Objective To detect the expression levels of Wnt antagonists, secreted frizzledrelated proteins(SFRPs) ,in different developmental stages of liver tissues, and investigate the effect of SFRP3 on the differentiation of embryonic liver stem cells in vitro. Methods Liver tissues were isolated from embryos 12. 5 days to postnatal 4 weeks in mice. The expression profiles of 5 SFPRs in liver tissue and FLSC14. 5 cells were detected by RT-PCR. ELSC14. 5 cells were infected with adenovirus SFRP3,and treated with dexamethasone and hepatic growth factor(HGF). Relative ALB-GLuc activity was measured at the 3rd, 6th,9th and 12th day after induction. RT-PCR and Western blot were used to detect the expression levels of hepatic related markers after induction for 12 days. PAS staining was performed to check glycogen storage function of induced hepatic cells. Results SFRP1 ,4 and 5 were uniformly expressed during liver development. However, SFRP2 and SFRP3 were only detected in F12. 5 through F16. 5 liver tissues and decreased quickly. SFRP2 had high expression level while SFRP3 had very low expression level in FLSC14. 5 cells. Differentiation of ELSC cells could be effectively induced in vitro by 0. 1 μmol/L dexamethasone+ 10 ng/mL HGF. As compared with control group,over-expression of SFRP3 diminished the expression of mature hepatic markers . ABB, CK18 , TAT, ApoB, and UGTIA , promoted the expression of hepatic stem markers , DLK and AFP , and inhibited glycogen storage function of induced hepatic cells. Conclusion SFRP3 is decreased during liver formation and inhibits the differentiation of embryonic liver stem cells in vitro , indicating that the down-regulation of SFRP3 may be required in liver development and cell differentiation.%目的 检测不同发育阶段的肝组织中Wnt信号拮抗因子分泌性卷曲相关蛋白(secreted frizzled-related protein,SFRP)的表达,并探讨SFRP3对胚胎肝干细胞体外成熟分化的影响.方法 分离胚胎12.5 d至出生后4周共9个阶段的小鼠肝组织,RT-PCR检测5种SFRPs在肝组织及胚胎肝干细胞14.5(embryonic liver stem cells,ELSC)中的表达水平.腺病毒SFRP3感染小鼠胚胎肝干细胞,地塞米松(dexamethasone,Dex)联合肝细胞生长因子(hepatic growth factor,HGF)诱导肝细胞分化,诱导后第3、6、9、12天检测白蛋白启动子荧光素酶报告基因(ablumin promoter-Gaussia Luciferase,ALB-Gluc)活性,诱导后第12天,RT-PCR和Western blot检测肝脏相关基因表达,PAS染色检测糖原合成功能.结果 SFRP1、4、5在不同阶段肝组织中的表达无明显差异,SFRP2和SFRP3只在胚胎期肝组织中表达,随后很快消失.ELSC14.5细胞中,SFRP2高表达而SFRP3表达很低.Dex联合HGF可有效诱导ELSC14.5细胞的体外成熟分化,与对照组相比,SFRP3过表达可降低成熟肝细胞标志白蛋白(albumin,ALB)、细胞角蛋白18(cytokeratin 18,CK18)、酪氨酸转氨酶(tyrosine aminotransferase,TAT)、载脂蛋白B(apolipoprotein B,ApoB)及尿苷二磷酸葡萄糖醛酸转移酶1A(UDP-glucuronosyl transferase,UGT1A)的水平,增强肝干细胞标志Delta蛋白(Delta-like protein,DLK)和甲胎蛋白(alpha fetoprotein,AFP)的表达,并抑制糖原合成功能.结论 SPRF3在肝脏发育过程中表达水平迅速降低,并显著抑制胚胎来源的肝干细胞体外成熟分化,其下调可能是肝脏发育和肝细胞成熟分化的一个重要因素.
展开▼
