| 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 ? 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. |
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