天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (5): 449-453.doi: 10.11958/20190784

• 细胞与分子生物学 •    下一篇

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

李敏敏 1，李宽 1,2，孙昕 1,2，吴琦 1,2，陈怀永 1,2△   

  1. 基金项目：国家自然科学基金面上资助项目（31471121，81773394）；天津市科委基础项目（17JCYBJC24700，18ZXDBSY00150）；天津市卫 计委攻关项目（16KG163，16KG164） 作者单位：1天津医科大学海河临床学院（邮编300070）；2天津大学海河医院 作者简介：李敏敏（1992），女，硕士在读，主要从事肺损伤与修复机制研究 △通讯作者 E-mail：huaiyong.chen@foxmail.com
  • 收稿日期:2019-03-18 修回日期:2019-04-03 出版日期:2019-05-15 发布日期:2019-05-15
  • 通讯作者: 陈怀永 E-mail:7098326@qq.com
  • 基金资助:
    国家自然科学基金面上项目;天津市科委基础项目;天津市卫计委攻关项目

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

LI Min-min1, LI Kuan1,2, SUN Xin1,2, WU Qi1,2, CHEN Huai-yong1,2△   

  1. 1 Department of Basic Medicine, Haihe Clinical College of Tianjin Medical University, Tianjin 300070, China; 2 Haihe Hospital, Affiliated to Tianjin University △Corresponding Author E-mail: huaiyong.chen@foxmail.com
  • Received:2019-03-18 Revised:2019-04-03 Published:2019-05-15 Online:2019-05-15

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摘要： 李敏敏 1，李宽 1,2，孙昕 1,2，吴琦 1,2，陈怀永 摘要：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（MLg）混合培养。细胞 设0、4、20、100 nmol/L PF-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Rps6kb1、Club细胞标志物细胞色素氧化酶（Cyp2f2）、纤毛细胞标志物乙酰化微管 蛋白（Act）和叉头框转录因子J1（Fox j1），杯状细胞标志物氯化物通道钙活化家族成员3（Clca3）叉头框转录因子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、 100 nmol/L PF-4708671浓度组Club细胞克隆数量均较0 nmol/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Act、Fox j1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。

关键词: mTOR/S6K1信号通路, 气道干细胞, 细胞增殖, 细胞分化

Abstract: Abstract: 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, Fox j1, 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.

Key words: mTOR/S6K1, airway stem/progenitor cells, cell proliferation, cell differentiation