摘要
背景:胎肝来源肝干细胞作为细胞移植的供体来源具有优势,但有鉴于干细胞特质,其培养困难,传代后易分化生长;且现有干细胞示踪定位技术欠成熟,这些均是制约肝干细胞移植的重要因素。目的:观察原代肝干细胞电转pEGFP-Cl质粒是否能获得稳定表达绿色荧光的肝干细胞。方法:联合机械分离和胶原酶消化法体外分离孕13.5dSD大鼠胚胎来源肝细胞,应用特异培养基培养分离细胞,随后通过半量换液法纯化出肝干细胞,采用细胞免疫荧光法对贴壁细胞进行鉴定,并用pEGFP-Cl质粒电转染原代肝干细胞后持续表达绿色荧光作示踪定位标志。结果与结论:原代培养的胎肝干细胞24h后可见细胞半贴壁,形态基本相同,呈圆形或卵圆形;第2天观察细胞为大小几乎一致的圆形;培养第3天出现一些由3,5个形态一致的细胞集落,细胞直径6～10μm;细胞集落不断增大,至第5天集落中细胞数量增至10个左右,集落由大小不等的致密圆形细胞组成,界限清楚;第8天后细胞呈上皮样铺展;第12天时,细胞变大呈煎蛋样摊开,形态不规则,细胞浆内可见颗粒,生长变得缓慢;传代后细胞扩增速度无明显变化,至第3代仍保持较均一的上皮细胞状;通过细胞免疫荧光法检测出培养第5天的贴壁细胞表达干细胞标志物CD34和胆管细胞标志物CK19;经筛选pEGFP-Cl质粒电转染后的原代肝干细胞能稳定表达绿色荧光。实验成功构建转染了绿色荧光蛋白的肝干细胞。
BACKGROUND：Fetus liver-derived liver stem cells,as cell transplantation donor,have advantages in source.However,difficult culture,easy to differentiate following passage and immature tracing localization technique restrict liver stem cell transplantation.OBJECTIVE：To observe whether primary cultured liver stem cells after tranfection by electroporation with pEGFP-C1 plasmid can stably express green fluorescent protein （GFP）.METHODS：Mechanical separation and enzymatic digestion method were used to isolate liver stem cells from fetal Sprague Dawley rat liver tissues following pregnancy for 13.5 days.Half-amount medium replacement was used for purify isolated fetal liver cells following cultured in specific medium.Immunofluorescence technique was used to identify adherent cells.The plasmid pEGFP-C1 was identified correctly by eletrophoresis and then transfected into fetal liver stem cells.RESULTS AND CONCLUSION：The isolated fetal liver stem cells adhered to the culture plastic and presented round or oval cells after 24-hour cultivation in vitro.Isolated cells were almost identical circular after 2-day cultivation.At 3 days,cells grew into a colony which was constructed by 3 or 5 cells,with cell diameter of 6-10 μm;at 5 days,cell colony became enlarged and was composed of 10 dense,round cells of various sizes with clear boundary.At 8 days,they grew like epithelium cell.At 12 days,cells became big,extended like fried egg,with irregular forms,particles in cytoplasm,grew slowly.Following passage,there were no significant changes in cell amplification speed.Cells still presented epithelium-like shape at the passage 3.The adhered cells at day 5 following primary incubation were positively for human stem cell factor receptor CD34 and cytokeratin 19 using immunofluorescence technique.Green fluorescence was observed in many stem cells which has been transfected by pEGFP-Cl.This study successfully established liver stem cells with GFP.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第23期4295-4298,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30872564)课题名称:表达产甲酸草酸杆菌草酸分解基因的肝干细胞移植治疗高草酸尿症的实验研究~~
