【作者】 李云；

【导师】 周祖平； 林万华；

【作者基本信息】 广西师范大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 目的:目前普遍认为可以在体内分化形成肝细胞和胆管上皮细胞并且具有自我更新能力的细胞为肝干细胞。然而,迄今为止已被报道的肝干细胞表面标志物较少,有关标志物尚待理清,同时关于肝干细胞的年龄相关性的研究目前尚处在初级阶段。本文的目的是:利用实验动物C57BL/6J小鼠,分离鉴定了真正具有分化和自我更新能力的肝脏干细胞亚群,为深入了解肝干细胞及其临床应用奠定基础。通过分析比较青年(2-3月龄)、中年(10-12月龄)和老年(22-24月龄)小鼠肝干细胞含量、细胞周期、分化和自我更新能力,旨在阐明肝干细胞功能的年龄相关性变化,并通过青年和老年组肝干细胞基因表达谱分析,为探究肝脏干细胞衰老调控机制及潜在调控因子奠定基础。方法:(1)小鼠肝脏细胞的分离与单细胞悬液的制备:断颈法处死C57BL/6J小鼠后,取肝脏组织并通过机械剪碎和胶原酶Ⅳ消化处理及细胞筛过滤获得单细胞悬液。(2)小鼠肝干细胞流式分析及分选:新鲜制备的肝脏单细胞悬液经抗表面标志蛋白抗体染色孵育后,通过流式细胞分析仪分析潜在的肝干细胞靶细胞群表型及含量,通过流式细胞分选仪分选靶细胞群。(3)肝干细胞的体外增殖与分化:将分选得到的靶细胞群经完全培养基诱导培养,培养结束后用抗标志蛋白荧光抗体染色,通过荧光显微镜观察分化及增殖状况。(4)细胞周期测定:新鲜分离的细胞首先经抗表面标志蛋白抗体染色,然后经Fixation/Permeabilization固定渗透,再进行7-AAD染色,通过流式细胞分析仪分析肝干细胞的周期分布。(5)基因表达检测:分离得到的靶细胞提取总RNA,检测RNA质量与纯度,逆转录合成cDNA,制备基因芯片。(6)基因表达谱分析:Go分析、pathway分析。(7)统计方法:采用SPSS19.0统计软件对实验数据进行“独立样本T检验”分析,统计结果以平均值±标准差表示。结果:(1)分离的肝脏组织制备成单细胞悬液,经抗表面标志蛋白荧光抗体组合染色孵育,经流式分析,小鼠肝脏组织细胞中可能存在三个潜在的肝干细胞亚群,分别为Lin-CD45-Sca-1+CD49f+、Lin-CD45-Sca-1-CD49f+、Lin-CD45-Sca-1+CD49f,在肝脏组织细胞中的比例分别为 1.17%±0.18%,0.13%±0.02%,0.57%±0.023%。(2)对潜在的三个干细胞亚群进行体外增殖、分化培养和免疫荧光鉴定,确定Lin-CD45-Sca-1-CD49f+、Lin-CD45-Sca-1+CD49f细胞群具有体外增殖和分化能力,同时进行周期分布比较,相比于肝脏组织细胞,Lin-CD45-Sca-1-CD49f+、Lin-CD45-Sca-1+CD49f靶细胞群中有更多的细胞进入到了细胞分裂期(3.64%±0.14%VS5.73±0.41%,P<0.01;3.64%±0.14%VS7.16%±1.04%,P<0.01),这也说明这两群细胞呈现出了更为活跃的增殖和分化的状态,故确定Lin-CD45-Sca-1-CD49f+、Lin-CD45-Sca-1+CD49f细胞群可能为潜在的肝干细胞群。(3)小鼠肝干细胞功能的年龄相关性变化:Lin-CD45-Sca-1-D49f+细胞以及Lin-CD45-Sca-1+CD49f细胞随着鼠龄的增加,该细胞的含量呈现增高的趋势;相对于青年鼠,中年、老年鼠中的 Lin-CD45-Sca-1-CD49f+、Lin-CD45-Sca-1+CD49f细胞群处于 S 和 G2/M 期的比例增加;青年小鼠 Lin-CD45-Sca-1-CD49f+、Lin-CD45-Sca-1+CD49f细胞群的分化能力明显高于老年小鼠Lin-CD45-Sca-1-CD49f+、Lin-CD45-Sca-1+CD49f细胞群的分化能力。(4)通过基因芯片结果分析,初步筛选出一些与年龄相关性有关的差异基因。结论:本研究从细胞表型、体外增殖与自我更新潜能、体外分化能力和细胞周期几个方面,基本确认了小鼠肝脏组织中Lin-CD45-Sca-1-CD49f+和Lin-CD45-Sca-1+CD49f细胞群为肝内潜在的前体细胞或祖细胞亚群。对肝干/祖细胞的年龄相关性研究发现靶细胞群在中年和老年组中的含量增加,但分化潜能降低,故衰老的肝脏组织功能退行性变化可能主要是由肝干细胞功能降低所致,而非干细胞含量的减少。肝干细胞衰老受到严格的表观遗传修饰,涉及的潜在调控因子往往与细胞的自我更新相关。同时通过基因芯片筛选与肝干细胞年龄相关性基因,提升对LSC/PCs衰老的行为变化及其调节机理的认识,为今后了解人肝干细胞衰老及其与年龄依赖性肝脏疾病发生的内在联系提供极具价值的资料和线索。更多还原

