小鼠原代气道上皮基底细胞分离、增殖与气液界面培养诱导的定向分化

Isolation and proliferation of primary airway epithelial basal cells in mice,and directed differentiation induced by ALI culture

目的探究小鼠原代气道上皮基底细胞分离、增殖和气液界面(ALI)培养诱导定向分化的方法。方法无菌获取小鼠气管,改良酶消化法分离基底细胞,免疫荧光鉴定。细胞传代,倒置显微镜观察细胞增殖生长,免疫荧光检测KRT5表达水平。气液界面(ALI)培养,HE染色、扫描电镜观察定向分化情况。结果改良酶消化法及增殖培养基培养,可分离基底细胞,免疫荧光检测KRT5阳性。基底细胞增殖效果好,已传至第八代(P8),但P5以后,细胞老化增多,免疫荧光强度变弱。ALI培养诱导的定向分化,HE染色观察P1~P3细胞层多、且高,P1~P4游离面可见纤毛;扫描电镜观察P1~P4细胞游离面特化结构数量多,P5~P8细胞游离面特化结构少。结论该方法可分离纯度、活性均高的基底细胞,并可传代增殖培养,P4之前基底细胞增殖和定向分化效果好,为进一步研究体外气道重塑相关实验提供了细胞支撑。

Objective To explore the methods of isolation,proliferation and air-liquid interface(ALI)culture fordirected differentiation of primary mouse airway epithelial basal cells.Methods Mouse trachea were obtained aseptically,and basal cells were isolated by modified enzyme digestionand identified by immunofluorescence.The cells were passaged and observed by inverted microscope for proliferation and growth,and the expression level of KRT5 was detected by immunofluorescence.The cells were cultured at ALI,and HE staining and scanning electron microscopy were used to observe the directed differentiation.Results Modified enzyme digestion method and proliferation medium culture could isolate basal cells with positive KRT5 tested by immunofluorescence.The basal cells had been passed to the eighth generation(P8),and after the fifth generation(P5),the aging of the cells increased,and the immunofluorescence intensity became weaker.In ALI culture,the HE staining results showed that the P1~P3 cells have multiple high layers,and the cilia could be seen on the free surface of P1~P4;Scanning electron microscope results indicated that the free surface of P1~P4 cells had many specialised structures,while those structures were not so many on the free surface of P5~P8 cells.Conclusion The methods above can isolate the basal cells with high purity and activity,and the proliferated cells before P4 has good proliferation and directional differentiation ability.This work provides the cellular support for the further study of the experiments related to airway remodelling in vitro.