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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4 mmol/L新霉素处理后,在不同浓度(0、25、100、200 ng/mL)Wnt3a的培养液中继续培养1 d,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4 mmol/L新霉素处理后,在含25 ng/mL Wnt3a的培养液中培养6 d,分别在空白对照组,50μmol/L DAPT、0.1%DMSO、50μmol/L DAPT+25 ng/mL Wnt3a培养液中继续培养7 d,共培养14 d。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25 ng/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。
OBJECTIVE: To investigate the effect of Wnt3a on the regeneration of oval vesicle cells by interacting with DMSO via Wnt3a activating classic WNT signaling in vitro. Methods Forty mice were randomly divided into 8 groups. In the first part of the experiment, the mouse oval sacs were treated with 4 mmol / L neomycin for 1 day in culture medium containing different concentrations of Wnt3a (0, 25, 100, 200 ng / mL) catenin, hair cell cilia and nucleus immunofluorescence staining. In the second part of the experiment, the mouse oval sacs were treated with 4 mmol / L neomycin and cultured in 25 ng / mL Wnt3a medium for 6 days. The cells were cultured in blank control, 50 μmol / L DAPT, 0.1% DMSO, 50μmol / L DAPT + 25 ng / mL Wnt3a medium for 7 days and cultured for 14 days. 10 μmol / L Brdu was added to all cultures. Brdu, Myocin7a and nuclei were immunofluorescently stained after incubation. Results In the first part of the experiment, β-catenin was found to be positively expressed in cytoplasm of supporting cells in 25 ng / mL Wnt3a group. The number of β-catenin positive cells was significantly different from other groups (P <0.01). The second part of the experiment: Myosin7a-positive cells can be seen in the DMSO group, a large number of Brdu-positive cells can be seen in the control group, Wnt3a + DAPT group and DATP group but no Myosin7a-positive cells, DMSO group Myosin7a positive cells with the other three groups The differences between groups were significant (P <0.01). Conclusion Sequential combination of Wnt3a and DMSO can activate the canonical WNT signaling pathway to induce intracytoplasmic cells to transdifferentiate into hair cell-like cells.