解剖学报

解剖学报 ›› 2022, Vol. 53 ›› Issue (4): 461-469.doi: 10.16098/j.issn.0529-1356.2022.04.009

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Wnt5b过表达对小鼠胚胎肝干细胞分化的影响及其对小鼠慢性肝损伤的修复作用

姜浩1 谢卓君1 张昊东1 周红1 罗庆1 康权1,2*    

  1. 1.重庆医科大学附属儿童医院普通外科创伤外科，儿童发育疾病研究教育部重点实验室，国家儿童健康与疾病临床医学研究中心，儿童发育重大疾病国家国际科技合作基地，儿科学重庆市重点实验室，重庆 400014;  2.重庆医科大学附属儿童医院儿科研究所，重庆 400014
  • 收稿日期:2021-09-27 修回日期:2021-12-07 出版日期:2022-08-06 发布日期:2022-09-11
  • 通讯作者: 康权 E-mail:564799351@qq.com
  • 基金资助:
    重庆市自然科学基金面上项目

Effect of Wnt5b overexpression on the differentiation of embryonic liver stem cells in mice and its repair effect on chronic liver injury in mice

JIANG  Hao1 XIE  Zhuo-jun1  ZHANG  Hao-dong1  ZHOU  Hong1  LUO  Qing1  KANG  Quan1,2*   

  1. 1. Department of General and Trauma Surgery, Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China; 2. Department of Pediatric Reasearch Institute, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China
  • Received:2021-09-27 Revised:2021-12-07 Online:2022-08-06 Published:2022-09-11
  • Contact: KANG Quan E-mail:564799351@qq.com

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摘要：

目的  探讨重组腺病毒介导的无翅型MMTV整合位点家族成员5b(Wnt5b）基因过表达对小鼠胚胎肝干细胞分化的影响及对小鼠慢性肝损伤的修复作用。  方法  将表达Wnt5b和绿色荧光蛋白(GFP) 的重组腺病毒，分别感染小鼠胚胎肝干细胞HP14-19，诱导其分化，采用Real-time PCR、Western blotting验证Wnt5b的表达，吲哚菁绿（ICG）摄取实验、高碘酸-希夫（PAS）染色检测HP14-19成肝分化能力，Real-time PCR检测肝细胞标志物白蛋白(Alb) 和细胞角蛋白18(CK18)的表达；以48只雄性BALB/c小鼠为实验对象，随机分为对照组、模型组、干细胞治疗组及Wnt5b修饰的干细胞治疗组，采用CCl4建立慢性肝损伤模型，干细胞治疗2周后取材，进行肝脏病理染色及免疫组织化学评估肝脏修复情况。   结果  Real-time PCR及Western blotting检测均显示，Wnt5b在HP14-19中成功过表达（P<0.001）；Real-time PCR检测Wnt5b组细胞Alb、CK18的mRNA表达较GFP组及对照组均升高（P<0.001）； ICG摄取实验、PAS染色提示，诱导7 d后Wnt5b组细胞ICG摄取能力及糖原合成能力均较GFP组显著增强（P<0.01）；肝脏HE染色表明，模型组肝损伤明显，肝脏组织结构紊乱，干细胞组肝脏组织结构部分恢复，Wnt5b组肝组织恢复优于干细胞组；Masson染色显示，模型组有明显的纤维增生，Wnt5b组蓝染纤维较干细胞组更少（P<0.05）；免疫组织化学显示Ⅰ型胶原蛋白（ColⅠ）、髓过氧化物酶（MPO）的表达在模型组明显升高，上述指标在Wnt5b组的表达水平较胎肝干细胞治疗组显著（P<0.05）。   结论  Wnt5b可诱导小鼠胎肝干细胞向成熟肝细胞样细胞分化，且此基因修饰的胎肝干细胞对慢性肝损伤小鼠肝脏的修复作用较胚胎肝干细胞更有优势。

关键词: 无翅型MMTV整合位点家族成员5b, 胎肝干细胞, 慢性肝损伤, 肝纤维化, 实时定量聚合酶链反应, 小鼠

Abstract:

Objective  To investigate the effect of wingless-type MMTV integration site family member 5b(Wnt5b) gene overexpression mediated by recombinant adenovirus on the differentiation of mouse embryonic liver stem cells and repair of chronic liver injury in mice.    Methods  Recombinant adenoviruses expressing Wnt5b and green fluorescent protein (GFP) were applied respectively to infect mouse fetal liver stem cells HP14-19, and induced its differentiation and verified the expression of Wnt5b through Real-time PCR and Western blotting. It also applied indocyanine grean(ICG) uptake experiment and periodic acid-schiff(PAS) staining to detect the differentiation ability of HP14-19 into hepatocyte-like cells. The Real-time PCR was chosen to detect hepatocyte markers albumin (Alb) and cytokeratin 18 (CK18) expression. Forty-eight experimental male BALB/c mice were randomly divided into control group, model group, stem cell treatment group and Wnt5b modified stem cell treatment group. The carbon tetrachloride(CCl4) was selected to establish a chronic liver injury model. After 2 weeks of stem cell treatment, liver pathological staining and immunohistochemistry were used to evaluate liver repair.  Results  Both Real-time PCR and Western blotting detection showed that Wnt5b was successfully overexpressed in HP14-19（P<0.001）. The mRNA expressions of Alb and CK18 in the Wnt5b group were higher than those in the GFP group and control group by Real-time PCR（P<0.001）. ICG uptake test and PAS staining showed that the ICG uptake capacity and glycogen synthesis capacity of Wnt5b group increased significantly compared with the GFP group after 7 days of induction（P<0.01）. Liver HE staining result  showed that the model group had an obvious liver injury and liver tissue structure disordered. The stem cell group liver tissue structure partially recovered, and the liver tissue recovery in the Wnt5b group was better than that in the stem cell group. Masson staining showed that the model group had obvious fiber hyperplasia, and the Wnt5b group had less blue-stained fiber than the stem cell group（P<0.05）. The immunohistochemistry showed collagen type Ⅰ(ColⅠ) and myeloperoxidase (MPO) increased significantly in the  model group, and the repair effects of these indexes were significant in the Wnt5b group than in the fetal stem cell group（P<0.05）.    Conclusion  Wnt5b can induce mouse fetal liver stem cells to differentiate into mature hepatocyte-like cells. Also, the genetically modified fetal liver stem cells have more advantages in repairing the liver of chronic liver injury mice than fetal liver stem cells.

Key words: Wingless-type MMTV integration site family member 5b, Fetal liver stem cell, Chronic liver injury, Liver fibrosis, Real-time PCR, Mouse

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