摘要
目的了解小鼠胚胎肝脏发育的过程和体外干细胞向肝细胞定向分化与NF-κB表达的关系,探讨干细胞向肝细胞定向分化的分子机制。方法分离培养扩增小鼠原始生殖嵴细胞,向培养液中分别加入20μg/L的β-神经细胞生长因子（β-NGF）、0.5g/L新生鼠肝脏提取液和胎肝细胞上清液,体外诱导小鼠生殖嵴来源的胚胎干细胞（PGCs）向肝样细胞定向分化。观察细胞的形态学变化;免疫细胞荧光法检测肝样分化细胞内α1-抗胰蛋白酶（AAT）及NF-κB的表达,Real-Time PCR方法检测诱导分化过程中NF-κB mRNA的表达。免疫组化法检测胚胎发育11.5、14.5、18.5d的肝脏组织中NF-κB的表达。结果 PGCs培养液中加入β-NGF、新生鼠肝脏提取液和胎肝细胞上清液可见部分细胞变为圆形或卵圆形;诱导12d后,圆形或卵圆形细胞呈ATT阳性表达;在诱导分化过程中NF-κB的表达逐渐增强,伴随肝细胞的成熟而呈阳性表达并由胞质转入胞核。Real-Time PCR方法检测结果显示,在诱导分化过程中NF-κB的表达升高（t=50.229～75.344,P〈0.01）。在胎肝发育过程中随肝原细胞向肝成熟细胞转化,肝细胞胞质中NF-κB的表达逐渐增强,并向细胞核内转移。结论 NF-κB在PGCs体外向肝细胞分化和体内肝发生过程中变化一致。
Objective To study the development process of embryonic liver in mice,and the association of NF-κB expression with differentiation of mouse primordial germ cells（PGCs）into liver cells,and explore the molecular mechanism of differentiation of stem cells into hepatocytes. Methods The PGCs from the gonadal ridges of the mouse embryos were cultured and proliferated in vitro,20μg/Lβ-nerve growth factor（β-NGF）,0.5g/L newborn mouse liver extract and fetal hepatocyte supernatant were added into mouse embryonic stem cells,respectively,and the morphological changes of the mouse PGCs were observed.Expressions of alpha-1anti-trypsin（AAT）and NF-κB protein were detected using immunofluorescence.Employing Real-Time PCR,NF-κB mRNA expression was detected.Expression of NF-κB in the liver during 11.5-,14.5-and 18.5-day embryonic development was measured using immunohistochemistry method. Results After induction of 20μg/Lβ-NGF,0.5g/L newborn mouse liver extract and fetal liver cell supernatant,some PGCs became round or oval,in 12 dinduction,the cells showed positive ATT expression,which gradually enhanced in the process of induced differentiation,and with the mature of liver cells,the expression was brought from cytoplasm over nucleus.Real-Time PCR results showed that in the course of inducing differentiation,the expression of NF-κB elevated.In fetal liver development,with the transformation of hepatic progenitor cells into hepatic mature cells,the expression of NF-κB in hepatocyte cytoplasm gradually boosted and transferred to the nucleus. Conclusion The changes in the course of differentiation of NF-κB primordial germ cells into liver cells in vitro are in line with that in vivo.
出处
《青岛大学医学院学报》
CAS
2016年第4期379-381,385,共4页
Acta Academiae Medicinae Qingdao Universitatis
基金
山东省自然科学基金资助课题(ZR2012CM008)
