minority nucei. CONCLUSION: ①It was at the end of 3rd week that part of endoderm cells in foregut ventral were specialized to hepatic stem cells. ②The undifferentiated hepatic stem cells could be drawn to develop to the liver stem cells with bi-directional differentiation potentials by using specific markers for studying human embryonic liver stem cells. According to the corresponding relation of embryonic age between human and rats,the time studying the rat hepatic stem cells could be calculated. ③HGF,IGF-I,TGFβ1 and their receptors promoted the early development of human embryonic liver.更多还原