【作者】 王洪山；

【导师】 秦新裕； 牛伟新； 宋陆军；

【作者基本信息】 复旦大学 ， 普外科， 2008， 博士

【摘要】 目的研究正常成体小鼠肝细胞的增殖能力和分化潜能,分离正常成体小鼠肝脏内可能存在的干细胞或祖细胞并建立体外培养的细胞模型,研究其基本的生物学特性。方法应用改良的Seglen二步法灌注和离心分离肝脏细胞,将肝细胞初步分为肝脏实质细胞(parenchymal hepatocytes,PH)部分和富含AHPC(adult hepaticprogenitor cells,AHPC)部分,对两部分的细胞用添加胎牛血清(FBS)的改良DMEM培养基进行培养,持续观察超过60天,分析两部分中肝细胞的形态学差异,以及通过细胞增殖和克隆的形成情况分析两部分中肝细胞增殖能力的差异。应用免疫荧光技术对具有高增殖能力细胞及其形成的克隆进行Albumin、AFP、CK19、c-kit、CD45、CD34、Oct-4、Desmin、CD16、Thy-1和nestin等染色,分析细胞标记物的表达和克隆内细胞的成熟分化情况。利用形态学观察、记录细胞增殖状况,以及免疫荧光染色技术初步分析肝脏非实质细胞(nonparenchymalcell,NPC)的生长对于AHPC活化、增殖和分化的影响。另外,尝试了AHPC克隆的传代培养。结果本研究中两部分肝细胞均获得较高的产量和活性(＞90%),完全满足实验的需要。PH部分和富含AHPC部分的肝细胞大小分别为(37.03±6.65)μm和(22.63±2.04)μm,存在明显的统计学差异(p＜0.05),但在大小分布比例上存在小部分的重叠。两部分的贴壁细胞中均含有NPC污染,其中富含AHPC部分较多,占贴壁细胞总数的70.3%,NPC增殖后肝细胞活化并开始增殖,所有的肝细胞克隆均表达肝星状细胞标记物Desmin。富含AHPC部分的肝细胞增殖能力明显较PH部分高,两部分的克隆形成率分别为21.45%±1.25%和0.28%±0.09%(p＜0.001)。富含AHPC部分中,约13.5%的贴壁肝细胞在接种后第2～3天活化并迅速增殖,第4～5天形成小的细胞克隆,极少数细胞(0.5%～1%)在接种后第3天即可形成克隆;培养30天后克隆内出现类似成熟的肝细胞,细胞克隆可持续扩增超过60天,最大克隆面积达到0.64mm~2,细胞平均增殖超过10个周期。贴壁后24小时,所有的肝细胞均强阳性表达肝细胞标记物Albumin,不表达AFP和CK19,培养第5天细胞克隆开始表达Albumin和AFP,第30天克履诓糠窒赴泶锏ü芟赴昙俏顲K19,同时发现Albumin阴性细胞。通过免疫荧光双染发现,培养第30天,AHPC克隆内同时存在Albumin阳性和AFP阳性、Albumin阳性和AFP阴性、Albumin阳性和CK19阴性、CK19阳性和AFP阴性、CK19阳性和AFP阳性的细胞。另外,AHPC可以传代培养超过60天,传代培养中,贴壁的AHPC克隆内细胞较小,核浆比率大,呈上皮样细胞的形态,部分细胞仍然具有独立形成克隆的能力,但观察发现,AHPC克隆解离为细胞团进行传代培养中细胞的增殖比解离为单细胞传代好。结论1.在正常成体小鼠肝脏内存在一种肝脏组织特异性的成体肝脏祖细胞(AHPC),并已成功建立了体外培养的细胞模型。2.小鼠AHPC体外培养活化后可持续克隆性增殖超过60天,并可连续传代培养,具有向肝细胞和胆管细胞分化的双向分化潜能。3.肝脏非实质细胞(NPC)的生长可以促进AHPC的增殖、成熟和分化。4.与国外文献报导的肝脏祖细胞相比,小鼠AHPC体外培养中细胞表型不同,具有较高的增殖能力和明显的双向分化潜能,为肝细胞移植、肝脏发育和肝病等研究提供了一种新的肝脏干细胞模型和研究工具。更多还原

