微粒体Ⅱ相代谢酶基因在小鼠胚胎干细胞定向分化肝细胞中的表达  

Gene expressions of phase Ⅱ enzymes in mouse embryonic stem cellderived hepatocytes

作　　者：郭美媛[1] 朱丹雁[1] 杜悦[1] 楼宜嘉[1] 

机构地区：[1]浙江大学药学院药理毒理与生化药学研究所,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2007年第3期229-235,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金项目(No.30672564;No.30472112;No.30171121);浙江省自然科学基金项目(No.Y206473).

摘　　要：目的:评价微粒体Ⅱ相代谢酶基因在小鼠胚胎干细胞(embryonic stem cells,ES细胞)定向分化肝细胞中的表达,为进一步探索酶蛋白表达及其催化活性提供依据。方法:ES细胞体外悬滴培养定向分化成肝细胞表型,RT-PCR法检测肝细胞发育依赖性基因AFP、ALB、CYP7a1的表达,免疫细胞化学法确认成熟肝细胞特异性标记物白蛋白(ALB)表达,RT-PCR法检测上述肝细胞中Ⅱ相代谢酶尿苷二磷酸葡萄糖醛酸结合酶系(UGTs)和微粒体谷胱甘肽S-转移酶1(mGST1)基因表达。结果:ES细胞在定向分化至d8时,肝细胞发育依赖性基因AFP和ALB有较高表达,d18时AFP表达甚微而ALB表达显著增加,并伴有成熟肝细胞特异性基因CYP7a1的表达。定向分化呈肝细胞表型时,成熟肝细胞特异性蛋白ALB呈强阳性表达。分化过程中Ⅱ相代谢酶基因UGT1a1、UGT1a9在分化前后均稳定表达;UGT1a6表达呈发育依赖性;UGT2b5未见表达;mGST1在分化前后均有少量表达,但在成熟肝细胞中表达显著增加,与成熟小鼠肝相似。结论:ES细胞体外悬滴培养可定向分化为成熟肝细胞;Ⅱ相代谢酶UGTs、mGST1基因在分化成熟肝细胞表达与小鼠肝细胞相似,可望为Ⅱ相代谢酶代谢深层次研究提供高效模型。Objective： To elucidate gene expressions of phase Ⅱ enzymes in the mouse embryonic stem （ES） cell-derived hepatocytes. Methods： Embryoid body （EB） formation cultures were applied in directed differentiation of ES to hepatic-like cells. The expressions of hepatic-specific genes, including AFP, ALB, Cyp7a1, were detected by RT-PCR during the differentiation course. Albumin was detected by immunocyto-chemistry. The gene expressions of mGST1 and UGTs family,including UGT1a1,UGT1a6,UGT1a9 and UGT2b5,were investigated using RT-PCR. Results： A notable gene expression of AFP and ALB was observed on d 8. On d 18, AFP gene failed to express, while ALB and Cyp7a1 genes were detected. Albumin-positive staining was detected in hepatic-like cells. Phase Ⅱ enzyme genes expressed in variance during the differentiation course ,UGTlal and UGT1a9 were expressed stably,UGT1a6 expression increased gradually,and UGT2b5 failed to express. Little mGST1 gene expression could been detected in the early course until d 18. In addition, all the enzymes gene expressions in the derived hepatocytes on d 18 were similar to those from mature mouse hepatocytes. Conclusions： Mouse ES cell-derived mature hepatocytes express phase Ⅱ enzyme UGTs and mGST1 genes similar to those in mature hepatocytes. The system may offer an alternative animal testing model related to phase Ⅱ enzymes in further research.

关 键 词：葡萄糖醛酸基转移酶 肝细胞 细胞分化 胚胎/细胞学 干细胞/生理学 细胞 培养的 谷胱甘肽转移酶 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]