全反式维A酸对骨形态发生蛋白9诱导小鼠肝祖细胞成熟分化的作用及机制

Effect of all-trans retinoic acid on maturation and differentiation of mouse hepatic progenitor cells induced by bone morphogenetic protein 9 and its mechanism

目的探讨全反式维A酸(ATRA)对骨形态发生蛋白9(BMP9)诱导肝祖细胞(HPCs)成熟分化的作用。方法BMP9、BMP9+1μmol/L ATRA及BMP9+10μmol/L ATRA分别作用于肝祖细胞14-19(HP14-19),荧光素酶报告基因检测清蛋白启动的荧光素酶(ALB-Glus)表达情况,Real-time PCR检测肝脏相关基因ALB、细胞角蛋白18(CK18)、酪氨酸转氨酶(TAT)、载脂蛋白B(ApoB)的mRNA表达水平,免疫荧光检测Alb、尿苷二磷酸葡萄糖醛酸转移酶1A(UGT1A)的表达、高磺酸-希夫(PAS)染色和吲哚菁绿(ICG)摄取实验检测肝细胞的代谢及糖原合成功能,Real-time PCR及Western blotting检测维A酸(RA)信号通路相关分子维A酸受体(RAR)α、RARβ、RARγ及BMP9信号相关分子Samd1、Samd5、Samd8的表达。Ad-siRARα、Ad-siRARβ、Ad-siRARγ感染细胞,BMP9+10μmol/L ATRA处理,观察细胞形态及PAS染色结果,检测ALB、CK18、TAT、ApoB的mRNA水平表达。结果BMP9可显著诱导HP14-19的成熟分化,细胞形态呈现多角形铺路石样,Alb、CK18、ApoB及UGT1A表达显著上调,部分细胞具有代谢解毒及糖原合成功能。BMP9+1μmol/L ATRA处理后,细胞形态更加成熟,体积增大,ALB、CK18、ApoB及UGT1A表达较BMP9组显著上调(P<0.05),ICG和PAS染色阳性细胞数增多,与之相比,BMP9+10μmol/L ATRA的细胞呈现长梭形、纺锤形及多角形等多种形态,肝细胞相关标志表达降低,ICG和PAS染色阳性细胞数减少。1μmol/L ATRA显著增高RARα、RARβ、RARγ的表达,10μmol/L ATRA组较1μmol/L ATRA组仅RARα表达增高,BMP9不影响Samd1、Samd5、Samd8的表达水平,但上调其磷酸化。Ad-siRARα可改善10μmol/L ATRA诱导的细胞形态及PAS染色,Alb、CK18的表达显著上调(P<0.05)。结论1μmol/L ATRA可促进BMP9诱导的肝祖细胞成熟分化,10μmol/L ATRA会导致细胞分化减弱,过量ATRA可能过度激活RARα信号,影响肝祖细胞分化。

Objective To investigate the effect of 1μmol/L and 10μmol/L all trans retinoic acid(ATRA)on bone morphogenetic protein 9(BMP9)-induced maturation and differentiation of hepatic progenitor cells.Methods BMP9,BMP9+1μmol/L ATRA and BMP9+10μmol/L ATRA acted on HP14-19,respectively.The expression of albumin-drive gussid(LAB-Glus)was detected by luciferase reporter gene.The mRNA levels of ALB,cytokeratin 18(CK18),tyrosine aminotransferase(TAT),apolipoprotein B(ApoB)were detected by Real-time PCR.The expressions of ALB and uridine diphosphate glucuronosyltransferase 1 A(UGT1 A)were detected by immunofluorescence.Periodic acid-schiff(PAS)staining and indocyanine green(ICG)uptake assay were used to detect the metabolism and glycogen synthesis of hepatocytes.Real-time PCR and Western blotting were used to detect the expression of retinoic acid receptor(RAR)α,RARβ、RARγand BMP9 signal related molecules Samd1,Samd 5 and Samd 8.Ad-siRARα、Ad-siRARβ、Ad-siRARγinfected cells were treated with BMP9+10μmol/L ATRA,the cell morphology and PAS staining result were observed,the mRNA levels of ALB,CK18,TAT and ApoB were detected by Real-time PCR.Results BMP9 could significantly induce the maturation and differentiation of HP14-19 cells.The morphology of HP14-19 cells looked like polygonal paving stone.The expressions of ALB,CK18,ApoB and UGT1 A were significantly up-regulated.Some cells had the function of metabolic detoxification and glycogen synthesis.Compared with the BMP9 group,BMP9+1μmol/L ATRA group had more mature morphology and larger volume.The expressions of Alb,CK18,ApoB and UGT1 A were up-regulated significantly(P<0.05).The number of ICG and PAS positive cells increased.Compared with the BMP9+1μmol/L ATRA group,BMP9+10μmol/L ATRA group showed long spindle,spindle and polygonal shapes,and the expression of hepatocyte related markers decreased,and the number of ICG and PAS positive cells decreased.ATRA(1μmol/L)significantly increased the expression of RARα,RARβand RARγ.Compared with the 1μmol/L ATRA group,10μmol/L ATRA group only increased the expression of RARα.BMP9 did not affect the expression levels of Samd1,Samd5 and Samd8,but up-regulated their phosphorylation.Ad-siRARαcould improve cell morphology and PAS staining induced by 10μmol/L ATRA,while increased the expression of Alb and CK18(P<0.05).Conclusion ATRA(1μmol/L)can promote the maturation and differentiation of hepatic progenitor cells(HPCs)induced by BMP9,while 10μmol/L ATRA can weaken the differentiation of hepatic progenitor cells.Excessive ATRA may over activate RARαsignal to affect the differentiation of hepatic progenitor cells.