【Abstract】 ObjectiveThis study is aimed to isolate and culture candidate cells of liver stem cells or progenitor cells from normal adult mouse liver,by investigating their abilitys to proliferate and potentials to differentiate in vitro,and to establish a stable cell model cultured in vitro,followed by characterizing the fundamental properties of those cells.MethodsMouse hepatocytes,isolated by two-step collagenase perfusion and mechanical centrifugation,were separated into two fractions as fraction of PH(parenchymal hepatocytes,PH) and fraction with enriched AHPC(adult hepatic progenitor cells, AHPC).Two fraction cells were cultured in modified DMEM more than 60 days,and observed by phase contrast microscopy.It was clarified that the morphological and proliferating difference between those hepatocytes from two fractions.The expression of cell markers,such as Albumin,AFP,CK19,c-kit,CD45,CD34,Oct-4,Desmin, CD 16,Thy-1 and nestin in high proliferating cells and their colonies,was illuminated by immunofluorescence at different culture days,to analyze the origin of those cells and their potentials to differentiate.Besides,it was primarily investigated that the impact of NPC(nonparenchymal cells,NPC) contamination and growth on the activation,proliferation and differentiation of AHPC during primary culture,by immunofluorescence and noticing the morphological changes and growing state of NPC and AHPC.In addition,efforts were made to investigate the subculture possilbity of AHPC colonies.ResultsThe total cell quantity and cell vitality of hepatocytes were relatively high in two fractions,with cell vitality more than 90%.The cell size of fraction with enriched AHPC was(22.63±2.04)μm,and PH fraction was(37.03±6.65)μm,with a significant statistical difference(p＜0.05).The percentage of different cell size, however,showed two fractions shared some cells with certain sizes.There was a contamination of HPC in both fractions,and the one of enriched AHPC had more HPC(about 73%of attached cells).In the primary culture,HPC firstly began to divide and proliferate,then attached hepatocytes showed being activated,and all formed colonies expressed Desmin,a relatively specific cell marker of hepatic stellate cell(HSC).The fraction with enriched AttPC manifested a definitely larger colony-forming rate than PH ones,respectively 21.45%±1.25%and 0.28%±0.09%(p＜0.001).It was found that a subpopulation of hepatocytes in the fraction with enriched AHPC(about 13.5%) began to proliferate significantly and to form colonies on being activated after 2～3 days in culture.The colonies could expand continually more than 2 months,with the occupied area of some reaching 0.64mm~2 and cell dividing more than 10 rounds.Some larger cells,morphologically like mature hepatocytes,appeared in colonies around day30.At 24 hours after attachment,all hepatocytes expressed Albumin,but were negative for AFP,CK19,Oct-4,CD34, CD45,Thy-1,nestin and c-kit.After being activated,the proliferating cells expressed AFP and Albumin at day5,no CK19 expression.But at day30,the expanding colonies expressed CK19,and some cells within colonies manifested Albumin negative. Immunofluorescence double staining showed some cells with specific pattern of expression in colonies at day30,such as Albumin~+AFP~-,Albumin~+AFP~+, Albumin~+CK19~+,CK19~+AFP~+,CK19~+AFP~-.All formed colonies didn’t express Oct-4, CD34,CD45,Thy-1,nestin and c-kit during being cultured.Besides,AHPC colonies could be continuously subcultured for more than 60 days without losing proliferating ability.Attachment of colony aggregates showed a better proliferating performance than those of detached single cells of colonies.Attached AHPC colonies off-spring cells were small with epithelial cell-like appearance and a relatively larger nucleus plasma ratio.Conclusion1.There exists a kind of liver specific progenitor cells in adult normal mouse, named adult hepatic progenitor cells(AHPC),and mouse AHPC cell model cultured in vitro has been successfully established.2.Mouse AHPC is capable of proliferation,and holds bipotentials to differentiate into hepatocytes and duct cells.In addition,it can be subcultured for more than 60 days without losing proliferating ability.3.The contamination and growth of NPC can promote AHPC to activate and proliferate at early culture days,and however NPC may play a part role in inducing progeny cells in AHPC colonies to differentiate at later days. 4.By comparing with other reported hepatic progenitor cells isolated from normal rats and mouse,the mouse AHPC isolated in this study shows a relatively higher ability to proliferate with differently expressing characters,and holds definite two differentiating potentials.Mouse AHPC may be useful tools as new in vitro cell models to investigate liver diseases,liver development and regeneration,and can facilitate the study of liver stem cells that can be used in cell transplantation.更多还原