离体培养时Wnt3a对椭圆囊毛细胞再生的影响

目的 在小鼠椭圆囊体外培养模型上，通过Wnt3a激活经典WNT信号，与DMSO共同作用，研究其对椭圆囊毛细胞再生的影响。方法 40只小鼠随机分成8组。实验第一部分：小鼠椭圆囊经4mmol/L新霉素处理后，在不同浓度(0、25、100、200ng/mL)Wnt3a的培养液中继续培养1d，培养结束后对β-catenin，毛细胞纤毛及细胞核进行免疫荧光染色。实验第二部分：小鼠椭圆囊经4mmol/L新霉素处理后，在含25ng/mL Wnt3a的培养液中培养6d，分别在空白对照组,50μmol/L DAPT、0.1%DMSO、50μmol/L DAPT+25ng/mL Wnt3a培养液中继续培养7d，共培养14d。在所有培养过程中均加入10μmol/L Brdu。培养结束后Brdu，Myocin7a及细胞核进行免疫荧光染色。结果 实验第一部分：在25ng/mL Wnt3a组中可见β-catenin在支持细胞胞浆中有阳性表达，β-catenin阳性细胞数与其他各组比较差异具有统计学意义(P<0.01)。实验第二部分：DMSO组中可见Myosin7a染色阳性细胞，对照组、Wnt3a+DAPT组及DATP组中可见大量Brdu阳性细胞而未见Myosin7a染色阳性细胞，DMSO组中Myosin7a阳性细胞数与其他3组相比较，组间差异具有显著性(P<0.01)。结论 顺序联合应用Wnt3a和DMSO可通过激活经典WNT信号途径,促使椭圆囊内细胞向毛细胞样细胞转分化。

Effects of Wnt3a on utricle hair cells regeneration in vitro

Objective To investigate the effects of cranial WNT signal pathway combined with DMSO on regeneration of mouse utricle hair cells in vitro. Methods 40 mice were divided into 8 groups randomly. In 1st part: Utricles were treated with 4mmol/L neomycin, then cultured in culture medium with different concentration of Wnt3a (0, 25, 100, 200ng/mL) for 1day. After that, β-catenin, hair cell cilia and nuclei were labeled with immunofluorescence staining. In 2nd part: Utricles were treated with 4mmol/L neomycin, and then cultured in culture medium with 25ng/mL Wnt3a for 6day. After that, utricles were cultured in control and culture medium with 50μmol/L DAPT, 0.1%DMSO, 50μmol/L DAPT+25ng/mL Wnt3a respectively. 10μmol/L Brdu was added in culture medium in every phase in this part. After culturing, Brdu, Myocin7a and nuclei were labeled with immunofluorescence staining. Results In 1st part, β-catenin was expressed in support cell cytoplasm in 25ng/mL Wnt3a group, but not in 0, 100, 200ng/mL Wnt3a groups. β-catenin positive cells were calculated and compared with other groups. There was significant difference between 25ng/mL Wnt3a group and other groups (0, 100, 200ng/mL)(P<0.01). In 2nd part, Myosin7a positive cells were identified in DMSO group. There was no Myosin7a positive cells but Brdu positive cells in other groups. Myosin7a positive cells were calculated and compared with other groups. There was significant difference between DMSO group and the control, 50μmol/L DAPT, 50μmol/L DAPT+25ng/mL Wnt3a groups(P<0.01). Conclusion Wnt3a combined with DMSO promotes transdifferentiation to hair cell like cells in utricle by activating cranial WNT signal pathway.