Wnt/β-catenin信号通路对斑马鱼侧线毛细胞再生影响的实验研究

Role of Wnt/β-catenin signaling in zebrafish lateral line hair cell regeneration

目的探讨Wnt/β-catenin信号通路对斑马鱼侧线毛细胞再生影响。方法利用模式动物斑马鱼侧线毛细胞可再生的优势,通过流式细胞分选助细胞类群、荧光定量PCR、整胚原位杂交以及外源添加Wnt/β-catenin信号通路小分子化合物抑制剂和增强剂的方法,观察顺铂诱导毛细胞损伤过程中Wnt/β-catenin信号通路各因子的时空表达情况及毛细胞再生情况变化,为探究经典信号因子Wnt/β-catenin信号通路对毛细胞再生的影响提供在体实验依据。结果反转录PCR(reverse transcription PCR,RTPCR)与定量PCR(quantitative PCR,Q-PCR)结果显示,与对照组相比,顺铂处理组Wnt/β-catenin信号通路配体wnt2,wnt3a和关键因子ctnnb1(beta-catenin)在助细胞类群中表达量升高(P<0.05)。整胚原位杂交结果表明Wnt/β-catenin信号通路拮抗因子dkk1a和dkk2在助细胞类群表达量下降。外源添加抑制剂,低浓度连续浸泡48小时再生毛细胞数目可达到正常组40%左右,高浓度连续浸泡48小时毛细胞再生数目仅为正常组10%左右。而在毛细胞再生前12小时时间窗添加抑制剂也可观察到毛细胞再生数目减少(P<0.0001)。外源添加增强剂,毛细胞再生数目相较对照组无显著变化(P>0.05)。结论通过分选助细胞各因子表达变化、整胚原位杂交显示时空图谱以及外源添加抑制剂与增强剂,得出结论Wnt/β-catenin信号对斑马鱼侧线毛细胞再生是必需非充分条件。

OBJECTIVE To explore the role of Wnt/β-catenin signaling in hair cell regeneration using zebrafish lateral line system. METHODS Zebrafish larval were incubated in cisplatin solution to induce lateral line hair cells loss and then regenerated for 48 hrs. Supporting cells were sorted, and QPCR was taken to detect the expression change of Wnt/β-catenin signaling factors. Whole mount in situ hybridization was used to show the expression pattern of dkks. Wnt/β-catenin signalinginhibitors（FH535 and XAV939） and enhancer（BIO） was added to the medium to observe the influences on hair cell regeneration. RESULTS RT-PCR and Q-PCR showed that the expressions of wnt2, wnt3 a and ctnnbl in sorting supporting cells were elevated（P〈0.05）. Whole mount in situ hybridization showed that the expression of dkkla and dkk2 in lateral lines sub-supporting cellsreduced. The addition of Wnt/β-catenin signaling inhibitors reduced the regenerated hair cells to40% of normal, and even to 10% when the concentration of inhibitor was elevated.And the first 12 hrs Wnt/β-catenin signaling inhibition also led to the reduced regenerated hair cells（P〈0.001）. However,regeneratedhair cells have no significant change compared between BIO-treated and nontreated group（P〈0.05）. CONCLUSION Wnt/β-catenin signaling is necessary whilenot sufficient for zebrafish lateral line hair cell regeneration.