CAJ | 학술논문

目的探讨人外周血单核细胞向肝样细胞潜能及单核细胞衍生的肝样细胞移植的有效示踪剂,并进一步观察受体鼠移植后肝样细胞存活、分布与转归情况.方法分别设立A、B两组.A组为阴性对照组,在细胞培养过程中不添加诱导细胞分化肝细胞生长因子(HGF)和成纤维细胞生长因子-4 (FGF-4)；B组应用HGF和FGF-4体外联合诱导人外周血单核细胞向肝干细胞分化.鉴定和判断细胞分化的变化,采用免疫荧光细胞化学染色法检测细胞甲胎蛋白(alpha fetoprotein,AFP)和细胞角蛋白19(cytokeratin 19,CK19)表达.以红色荧光染料PKH26标记单核细胞衍生的肝样细胞,尾静脉注入受体鼠体内作为实验组.对照组注射等量生理盐水.分别于移植2周后取肝组织,通过荧光显微镜观察实验组肝样细胞向肝脏迁移与转归的情况.采用免疫荧光细胞化学染色法检测移植的肝样细胞白蛋白(albumin,ALB)的表达.结果 A组细胞随培养时间延长,单核细胞由刚分离出的较均一的圆形逐渐变得不规则,呈条索状、纤维样改变,少部分细胞呈圆形,细胞发生凋亡,裂解.B组细胞随诱导分化培养时间延长,细胞变大、变圆,呈集落生长,增殖活性强.B组有AFP和CK19阳性表达,而A组则没有表达.动物实验组受体鼠肝组织冰冻切片可见大量PKH26标记阳性的肝样细胞,对照组未发现PKH26标记阳性的肝样细胞.移植2周后行免疫荧光细胞化学染色法检测实验组肝样细胞ALB表达阳性.结论在HGF和FGF-4体外联合诱导条件下,人外周血单核细胞具有向肝样细胞分化的潜能.并且,PKH26可作为肝样细胞移植的一种有效示踪剂.移居肝组织的肝样细胞2周后开始向成熟肝细胞分化,发挥肝细胞功能,表达白蛋白.
목적 탐토인외주혈단핵세포향간양세포잠능급단핵세포연생적간양세포이식적유효시종제,병진일보관찰수체서이식후간양세포존활、분포여전귀정황.방법 분별설립A、B량조.A조위음성대조조,재세포배양과정중불첨가유도세포분화간세포생장인자(HGF)화성섬유세포생장인자-4 (FGF-4)；B조응용HGF화FGF-4체외연합유도인외주혈단핵세포향간간세포분화.감정화판단세포분화적변화,채용면역형광세포화학염색법검측세포갑태단백(alpha fetoprotein,AFP)화세포각단백19(cytokeratin 19,CK19)표체.이홍색형광염료PKH26표기단핵세포연생적간양세포,미정맥주입수체서체내작위실험조.대조조주사등량생리염수.분별우이식2주후취간조직,통과형광현미경관찰실험조간양세포향간장천이여전귀적정황.채용면역형광세포화학염색법검측이식적간양세포백단백(albumin,ALB)적표체.결과 A조세포수배양시간연장,단핵세포유강분리출적교균일적원형축점변득불규칙,정조색상、섬유양개변,소부분세포정원형,세포발생조망,렬해.B조세포수유도분화배양시간연장,세포변대、변원,정집락생장,증식활성강.B조유AFP화CK19양성표체,이A조칙몰유표체.동물실험조수체서간조직빙동절편가견대량PKH26표기양성적간양세포,대조조미발현PKH26표기양성적간양세포.이식2주후행면역형광세포화학염색법검측실험조간양세포ALB표체양성.결론 재HGF화FGF-4체외연합유도조건하,인외주혈단핵세포구유향간양세포분화적잠능.병차,PKH26가작위간양세포이식적일충유효시종제.이거간조직적간양세포2주후개시향성숙간세포분화,발휘간세포공능,표체백단백.
Objective To induce human peripheral blood mononuclear cells differentiate into hepatocyte-like cells by hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) in vitro and determine whether PKH26 could be used to serve as an effective tracer for the cells,and observe the ability of transplanted hepatocyte-like cells differentiate into hepatic cells in nude mice.Methods Group A and B were set up respectively.In Group A,mononuclear cells were cultivated without hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) in cell culture.They were used as negative control group.In Group B,mononuclear cells were cultured with the administration of both HGF and FGF-4 to induce the differentiation into liver hepatocyte-like cells.The changes in cell morphology were observed and the expressions of AFP and CK 19 were detected by immunocytochemical staining in two groups at different times after induction.The hepatocyte-like cells differentiated from human peripheral blood mononuclear cells labeled by the fluorescent dye PKH26 injected into caudal vein in nude mice is experimental group.The nude mice injected with equal amount of normal saline in control group.The migration of the labeled cells into the liver are observed by the fluorescence microscope in the hepatic tissue sections of nude mice and the expressions of ALB were detected by immunocytochemical staining two weeks after the cells transplantation.Results Cells in group B have a strong proliferative activity.It becomes large and oval,grows in colonies following induction.Cells in group A that showed spherical shape when peripheral blood mononuclear cells were just isolated are gradually becoming inconformity in morphology,spindle or fibroid,and a few cells are round:cells developed apoptosis and cracked following incubation.The expressions of AFP and CK19 were positive after induction in group B as detected by immunocytochemicat staining.Inversely,the expressions of AFP and CK19 were negative in group A after incubation.The experimental group showed numerous PKH26 labeled cells in the hepatic tissue sections of nude mice.But the control group did not show PKH26 labeled cells.The expressions of ALB were positive in the experimental group as detected by immunocytochemical staining after two weeks of the cells transplantation.Conclusion Human peripheral blood mononuclear cells have the potential to differentiate into hepatocyte-like cells under the induction of HGF and FGF-4.Additionally,PKH26 is an effective tracer in hepatocyte-like cell transplantation.The hepatocyte-like cells settled in hepatic tissue begin to differentiate into mature hepatocyte after two weeks of the cells transplantation.It plays hepatic cells function and expresses alhnmin.