高粗榧碱诱导吉非替尼抗性非小细胞肺癌凋亡机理研究

发布时间：2018-03-10 03:27

本文选题：Gefitinib　切入点：非小细胞肺癌　出处：《清华大学》2016年博士论文　论文类型：学位论文

【摘要】：肺癌是目前全球罹患癌症患者中人数最多的癌症。肺癌分为两类:小细胞肺癌和(SCLC)非小细胞肺癌(NSCLC),其中NSCLC占到肺癌患者的80%。已有许多有效的药物如PD-1和EGFR激酶抑制剂Gefitinib(吉非替尼)和Erlotinib(厄洛替尼)等用于治疗肺癌患者,但是利用Gefitinib治疗一段时间(6~12个月)一般会产生耐药性,然而其中原因目前却知之甚少。EGFR的第790位苏氨酸突变为甲硫氨酸(T790M)和IL-6的异常表达是目前已知的原因。单一的抑制EGFR活性的治疗在NSCLC治疗中是不够的,而第二代EGFR TKIs效果有限。因此需要开发新的药物来最大程度的缓解治疗中的这种耐药性,而丰富的自然资源为我们挖掘开发新的治疗药物提供了巨大的资源库。高粗榧碱(HHT)是一种来源于三尖杉的化合物,我们对其在吉非替尼抗性非小细胞肺癌细胞中的作用和可能作用机理进行了研究。我们发现,HHT可以有效地抑制A549和H1975的体外增殖、细胞活力和软琼脂克隆形成的能力,但是这些抑制能力都未影响EGFR。实验中发现HHT诱导了线粒体途径的细胞凋亡,并且凋亡抑制蛋白MCL1和Survivin表达降低,而这两个蛋白基因的启动子区都有STAT3的结合区。进一步实验发现HHT通过抑制JAK1的活性抑制了STAT3的磷酸化激活。IL-6是赋予细胞耐药性一个重要的细胞因子。实验中发现,HHT以剂量和时间依赖的形式,可逆性地抑制IL-6诱导的STAT3激活。多西紫杉醇(DTX)是治疗NSCLC的二线药物,实验中我们发现联合使用这两种作用机制不同的药物可以更加有效地抑制非小细胞肺癌细胞的增殖而诱导凋亡。这也为临床治疗提供了新的治疗组合。此外,我们以H1975细胞异种移植瘤模型在体内环境下评估HHT治疗吉非替尼抗性非小细胞肺癌的效果。结果显示,与对照处理组和吉非替尼处理组可以更加有效地抑制肿瘤的生长,而并未显示出明显的副作用。在分子水平上,体内环境下HHT同样有效的抑制了STAT3的激活,而并未影响ERK和AKT的磷酸化,对肿瘤组织的组织免疫荧光实验进一步确认了上面的结果。以上结果证明了HHT在治疗Gefitinib抗性NSCLC方面展现出巨大的潜能。另外,为了改善抗肿瘤药物的传递方式,我们设计、合成并评估了一种星形纳米载药体系TAPP-PCL-b-TPGS,实现了高效载药、可控释放的同时,将化疗与光动力治疗联合起来,为DTX的传递提供了借鉴。
[Abstract]:Lung cancer is the most common cancer in the world. Lung cancer is divided into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in which NSCLC accounts for 80% of lung cancer patients. There are many effective drugs such as PD-1 and EGFR kinase inhibition. Gefitinib (gefitinib) and Erlotinib (erlotinib) are used to treat lung cancer patients. But treatment with Gefitinib for a period of 6 to 12 months generally produces drug resistance. However, the reasons for the abnormal expression of T790 threonine mutated to methionine (T790M) and IL-6 are known to be the reasons. The single therapy of inhibiting the activity of EGFR is not enough in the treatment of NSCLC. The second generation of EGFR TKIs has limited effectiveness, so new drugs need to be developed to maximize drug resistance in treatment. And the rich natural resources provide us with a huge pool of resources for the development of new therapeutic drugs. HHT is a compound derived from Sequoia sinensis. We have studied its role and possible mechanism in gifetini resistant non-small cell lung cancer cells. We found that HHT can effectively inhibit the proliferation, cell viability and the ability of soft Agar clone formation of A549 and H1975. However, none of these inhibition effects on EGFR. HHT induced apoptosis of mitochondria pathway, and the expression of MCL1 and Survivin decreased. Furthermore, it was found that HHT inhibits the phosphorylation of STAT3 by inhibiting the activity of JAK1. IL-6 is an important cytokine that endows cell resistance to drug resistance. Now HHT takes the form of dose and time dependence, Reversible inhibition of STAT3 activation induced by IL-6. Docetaxel is a second-line drug for the treatment of NSCLC. In our experiment, we found that the combination of these two drugs can inhibit the proliferation of non-small cell lung cancer cells and induce apoptosis more effectively. This also provides a new treatment combination for clinical treatment. We used H1975 cell xenograft tumor model in vivo to evaluate the efficacy of HHT in the treatment of gifetini resistant non-small cell lung cancer. The results showed that compared with the control group and the gifetini treatment group, the tumor growth was inhibited more effectively. At the molecular level, HHT also effectively inhibited the activation of STAT3, but did not affect the phosphorylation of ERK and AKT. The results of the tissue immunofluorescence assay on tumor tissue confirm the above results. These results demonstrate that HHT has great potential in treating Gefitinib resistant NSCLC. In addition, in order to improve the delivery of anti-tumor drugs, we designed, A star-shaped nano-drug carrier system TAPP-PCL-b-TPGSwas synthesized and evaluated. It realized high efficiency drug loading, controlled release and combined chemotherapy with photodynamic therapy, which provided a reference for the transmission of DTX.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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