PKH26标记单核细胞衍生的肝样细胞体内示踪研究  

Study on hepatocyte-like cells differentiated from human peripheral blood mononuclear cells labeled by the Fluorescent Dye PKH26 in vivo

作　　者：余幼林 施宝民[1] 王秀艳[1] 苏忠学[1] 卢晓飞[1] 张振海[1] 孙小刚[1] 梁锋[1] 

机构地区：[1]山东大学附属省立医院肝胆胰外科,济南250021

出　　处：《中华肝胆外科杂志》2012年第7期548-551,共4页Chinese Journal of Hepatobiliary Surgery

基　　金：国家自然科学基金资助项目（30972922）

摘　　要：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FGF4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（alphafetoprotein，AFP）和细胞角蛋白19（cytokeratin19，CK19）表达。以红色荧光染料PKH26标记单核细胞衍生的肝样细胞，尾静脉注人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。Objective To induce human peripheral blood mononuclear cells differentiate into hepatocyte-like cells by hepatoeyte 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 ob- serve 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 hepatoeyte 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 adminis- tration of both HGF and FGF-4 to induce the differentiation into liver hepatocyte-like cells. The chan- ges in cell morphology were observed and the expressions of AFP and CK19 were detected by immuno- cytochemical staining in two groups at different times after induction. The hepatocyte-like cells differ- entiated 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 nor- mal saline in control group. The migration of the labeled cells into the liver are observed by the fluo- rescence microscope in the hepatic tissue sections of nude mice and the expressions of ALB were detec- ted 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; ceils developed apoptosis and cracked following incubation. The expressions of AFP and CK19 were positive after induction in group B as detected by immunocytochemical staining. Inversely, the expres- sions of AFP and CK19 were

关 键 词：外周血单核细胞 细胞分化 肝样细胞 移植 裸鼠肝脏 

分 类 号：R657.3[医药卫生—外科学]