骨形成蛋白家族成员2、7对小鼠胚胎肝干细胞分化的体外研究  

The effecf of bone morphogenetic proteins 2,7 in inducing murine embryonic stem cells into hepatic cells in vitro

作　　者：陈聪[1] 康权[1] 罗庆[1] 迭小红[1] 田雯[1] 

机构地区：[1]重庆儿童发育疾病研究省部共建教育部重点实验室、儿科学重庆市重点实验室、重庆市儿童发育重大疾病诊治与预防国际科技合作基地、重庆医科大学附属儿童医院肝胆外科,400014

出　　处：《解放军医学杂志》2013年第4期260-264,共5页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(30973062、81172545);重庆市自然科学基金(CSTC2012jjA10120);重庆医科大学校际重点课题(XBZD201015)~~

摘　　要：目的探讨骨形成蛋白2、7(BMP2、BMP7)对小鼠胚胎肝干细胞(HP14.5)定向诱导分化为肝细胞样细胞的影响。方法将表达BMP2、BMP7、肝细胞生长因子(HGF)和绿色荧光蛋白(GFP)的重组腺病毒作为4个组分别感染HP14.5,诱导其分化,在病毒感染后的第1、4、7天通过检测荧光素酶报告基因读数,在第7天通过细胞免疫荧光染色,观察肝细胞标志物白蛋白(ALB)的表达情况;并在第4、7、10天通过PAS染色、尿素氮合成功能检测,观察诱导后HP14.5向肝细胞方向的分化成熟度。结果 BMP2组、HGF组荧光素酶读数较GFP对照组明显上升;免疫荧光染色显示,诱导7d后BMP2组、HGF组细胞质内表达肝细胞特有的ALB,而GFP对照组几乎无表达;糖原染色可见BMP2组、HGF组胞质存在紫红色颗粒,呈阳性反应;尿素合成功能检测显示BMP2组、HGF组培养液中尿素氮随时间而升高。BMP7组诱导后,细胞免疫荧光染色、荧光素酶活性、PAS染色和尿素合成检测均呈阴性或弱阳性反应。结论 BMP2具有一定的诱导HP14.5向成熟肝细胞分化的作用,并初步具备肝细胞的合成分泌功能,而BMP7对其无诱导作用。Objective To explore the effect of recombinant adenovirus-mediated bone morphogenetic proteins 2, 7 （Adv- BMP2, Adv-BMP7） in inducing transformation of murine embryonic hepatic progenitor cells to mature hepatic-like cells. Methods HP14.5 cells were divided into 4 groups, and then infected by recombinant adenovirus expressing BMP2, BMP7, hepatocyte growth factor （HGF）, and green fluorescent protein （GFP）, respectively. For investigating the differential regulation of HP 14.5 cells, the luciferase report gene was detected at the 1st, 4th and 7th day post infection, the expression of hepatocyte marker albumin （ALB） was detected at the 7th day after infection by cellular immunofluorescence assay. The maturation and differentiation of HP 14.5 cells were examined by PAS staining and urea nitrogen synthesis of the cells at day 4, 7 and 10 post-infection. Results The expression of ALB with BMP2 and HGF increased significantly compared to that in GFP control group tested by luciferase report gene; cellular immunofluorescence assay indicated that the specific marker of mature hepatocyte ALB was strong expressed at day 7 post-infection, while a negative result was observed in the GFP control group; HP14.5 cells infected with BMP2 and HGF have also acquired functional characteristics of hepatocytes which synthesized and secreted urea nitrogen, and stored glycogen. However, less inductive activity was found in BMP7 group. Conclusion BMP2 may induce the differentiation of HP14.5 cells into mature hepatocyte-like cells with initial synthesis and secretion, but BMP7 may have no such a capability.

关 键 词：骨形态发生蛋白质类 胚胎肝干细胞 细胞分化 重组腺病毒 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]