【Abstract】 Objectives:It is generally believed that the liver stem cells are eells that can be differentiated into hepatic and cholangiocyte in vivo.However,surface markers of the liver stem cells have been reported to be less,and the relevant biomarkers are still to be sorted out,and the age-related study of the liver stem cells is still in the primary stage.The purpose of this paper is to isolate and identify subsets of liver stem cells which have the ability of differentiation and self-renewal,aiming to lay the foundation of insight and clinical application about liver stem cells in C57BL/6 mice.By analyzing and comparing content,cell cycle,diflferentiation and self－renewal ability of liver stem cells in young(2-3 months old),middle-aged(10-12 months old)and aged(22-24 months)mice,seek to illuminate age-related changes in liver stem cells,and by analyzing gene expression profile of liver stem cells in the young and old groups,to lay the foundation for exploring liver stem cell senescence regulation mechanism and potential regulatory factors.Methods:(1)Isolation of mouse liver cells:mice were killed by cervical dislocation and then took liver out,single cell suspension was prepared by mechanical shear method,collagenase IV digestion and cell filtration.(2)Analysis and sorting of mouse liver stem cells by Flow cytometry:freshly isolated cells were first stained with four kinds of fluorescent-labeled antibodies against relevant surface marker proteins,then analyzed phenotype and content of potential liver stem cells by flow cytometry,sorted targeted cell population.(3)The ability of differentiation and proliferation ability in liver stem cells:sorted liver stem cells cultured in complete medium,then stained with fluorescent-labeled antibodies against relevant marker proteins for observe the condition of differentiation and proliferation by fluorescence microscopy.(4)The measure of cell cycle:isolated cells was stained firstly with fluorescent-labeled antibodies against relevant marker proteins,then fixed and permeated by Fixation/Permeabilization.stained again with 7-AAD,finally analysis of the distribution of cell cycle about liver stem cells by flow cytometry.(5)Detection of gene expression:total RNA was extracted from the isolated target cell and detected the RNA quality and purity,then synthesized cDNA and prepared gene chip.(6)Gene expression analysis:GO analysis,Pathway analysis.(7)Statistical methods:T test was applied for all date analysis using SPSS 19.0 statistical software,the data was showed as Mean 士 standard deviation.Result:(1)Separation of liver tissues were prepared into single cell suspension and incubated by fluorescent-labeled antibodies against relevant surface marker proteins.Analyzing by flow cytometry,mouse liver cells may exist three potential liver stem cell subsets,respectively Lin-CD45-Sca-1+CD49f+、Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f,the ratio was respectively 1.17%±0.18%,0.13%±0.02%and 0.57%±0.023%.(2)Lin-CD45-Sca-1-CD49f and Lin CD45-Sca-1+CD49f cells have ability of proliferation and differentiation in vitro that identified by the culture about proliferation and differentiation in vitro of the three potential stem cell subsets.At the same time,Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f cells entered mitotic time were more than all liver cells(3.64%±0.14%VS5.73±0.41%,P<0.01;3.64%±0.14%VS7.16%±1.04%,P<0.01),it also shows that those cells were in the more proliferative and differentiated states,so Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f cells may be potential liver stem cells.(3)Function of age-related changes in Mouse liver stem cells:the content of Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f cells showed increasing tendency with the age of mice;compared to the young mice,Lin-CD45-Sca-1-CD49f+ and Lin-CD45-Sca-1+CD49f cells of middle-aged and aged mice had more cells in phase of S and G2/M;the increase in the proportion;The differentiation ability of Lin-CD45-Sca-1-D49f+ in young mice was significantly higher than that in aged mice.(4)Through the analysis of gene chip results,some differentially expressed genes related to age were screened out.Conclusion:This study has basically identified that Lin-CD45-Sca-1CD49f+ and Lin-CD45-Sca-1+CD49f cells were potential precursor cells or progenitor cell subsets in liver though analyzing follow aspects:the cell phenotype,the potential ability of proliferation,self-renewal and differentiation in vitro,as well as cell cycle and so on.The age-related study in liver stem/progenitor cells found the population of target cells in the middle-aged and aged group increased,but differentiation ability reduced,50 the degenerative changes of function in aging liver may mainly be caused by liver stem cells function lessening,not cell content reducing.Aging of liver stem cell is modified strictly by epigenetics,and potential regulatory factor is related to cell self－renewal ability.At the same time,screening out age-related gene through gene chip can improve the awareness of aged-behavior change and regulation mechanism of LSC/LPCs,provide valuable data and clue to understand internal relations between aging of human liver stem cells and age-dependent liver diseases for the future.更多还原