解剖学报

解剖学报 ›› 2022, Vol. 53 ›› Issue (5): 585-593.doi: 10.16098/j.issn.0529-1356.2022.05.007

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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

何昀1* 王银光2
  

  1. 1.重庆医科大学附属儿童医院胃肠新生儿外科，国家儿童健康与疾病临床医学研究中心，儿童发育疾病研究教育部重点实验室，儿科学重庆市重点实验室，重庆 400014;2.重庆市中医院肛肠科，重庆 400021
  • 收稿日期:2021-02-07 修回日期:2021-05-24 出版日期:2022-10-06 发布日期:2022-10-06
  • 通讯作者: 何昀 E-mail:dr_yunhe@hospital.cqmu.edu.cn
  • 基金资助:
    重庆市科委基础研究与前沿探索项目

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

HE  Yun1*  WANG  Yin-guang2   

  1. 1.Department of Pediatric Surgery of the Children’s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China; 2.Department of Anorecterology of Chongqing Traditional Chinese Medicine Hospital, Chongqing 400021,China
  • Received:2021-02-07 Revised:2021-05-24 Online:2022-10-06 Published:2022-10-06
  • Contact: HE Yun E-mail:dr_yunhe@hospital.cqmu.edu.cn

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摘要：

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。

关键词: 全反式维A酸, 骨形态发生蛋白9, 肝祖细胞, 高碘酸-希夫染色, 吲哚菁绿摄取, 小鼠

Abstract:

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.

Key words: All-trans retinoic acid, Bone morphogenetic protein 9, Hepatic progenitor cell, Periodic acid-Schiff staining, Indocyanine green , uptake, Mouse

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