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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (11): 1-7    DOI: 10.13523/j.cb.2305043
研究报告     
层粘连蛋白γ2诱导小鼠肝细胞AML12细胞发生上皮-间质转化
陈辉*(),张继舜
首都医科大学附属北京朝阳医院消化内科 北京 100043
Laminin γ2 Mediates Epithelial to mesenchymal Transition in Hepatocytes AML12 Cell Line
CHEN Hui*(),ZHANG Ji-shun
Digestive Department, Beijing Chaoyang Hospital, Capital Medical University,Beijing 100043,China
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摘要： 目的: 观察层粘连蛋白γ2(laminin γ2,LAMC2)对小鼠肝细胞AML12分化的作用,探讨转化生长因子-β1(TGF-β1)信号通路对AML12细胞LAMC2表达的影响。方法: 利用小鼠LAMC2慢病毒过表达载体转染AML12细胞,通过实时荧光定量PCR(qPCR)观察LAMC2对AML12细胞分化的影响。利用TGF-β1和TGF-β1通路抑制剂(SB431542)刺激AML12细胞,明确TGF-β1信号通路对LAMC2表达的作用。最后利用小干扰RNA(siRNA)抑制AML12细胞LAMC2表达,通过qPCR和Western blot检测TGF-β1预处理AML12细胞后对其分化的影响。结果: 过表达LAMC2显著上调AML12细胞间质细胞相关标志物α-SMA和1型胶原蛋白(Col1a1)mRNA水平,下调成熟肝细胞标志物白蛋白(ALB)mRNA水平。TGF-β1成功诱导小鼠肝细胞AML12细胞发生上皮-间质转化。TGF-β1上调AML12细胞Tgfbr1和LAMC2的mRNA表达水平,并且与TGF-β1的作用时间呈正相关,差异具有统计学意义(P<0.05)。TGF-β1诱导AML12细胞发生上皮-间质转化和促进LAMC2表达的作用,皆可被TGF-β1通路抑制剂SB431542阻断。重要的是,抑制肝细胞LAMC2表达可有效阻断TGF-β1诱导AML12细胞上皮-间质转化。结论: LAMC2参与TGF-β1诱导小鼠肝细胞AML12细胞上皮-间质转化,抑制LAMC2阻断TGF-β1诱导上皮-间质转化。
关键词： 肝细胞;  转化生长因子-β1;  层粘连蛋白;  肝纤维化;  上皮-间质转化    
Abstract: Objective: To investigate the effect of TGF-β1 on laminin γ2 (LAMC2) expression in mouse normal hepatocyte lines AML12, and the role of LAMC2 on the differentiation of AML12 cells. Methods: Lentiviral vector of LAMC2 (Lent-LAMC2) was transfected with AML12 cells, Lenti-NC was used as the control group, the transfection efficiency was detected by Western blot, and the role of LAMC2 on the AML12 differentiation was detected by qPCR analysis. The AML12 cells were stimulated with TGF-β1 (5 ng/mL) from 6 h to 24 h, and the mRNA expression of TGF-β1 and LAMC2 was detected by real-time PCR (qPCR). AML12 cells were stimulated with TGF-β1 or TGF-β1 + TGF-β inhibitor (SB431542) for 24 h, and the mRNA of LAMC2 was detected by qPCR analysis. Finally, small interfering RNA (siRNA) was used to inhibit the expression of LAMC2 in AML12 cells, and the differentiation of AML12 cells was detected by Western blot followed by stimulation of TGF-β1 at dose of 5 ng/mL. Results: Compared with the Lenti-NC vector group, the protein level of LAMC2 was significantly increased in the LAMC2 overexpressed cells, the expressions of α-SMA and Col1a1 mRNA were up-regulated (all P<0.05), and ALB expressions were significantly down-regulated (P< 0.05). With the prolongation of the AML12 cell time of TGF-β1, the mRNA expression of LAMC2 gradually increased, which was positively correlated with the stimulation time of TGF-β1 (P<0.05). The transcription level of LAMC2 in TGF-β1+SB431542 group was significantly lower than that in TGF-β1 group (P<0.05). Importantly, compared with the siNC group, the siLAMC2 group can effectively block the effect of TGF-β1 on the upregulation of expression in AML12 cells α-SMA and Col1a1. Conclusion: Laminin γ2 overexpression induces mesenchymal-like cell changes in AML12 cells. TGF-β1 promotes the expression of LAMC2 in AML12 cells, and LAMC2 specific siRNAs block the fibrogenic effect of TGF-β1 on AML12 cells.
Key words: Hepatocyte    TGF-β1    Laminin    Liver fibrosis    Epithelial-mesenchymal transition (EMT)
收稿日期: 2023-05-28 出版日期: 2023-12-01
ZTFLH:  Q26  
通讯作者: *陈辉     E-mail: chenhui_0516@126.com
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陈辉, 张继舜. 层粘连蛋白γ2诱导小鼠肝细胞AML12细胞发生上皮-间质转化[J]. 中国生物工程杂志, 2023, 43(11): 1-7.

CHEN Hui, ZHANG Ji-shun. Laminin γ2 Mediates Epithelial to mesenchymal Transition in Hepatocytes AML12 Cell Line. China Biotechnology, 2023, 43(11): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2305043    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I11/1

图1  TGF-β1对肝细胞AML12分化和LAMC2表达的影响 A. Cell morphological phenotype was observed under an inverted phases-contrast microscope. Expression of albumin (red immunofluorescence) and α-SMA (green immunofluorescence) was estimated by immunofluorescence analysis. DAPI staining (blue immunofluorescence) represents cell nuclei B.Analysis of ALB, Acta2, Col1a1 and LAMC2 transcripts in AML12 cells (** P<0.01,*** P< 0.001, **** P<0.000 1)
图2  TGF-β1对肝细胞AML12细胞Tgfbr1和LAMC2表达的影响
图3  TGF-β信号通路抑制剂阻断肝细胞LAMC2的表达
图4  LAMC2对肝细胞AML12细胞上皮-间质转化的作用
图5  干扰LAMC2抑制TGF-β1对肝细胞上皮-间质转化的作用
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