气管干细胞休眠及激活分子机制的研究

Commonly,adult stem cells remain dormant state in G0 phase and donot participate in proliferation. Under special condition or regulated by intrinsic factors, stem cells can be activated, recovery the ability to enter the cell cycle again, then proliferate and differentiate to specific cells. This can taken place in any phase of the body development as well as the processes of injury and recovery.But only a few stem cells can enter the cell cycle again and most cells exit the cell cycle after differentiation, the reason of which is still unknown. our previous studies fistly found that nomal tracheal stem cells remained dormant state, and transcriptional markers of stem cells (Oct3/4、Sox2 and Nanog) were negative. After 5Fu treatment, among the left G0 phase cells, Oct3/4、Sox2 and Nanog became positive, demonstrating the characteristics of activated stem cells. Our study intended to explore an effective method to distinguish activated cells from dormant stem cells, analyze the effect of the factors which keep cells in G0 phase, and reveal the mechanism of dormant stem cells in G0 phase entering into cell cycle again. Our study also explored the theory and technology of artificially regulating cells enter into the cell cycle, promoting the development of life sciences and regenerative medicine. The molecular mechanism of stem cells dormancy and activation has not been reported so far.

通常情况下，成体干细胞都处于休眠状态，停留在G0期，不参与增殖活动。在特殊环境或体内因子作用下，干细胞能被激活，重新进入细胞周期，完成细胞增殖，分化成特定组织需要细胞，此过程可发生在机体发育的各个阶段，以及损伤修复过程中。但是，为何大部分细胞随着分化一起退出细胞周期，而只有少数的干细胞可再次进入细胞周期，其机制至今尚不清楚。我们前期研究首次证明，稳态下，气管干细胞处于休眠状态，干细胞转录因子Oct3/4、Sox2、Nanog阴性，5Fu作用后，残存的G0期细胞出现Oct3/4、Sox2、Nanag阳性，呈现激活干细胞特征。本研究拟建立分辨率休眠及激活干细胞的可视化方法，明确G0期维持因子的作用，解析G0期维持因子调控干细胞休眠及再次进入细胞周期机理，探索人工调控进入细胞周期理论及技术，促进生命科学发展及在再生医学的应用。阐明气管干细胞休眠及激活分子机理研究国内外尚无报告。

气管干细胞休眠及激活分子机制的研究. .1 5-FU作用及无血清培养支气管HBE细胞株，G0期细胞被富集纯化，此时oct4、nanog、sox2一过性阳性，实验组加入增殖因子形成干细胞球，移植裸鼠形成肿物，形态学检查为畸胎瘤，提示干細胞富集纯化成功。..2 经DNA甲基化测序, 无处置对照组三者啟动子甲基化, 而实验组oct4,nanog变化不大， SOX2 DNA启动子由8.4% 降到4.8%，甲基化岛消失，去甲基化明确， SOX2基因激活，即DNA甲基化与基因沉默密切相关，非甲基化（unmethylated）的DNA区域一般与基因的活化（gene activation）相关。..3 DNA甲基转移酶DNMT1在Oct4DNA甲基化中起重要作用。在分化细胞含量升高。DNMT3a，DNMT3b在未分化干细胞中高表达，在分化细胞降低。..4 对照组气管上皮oct4阴性，polymeraseⅡ的 C 端 Ser5磷酸化阳性为分化细胞特点。oct4阳性，polymeraseⅡ的 C 端 Ser2磷酸化阴性时说明为休眠的干细胞。此时oct4阳性，polymeraseⅡ的 C 端 Ser2 磷酸化受抑制，ser2为阴性。β-catenin核内表达为干细胞激活。..5 5-Fu作用肺泡上皮，再生与Oct3/4关系密切。而支气管上上皮分化与Sox2关系密切。..6意义：a 用患者手朮后材料可避免异种排斥，b为干细胞修复气管肺提供新的实验及理论依据。c 通过研究为延长休眠期间达到延长患者寿命，或将癌干细胞赶出休眠期，将其杀灭提供战略性策略。