基于代谢重编程探讨攻毒治法靶向肺癌干细胞的研究

Lung cancer might be a disease related to stem cells. HIF-mediated metabolic reprogramming can promote lung cancer stem cells acquiring the ability of invasion and metastasis. Referring to early research, we proposed the hypothesis that the metabolic reprogramming driving by HIF effector molecule conform to the molecular biological microscopic behavior of the resurgence of toxin and spread of cancer among lung cancer patients, and we designed this research. By using a sophisticated cell-sorting technique, which is known as Immunomagnetic beads, we selected SP cells among lung cancer A549 clone and analysed the characteristics of them. Realgar nanoparticles, which plays an important role in eliminating toxin therapeutics, was divided into different dosage groups comparing to control groups. By using glucose oxidase method , immunofluorescence technique, Real-time PCR, Western blot, ELISA in Vitro and vivo tests, we would measure the differences in glucose metabolism among different groups, observe the expression of metabolic reprogramming key factor HIF-1, gene C-Myc and P53, protein PI3K, Akt and mTOR, and compare the levels of relative enzyme, such as GLUT1, PDK1, LDH, et al. In order to evaluate the effect of realgar nanoparticles that transform metabolic reprogramming by targeting HIF effector molecule. Then, we would explore the effector mechanisms of lung cancer stem cell and metabolic reprogramming in the metastasis of lung cancer. Through this research, we hope to discover more targets in the process of metastasis of lung cancer, to analyse the targetting regulatory mechanism of the metabolic reprogramming of lung cancer stem cells at he microscopic scale by using eliminating toxin therapeutics, in order to provide more experimental and theoretical foundations for eliminating toxin therapeutics in prevention and treatment of lung cancer metastasis.

肺癌可能是一种干细胞疾病，HIF介导的代谢重编程可推动肺癌干细胞获得侵袭转移能力，结合前期研究基础，提出了HIF效应分子驱动肺癌干细胞代谢重编程是肺癌患者余毒复燃、癌毒走窜的分子生物学微观表现之一的假说，并设计了该课题。研究选择免疫磁珠法分选肺癌干细胞，体内外实验通过葡萄糖氧化酶法、免疫荧光技术、荧光定量PCR、Western Blot、酶联免疫法分别测定攻毒治法之纳米雄黄不同剂量组及对照组的糖代谢差异、代谢重编程网络关键因子HIF-1、基因c-Myc、P53、蛋白PI3K、Akt、mTOR的表达情况及GLUT1、PDK1、LDH等酶的水平，评价纳米雄黄靶向肺癌干细胞HIF效应分子驱变代谢重编程的作用，进而探究肺癌干细胞、代谢重编程在肺癌转移过程中的效应机制，挖掘作用靶点，从细胞、分子水平解析攻毒治法对肺癌干细胞代谢重编程的靶向调节机制，以期为攻毒治法防治肺癌转移提供实验依据及理论支持。

肺癌干细胞是肺癌发生发展的“根源”，其存在特殊的代谢状态，Warburg效应的发生可导致肺癌干细胞侵袭转移。而在整个代谢网络中，HIF介导的代谢重编程是维持肺癌干细胞自我更新的关键，可推动肺癌干细胞获得侵袭转移的能力。“毒聚”贯穿肺癌发生发展过程的始终，余毒复燃可导致肺癌浸润转移，且攻毒法治是肿瘤治疗的有效方法之一，前期研究发现其代表药物纳米雄黄可有效抑制肺癌A549细胞迁移、侵袭及HIF-1的表达。基于此，我们进行了该研究，观察探讨了纳米雄黄调控HIF效应分子消减Warburg效应、驱变肺癌干细胞代谢重编程，达到延缓肺癌进展和转移的目的。我们采用了体内外实验相结合的方式，体外实验培养肺癌A549细胞分选肺癌干细胞，应用葡萄糖氧化酶法检测纳米雄黄对肺癌干细胞糖代谢的影响；体内实验BALB/c-nu接种肺癌干细胞建立肺癌小鼠模型，应用血糖测定法观察纳米雄黄对肺癌小鼠糖代谢的影响；体内外实验均应用PCR检测纳米雄黄对代谢重编程网络相关基因c-Myc、P53、HIF-1表达的影响，应用Western Blot检测纳米雄黄对代谢重编程网络相关蛋白PI3K、Akt、mTOR、HIF-1表达的影响，应用酶联免疫法检测纳米雄黄对代谢重编程网络相关酶GLUT1、PDK1、PFK、PKM、PDH、LDH表达的影响。结果表明：纳米雄黄可降低肺癌干细胞及肺癌小鼠的糖代谢能力，且呈时间、剂量依懒性；可抑制代谢重编程相关分子HIF-1、c-Myc、PI3K、Akt、mTOR、GLUT1、PDK1、PFK、PKM、PDH、LDH的表达及上调P53的表达。研究结果提示，纳米雄黄可靶向调控HIF效应分子消减肺癌干细胞Warburg 效应，进而驱变代谢重编程防治肺癌细胞侵袭转移，验证了HIF效应分子驱动肺癌干细胞代谢重编程是肺癌患者余毒复燃、癌毒走窜的分子生物学微观表现之一的假说，为攻毒治法防治肺癌转移提供了实验依据及理论支持。