Wnt通路对胰岛素样生长因子-1抑制小鼠毛细胞凋亡的作用

目的探讨胰岛素样生长因子-1(IGF-1)抑制小鼠毛细胞凋亡作用的调控机制。方法体外培养新生小鼠耳蜗基底膜。在分别含有正常对照培养液(A组)、0.5 mmol/L庆大霉素(GM)(B组)、0.5 mmol/L GM+1 ng/mL IGF(C组)、0.5 mmol/L GM+1 ng/mL IGF+5μg/mL wif(D组)、0.5 mmol/L GM+1 ng/mL IGF+10μg/mLwif(E组)、0.5 mmol/L GM+1 ng/mL IGF+15μg/mL wif(F组)的成分中培养24 h后收集细胞及固定。应用TUNEL法观察各组耳蜗毛细胞的凋亡情况。应用Western blot及Real-time PCR法观察Caspase-3的表达情况。结果 B组耳蜗毛细胞经刺激后,细胞凋亡数量及Caspase-3 mRNA和蛋白表达较A组明显上升(P<0.01),C组耳蜗毛细胞经IGF刺激后,细胞凋亡数量及Caspase-3mRNA和蛋白表达较B组明显下降(P<0.01),应用Wnt阻断剂(wif)后D、E及F组细胞凋亡数量及Caspase-3 mRNA和蛋白表达较C组升高,其中F组升高显著(P<0.01)。结论 IGF可能通过激活Wnt通路抑制毛细胞凋亡。

The effect of Wnt pathway on antiapoptosis activity by Insulin-like Growth Factor-1 in cochlear hair cells in rat

Purpose To investigate the gene regulation mechanism of Insulin-like Growth Factor-1(IGF-1) antiapoptosis activity on rat hair cells.Methods Basilar membrane of cochlea of newly born mice were cultured in vitro in six group: control culture solution(group A),0.5 mmol/L GM(gentamicin)(group B),0.5 mmol/L GM+1 ng/mL IGF-1(group C),0.5 mmol/L GM+1 ng/mL IGF-1+5 μg/mL wif(group D),0.5 mmol/L GM+1 ng/mL IGF-1+10 μg/mL wif(group E),0.5 mmol/L GM+1 ng/mL IGF-1+15 μg/mL wif(group F).After cultured,cochlear preparation were made by used TUNEL method,the apoptosis of cochlear hair cells was observed with laser scanning confocal microscope.Caspase-3 protein and m RNA was examed by western blot and real-time PCR.Results There was no apoptosis cell labeled by TUNEL in basilar membrane in group A.Apoptosis cells labeled by TUNEL in basal,in group B,was higher than that in group A(P<0.05).Apoptosis cells in group C was lower than that in group B(P<0.05).Apoptosis cells in group D,E,F were higher than that in group C,especialy in group F.The expression of Caspase-3 showed the same result.Conclusion Wntpathway plays an important role in the process of apoptosis in cochlear hair cells by IGF-1.