靶向性抑制非小细胞肺癌的新型人中性粒细胞防御素的设计合成及作用机制的研究

In spite of great advances in cancer therapy, there is considerable current interest in developing targeted cancer agents efficiently because of the development of resistance by cancer cells towards current anticancer drugs and the side-effects of chemotherapeutics. Our study previously had shown that new agents, which were the transformation of the amino acid sequence of human neutrophil defensins (HNP), exhibited a broad spectrum of cytotoxic activity against Nonsmall-cell lung cancer. However, it is unclear the mechanism . In this study, the HNP series peptide which is based on HNP sequences, increasing the CXCR4 motif and peptide ligands of antagonism transduction domain structure , can reveal the ability of more efficient and specifically targeted anticancer .The anti-tumor activity and the mechanism of action also can be researched in vitro and vivo. Cri Maestro In-vivo imaging Systems may be carried out to observe the distribution of the HNP series peptide in vivo. The influences of the HNP series peptide, which will effect on autophagy and apoptosis of tumor cells, can be clarified by Transmission electron microscopy (sem), laser confocal microscope, flow cytometry, immunofluorescence microscopy techniques and the Real - time PCR . We also analyze that different inhibitors (siRNA, early and late autophagy inhibitor) act on autophagic bodies. In conclusion , our study attempt to detailedly expound the mechanism of the HNP series peptide which effects on autophagy and apoptosis of tumor cells by experiments on the molecular , cellular and animal levels , and the HNP series peptide can serve as a new clue and a powerful theoretical basis of anticancer drugs for cancer treatment.

由于化疗药物存在副作用强和引发耐药性问题,故研发高效抗癌且有靶向性的药物是征服肺癌所面临的迫切要求。课题组前期研究发现：经序列改造的人中性粒细胞防御素（HNP）能很好抑制非小细胞肺癌细胞的生长,但机制尚不清楚。为提高HNP靶向性和抗癌性，本研究拟在HNP基础上增加CXCR4配体拮抗基序和肽转导结构域，并在此基础上探讨HNP系列肽的体内外抗癌活性及作用机制。为此本研究拟利用活体动物成像系统观察HNP系列肽在动物体内的分布情况；采用透射电镜、激光共聚焦显微镜、流式细胞术、免疫荧光显微技术、Real-time PCR等方法阐明HNP系列肽对细胞自噬及凋亡的影响；详细分析HNP系列肽联合不同抑制剂（siRNA、自噬早、晚期抑制剂）抑制自噬流后自噬及凋亡相关基因的变化情况。进而从分子、细胞、动物三个不同层面详细阐述HNP系列肽在诱导细胞自噬和凋亡过程中的作用机制，从而为研制高效抗癌药物提供理论依据。

由于化疗药物存在副作用强和引发耐药性等问题,故研发高效抗癌且有靶向性的药物是征服肺癌所面临的迫切要求。课题主持人在美国访学期间发现LL-37的衍生肽17BIPHE2在抗肿瘤活性、抑菌活性等方面均比本实验室研究的人中性粒细胞防御素HNP的活性高，因此在后期的研究过程中，采用17BIPHE2作为主要研究对象，通过研究发现：抗菌肽17BIPHE2可以抑制A549细胞增殖，人正常肺上皮BEAS-2B细胞不能被17BIPHE2抑制，说明17BIPHE2对肿瘤细胞有一定的靶向性。利用细胞迁移和侵袭实验以及流式细胞测定技术及MTT实验发现：抗菌肽17BIPHE2可通过抑制肺腺癌A549细胞迁移和侵袭，阻滞A549细胞的细胞周期，促进A549细胞凋亡，在体内外发挥抗肿瘤作用。流式细胞实验及WB测定抗菌肽17BIPHE2作用于A549细胞后凋亡相关蛋白等实验发现：抗菌肽17BIPHE2可降低肺腺癌 A549细胞内线粒体膜电位（△Ψm），使A549细胞内活性氧（ROS）、钙离子（Ca2+）升高，从而激活ERK，通过ERK调控凋亡相关蛋白Bax、Bcl-2的表达，调控A549细胞凋亡。移植瘤动物模型实验结果显示抗菌肽 17BIPHE2治疗后移植瘤体积明显缩小，体内实验表明，抗菌肽抑制移植瘤的作用途径是引起A549细胞凋亡。通过本研究阐述了17BIPHE2在诱导凋亡过程中的作用机制，从而为研制高效抗癌药物提供理论依据。