小鼠骨髓干细胞向肝细胞转化的体外实验研究  

In vitro study on differentiation of mouse bone marrow stem cells into hepatocytes

作　　者：施晓雷[1] 丁义涛[1] 仇毓东[1] 谢婷[1] 朱章华[1] 李雷[2] 陈蕾蕾[2] 

机构地区：[1]南京大学医学院附属南京鼓楼医院肝胆外科,南京市210008 [2]南京大学医学院附属南京鼓楼医院科研部,南京市210008

出　　处：《中华肝胆外科杂志》2006年第7期479-483,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：本实验受国家自然科学基金(NO:30371391);江苏省自然科学基金(NO:BK2003008)资助

摘　　要：目的建立以30 ng/ml FGF-4和30 ng/ml OSM 为主的诱导培养体系,研究体外骨髓干细胞向肝细胞的转化过程中细胞形态、结构、功能的变化。方法建立以30 ng/ml FGF,30 ng/mlOSM 为主的小鼠骨髓干细胞诱导培养体系。观察细胞形态和数量的变化;RT-PCR、Western blot、免疫荧光染色检测肝细胞特异性标记物的表达;通过 PAS 糖原染色、白蛋白 ELISA、尿素合成试验,检测细胞的合成和代谢功能。结果诱导12 d,观察到多极性的肝细胞样细胞,且逐渐增多、集落不断增大。在诱导过程中,不同的时间点可以分别检测到 HNF-3βmRNA、ALBmRNA、CK18mRNA、TTRmRNA、G-6-PasemRNA 和 TATmRNA 的表达。诱导21 d,细胞表达 HNF-3β、ALB、CK18蛋白;免疫荧光定位示 ALB 表达于胞浆和胞膜,而 CK18表达于胞浆。诱导21 d,细胞胞浆内可见红染的糖原颗粒;在诱导过程中,不同的时间点检测到细胞分泌 ALB、合成尿素的量的变化。结论我们建立的以30 ng/ml FGF、30 ng/ml OSM 为主的诱导培养体系,可以有效地促进骨髓干细胞体外定向转化为肝细胞。Objective To establish the directed differentiation media including 30 ng/ml FGF-4 and 30ng/ml OSM and reveal the differentiation of bone marrow cells （BMSCs） into hepatocytes by morphological examination and determination of rnRNA expression, protein expression and cell function. Methods The mouse BMSCs were cultured in the directed differentiation media including FGF-4 and OSM. Morphology of the hepatocytes form directed differentiation of BMSCs were identified. RNA expression of the hepatocytes was tested by RT-PCR and the protein expression by Western blotting and immunofluorescent assay. Synthetic and metabolic functions of the hepatocytes were investigated by albumin ELISA, periodic acid Shill staining and urea assay. Results Some epitheliumlike cells or polygonal cells appeared and increased in the course of the cell directed differentiation. HNF-313, ALB, CK18, TTR, G-6-Pase and TAT mRNA were expressed in the course of the directed differentiation. The directed differentiated cells expressed HNF-313, ALB and CK18 on day 21. ALB was found in the cytoplasm and cell membrane while CK18 scattered in the cytoplasm. The differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. Conclusions The differentiation media including FGF-4 and OSM is effective to differentiate mouse BMSCs into hepatoeytes.

关 键 词：骨髓干细胞 肝细胞 分化 诱导 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]