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+1 μmol/L ATRA及BMP9+10 μmol/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+10μmol/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+1 μmol/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+10 μmol/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。1 μmol/L ATRA显著增高RARα、RARβ、RARγ的表达，10 μmol/L ATRA组较1 μmol/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10 μmol/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  1 μmol/L ATRA可促进BMP9诱导的肝祖细胞成熟分化，10 μmol/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 1 μmol/L and 10 μmol/L all trans retinoic acid (ATRA) on bone morphogenetic protein 9 (BMP9) - induced maturation and differentiation of hepatic progenitor cells.    Methods BMP9, BMP9 + 1 μmol/L ATRA and BMP9 + 10 μmol/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 1A(UGT1A) 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、RARγ and BMP9 signal related molecules Samd1, Samd 5 and Samd 8. Ad-siRARα、Ad-siRARβ、Ad-siRARγ infected cells were treated with BMP9+10 μmol/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 UGT1A were significantly up-regulated. Some cells had the function of metabolic detoxification and glycogen synthesis. Compared with the BMP9 group, BMP9+1 μmol/L ATRA group had more mature morphology and larger volume. The expressions of Alb, CK18, ApoB and UGT1A were up-regulated significantly (P<0.05). The number of ICG and PAS positive cells increased. Compared with the BMP9+1 μmol/L ATRA group, BMP9 + 10 μmol/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(1 μmol/L) significantly increased the expression of RARα, RARβ and RARγ. Compared with the 1 μmol/L ATRA group, 10 μmol/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-siRARα could improve cell morphology and PAS staining induced by 10 μmol/L ATRA, while increased the expression of Alb and CK18（P＜0.05）.  Conclusion ATRA（1 μmol/L） can promote the maturation and differentiation of hepatic progenitor cells(HPCs) induced by BMP9, while 10 μmol/L ATRA can weaken the differentiation of hepatic progenitor cells. Excessive ATRA may over activate RARα signal to affect the differentiation of hepatic progenitor cells.