核糖体蛋白S6激酶1抑制剂对气道上皮干/祖细胞增殖及分化功能的影响

The effect of ribosomal protein S6 kinase 1 inhibitor on the proliferation and differentiation of airway epithelial stem/progenitor cells

目的研究mTOR信号通路下游元件核糖体蛋白S6激酶1(S6K1)抑制剂PF-4708671对气道干/祖细胞增殖与分化功能的影响。方法准备10只C57BL/6小鼠,采用流式细胞分选技术将气道干细胞(vClub)和气道祖细胞(Club)从小鼠气道分选出来;采用类器官培养技术分别把vClub细胞、Club细胞和成纤维细胞(M Lg)混合培养。细胞设0、 4、 20、 100nmol/LPF-4708671处理组,培养第8天时显微镜下观察克隆生长情况,统计克隆形成数量;第10天时荧光定量PCR检测S6K1激酶基因R ps6kb1、Club细胞标志物细胞色素氧化酶(Cyp2f)、纤2 毛细胞标志物乙酰化微管蛋白(Ac)和t 叉头框转录因子J1(Foxj),杯1 状细胞标志物氯化物通道钙活化家族成员3(Clca)叉3 头框转录因子a3(Foxa3)的mRNA表达情况。结果不同浓度的PF-4708671对vClub细胞克隆数量无明显影响(P> 0.05),而 4、 20、100nmol/LPF-4708671浓度组Club细胞克隆数量均较0nmol/L组减少(P< 0.05)。不同浓度的PF-4708671对vClub向Club细胞的分化无明显影响,其特征分子Cyp2f2在 mRNA表达水平差异方面无统计学意义。PF-4708671对Club细胞向纤毛细胞和杯状细胞的分化能力均无明显影响,4组间2种细胞的标志物A c、tFoxj1及 Clca3、Foxa3表达水平差异均无统计学意义(P> 0.05)。结论 mTOR/S6K1信号通路对小鼠气道干/祖细胞的增殖功能呈正调控作用,对其分化功能影响甚微。

Objective To evaluate the effect of PF-4708671, an inhibitor of mTOR downstream element ribosomal protein S6 kinase 1 (S6K1), on the proliferation and differentiation of mouse airway stem/progenitor cells. Methods A total of ten C57BL/6 mice were included in the present study. Mouse airway stem cells (vClub) and progenitor cells (Club) were isolated from lungs by fluorescent activated cell sorting. Organoid culture model was used to culture vClub cells, Club cells and supportive fibroblast MLg cells in transwells respectively. Stem/progenitor cells were maintained with PF-4708671 (0, 4, 20 and 100 nmol/L). Organoid cultures were imaged with inverted microscopy at day 8 after seeding. Stem cell-derived colonies were counted. Real-time PCR was conducted to analyze mRNA expression of S6K1 kinase gene Rps6kb1 , Club cell marker cytochrome P450 family 2 subfamily, polypeptide 2 ( Cyp2f2 ), ciliated cell marker acetylated tubulin ( Act ) and Forkhead box J1 ( Foxj1 ), goblet cell marker Chloride channel accessory 3 ( Clca3 ) and Forkhead box A3 ( Foxa3) at day 10 after seeding. Results The number of colonies generated by vClub cells showed no difference between the PF-4708671 treatment groups ( P >0.05). Also, numbers of colonies generated by Club cells were significantly decreased in PF-4708671 treatment groups (4, 20 and 100 nmol/L) compared with those of 0 nmol/L group ( P <0.05). Different concentrations of PF- 4708671 showed no significant effects on the differentiation of vClub into Club cells. There was no significant difference in mRNA expression level between characteristic molecular Cyp2f2 . PF-4708671 showed no significant effect on the differentiation ability of Club cells into ciliated cells or goblet cells. There were no significant differences in expression levels of ciliated cells Act , Foxj1 , Clca3 and Foxa3 between the four groups ( P >0.05). Conclusion mTOR/S6K1 signaling promotes the proliferation of mouse airway stem/progenitor cells, and has little effect on the differentiation.