TMTyP4光动力疗法诱导喉癌干细胞死亡中自噬应答/凋亡途径调控作用及机制研究

PDT is a specificity treatment for malignant tumor in recent years，however the death pathways of cancer stem cell and its mechanism after PDT is worth studying. On the basis of the previous experiments results which have indicated that CD133 + laryngeal cancer stem cells exhibit autophagy and apoptosis resistance after PDT , this project further is proposed by regulating cell autophagy to detect its related mechanism in the resistance of cancer stem cell in laryngeal cacinoma in the process of TMTyP4-mediated PDT .Applying TMTyP4 / PDT after using rapamycin to induce autophagy , the characteristic of CD133 + laryngeal cancer stem cells will be detected. The inhibition of autophagy by pharmacological inhibitors —chloroquine phosphateand and doting enzyme to silence the ATG7 gene by CRISP/Cas9. After the application of TMTyP4 / PDT in vitro experiments, we will detect autophagy related proteins expression, autophagy bubble formation by using electron microscopy and the changes of autophagy related upstream and downstream pathways Akt/mTOR and cell apoptosis . In vivo tests after autophagy has been inhibited the tumor growth and apoptosis will be confirmed . This topic would aid in the development of novel therapeutic approaches for CSC treatment and has clinical application prospects, which will provide a new direction for the treatment of laryngeal cancer.

PDT是近年来一种特异性的恶性肿瘤治疗方法，PDT后肿瘤干细胞的死亡途径及其机制是值得研究的热点问题。本项目在前期实验已经证实CD133+喉癌干细胞在经历PDT后出现细胞自噬、抵抗凋亡的基础上，进一步拟通过调控细胞自噬来检测其在CD l33+喉癌干细胞耐受TMTyP4/PDT治疗过程中的作用及相关机制。通过雷帕霉素诱导自噬后应用TMTyP4/PDT，检测CD133+喉癌干细胞干性的维持作用；通过抑制自噬（包括采用CRISP/Cas9基因定点酶切抑制ATG-7 的表达以及应用自噬抑制剂磷酸氯喹）来阻断CD133+喉癌干细胞自噬的发生，应用TMTyP4/PDT后体外实验检测自噬相关蛋白的表达、电镜检测自噬泡的形成，自噬相关上下游通路Akt/mTOR的变化及细胞凋亡的相关指标；动物体内试验证实自噬被抑制后肿瘤的生长及凋亡情况，本课题具有研究价值和临床应用前景，将为喉癌的治疗提供新的方向和方法。

PDT是近年来一种特异性的恶性肿瘤治疗方法，PDT后肿瘤干细胞的死亡途径及其机制是值得研究的热点问题。本实验通过调控细胞自噬来检测其在CD l33+喉癌干细胞耐受TMTyP4/PDT治疗过程中的作用及相关机制。1.本项目证实CD133+喉癌干细胞在经历PDT后出现细胞自噬、抵抗凋亡。2.进一步通过调控细胞自噬来检测其在CD l33+喉癌干细胞耐受TMTyP4/PDT治疗过程中的作用及相关机制。通过雷帕霉素（RAPA）诱导自噬后应用TMTyP4/PDT，发现RAPA+TMTyP4/PDT组CD l33+喉癌细胞的克隆形成能力强于对照组和TMTyP4/PDT组；RAPA+TMTyP4/PDT组CD l33+喉癌细胞发生较为明显的自噬，凋亡蛋白表达减少，LC3荧光蛋白表达增多。3.通过抑制自噬（包括采用抑制ATG-7 的表达以及应用自噬抑制剂磷酸氯喹CQ）来阻断CD133+喉癌干细胞自噬的发生。体外实验证实：CQ+PDT组可有效抑制细胞自噬，促进细胞凋亡。抑制ATG-7的表达后有效抑制细胞自噬，促进细胞凋亡。体内实验：抑制ATG-7的表达来阻断自噬可以有效提高PDT对荷瘤小鼠的治疗作用，可以有效诱导肿瘤细胞凋亡。4.本项目完成过程中发表论文9篇，其中SCI论文5篇，中文期刊4篇。（在投1篇SCI）论文。以此项目为基础申请省级课题2项，培养博士研究生2人。