转化生长因子β信号通路对小鼠肝大部分切除术后干细胞介导的肝再生的影响

Effect of transforming growth factor β signaling on the stem cell-mediated liver regeneration in mice after partial hepatectomy

目的·探讨抑制转化生长因子β(transforming growth factor β,TGF-β)通路对小鼠肝大部分切除术(partial hepatectomy,PH)后干细胞介导的肝再生过程的影响。方法·取18只C57BL/6雄性小鼠,建立小鼠肝大部分切除模型,监测术前(PH0)、术后第1日(PH1)、第3日(PH3)、第7日(PH7)的肝组织中TGF-β通路的信号分子,以及干细胞标志物甲胎蛋白(α-fetoprotein,AFP)和富含亮氨酸重复单位的G蛋白偶联受体5(leucine-rich repeat-containing G-protein coupled receptor 5,LGR5)的m RNA和蛋白表达情况。再取32只小鼠,分为抑制剂组[PH+SB-431542,10 mg/(kg·d)]和对照组(PH+生理盐水),并于术后第1日(PH1)和第3日(PH3)留取肝脏标本,使用荧光定量PCR、Western blotting以及免疫荧光染色检测TGF-β通路和干细胞标志物AFP、LGR5的变化情况。结果·肝大部分切除术后小鼠肝脏TGF-β1的m RNA和蛋白、磷酸化SMAD2(p-SMAD2)蛋白,以及Afp的mRNA和阳性细胞数量、Lgr5的m RNA水平均随时间推移逐渐上调,至PH3达到高峰(均P<0.05),到PH7时则恢复至术前水平。与对照组相比,抑制剂组小鼠PH3时的上述指标均被显著抑制(均P<0.05)。结论·TGF-β通路可能对小鼠肝大部分切除术后干细胞介导的肝再生起着一定的调控作用。

Objective · To explore the effect of inhibiting transforming growth factor β(TGF-β) signaling pathway on the stem cell-mediated liver regeneration in mice after partial hepatectomy (PH). Methods · Eighteen C57BL/6 male mice were selected to establish the model of hepatectomy. mRNA and protein levels of the signal molecules in TGF-β pathway, as well as stem cell markers α-fetoprotein (AFP) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) in the liver tissues were detected before (PH0) and on the 1st (PH1), 3rd (PH3) and 7th (PH7) day after operation. Then, additional 32 mice were assigned into inhibitor group [PH+SB-431542, 10 mg/(kg·d)] and control group (PH+normal saline). The mice were sacrificed on the 1st (PH1) and 3rd (PH3) day after operation and liver remnants were obtained. Realtime-PCR, Western blotting, and immunofluorescence staining were used to detect the expressing variation of TGF-β signaling pathway, AFP and LGR5. Results · The mRNA and protein expression of TGF-β1, phosphorylated SMAD2 (p-SMAD2) protein, Afp mRNA, the number of AFP immune-positive cells and Lgr5 mRNA were upregulated significantly after PH, peaked at PH3 (P<0.05) and recovered to pre-operative level at PH7. Compared with control group, the above indices were all obviously inhibited in inhibitor group at PH3 (P<0.05). Conclusion · TGF-β signaling pathway may regulate the stem cell-mediated liver regeneration in mice after PH.