小鼠成体肝脏祖细胞（AHPC）体外培养模型的建立和生物学特性研究

发布时间：2018-06-14 16:37

本文选题：小鼠 + 肝脏干细胞　；参考：《复旦大学》2008年博士论文

【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天克履诓糠窒赴鉤泶锏ü芟赴鉤昙俏榬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。
[Abstract]:objective
To study the proliferation and differentiation potential of normal adult mouse hepatocytes, to isolate the possible stem cells or progenitor cells in normal adult mice and to establish a cell model in vitro, and to study their basic biological characteristics.
Method
Liver cells were separated by improved Seglen two step method and centrifugation, and hepatocytes were preliminarily divided into liver parenchyma cells (parenchymal hepatocytes, PH) and AHPC (adult hepaticprogenitor cells, AHPC). The two parts of the cells were cultured with a modified DMEM medium adding fetal bovine serum (FBS), and continued to observe more than 60. Analysis of the morphological differences in the two parts of the liver, and analysis of the difference in the proliferation of hepatocytes in the two part by cell proliferation and cloning. Immunofluorescence technique was used to carry out Albumin, AFP, CK19, c-kit, CD45, CD34, Oct-4, Desmin, CD16, Thy-1 and nestin in the Clones of high proliferative cells and their formation. Color, analysis of the expression of cell markers and the maturation and differentiation of cells in clones. The effects of the growth of nonparenchymalcell (NPC) on the activation, proliferation and differentiation of AHPC were preliminarily analyzed by morphological observation, and the immunofluorescence staining technique was used to analyze the proliferation and differentiation of non parenchymal cells in the liver. In addition, the transmission of AHPC clones was tried. Raise.
Result
In the two parts of this study, the two parts of the liver cells obtained high yield and activity (> 90%). They fully met the needs of the experiment and the size of the liver cells rich in the AHPC part were (37.03 + 6.65) m and (22.63 + 2.04) m respectively. There was a significant statistical difference (P < 0.05), but there was a small overlap in the proportion of the size distribution. The two part of the paste was attached. The parietal cells contained NPC pollution, which were rich in AHPC and accounted for 70.3% of the total number of adherent cells. After NPC proliferation, hepatocytes were activated and began to proliferate. All the hepatocyte clones expressed the hepatic stellate cell marker Desmin. rich in AHPC part of the hepatocytes, which was higher than that of PH, and the clone formation rate of the two parts was 21.45, respectively. % + 1.25% and 0.28% + 0.09% (P < 0.001). In the rich AHPC part, about 13.5% of the adherent hepatocytes were activated and proliferated at second to 3 days after inoculation, and small cell clones were formed on fourth to 5 days, and a few cells (0.5% ~ 1%) could form clones in 1.25% days after inoculation, and there were similar mature hepatocytes in clones and clones could be cloned after 30 days. For more than 60 days, the maximum clone area reached 0.64mm~2, and the cell proliferation was more than 10 cycles. After 24 hours of adherence, all liver cells strongly positive expression of liver cell marker Albumin, not AFP and CK19, and the cell clone began to express Albumin and AFP for fifth days. The thirtieth days of the thirtieth days At the same time, Albumin negative cells were found. By immunofluorescence double staining, it was found for thirtieth days that there were Albumin positive and AFP positive in AHPC clones, Albumin positive and AFP negative, Albumin positive and CK19 negative, CK19 positive and AFP negative, CK19 positive and AFP positive cells. The cells of the AHPC clones in the wall were small, the ratio of the nuclear plasma was large and the morphology of the epithelioid cells. Some cells still had the ability to form clones independently. However, it was found that the proliferation of AHPC clones in the subculture of cell groups was better than that of single cell subculture.
conclusion
1. in the liver of normal adult mice, a liver specific adult liver progenitor cell (AHPC) was established, and a cell model was established successfully in vitro.
2. the mouse AHPC was cultured and activated in vitro for more than 60 days, and could be cultured continuously, with the differentiation potential of hepatocyte and bile duct.
3. the growth of hepatic non parenchymal cells (NPC) can promote the proliferation, maturation and differentiation of AHPC.
4. compared with the liver progenitor cells reported in foreign literature, the cell phenotype of mouse AHPC in vitro is different. It has high proliferation ability and obvious bidirectional differentiation potential. It provides a new liver stem cell model and research tool for liver transplantation, liver development and liver disease.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【参考文献】