温下方干预肺癌微环境整合素β1介导的粘附耐药信号转导通路研究

Lung cancer microenvironment is the locus where adhesion drug resistance occurs; adhesion drug resistance is the main factor which leads to a less effective lung cancer therapy. Integrinβ1 is an important component of the microenvironment, it mediates the adhesion between the tumor cells in addition to adjusting many signal pathways and prohibiting the apoptosis of tumor cells in order to maintain the resistance phenotype of tumor cells. Earlier studies in vitro and in vivo have proved that the combination of Wenxia Formula with DDP has an obvious effect of enhancing sensitivity, attenuation and inducing apoptosis. On the basis of the former studies, we propose the opinion "Prohibiting over expression of the lung cancer microenvironment Integrinβ1, reducing the FAK activation and PI3K-Akt signal pathway, which are induced by Integrinβ1 are the scientific basis of Wenxia Formula's Intervention on adhesion drug resistance. .This study intends to establish a three-dimensional cultured cell model and Integrinβ1's gene over expression and immunodeficiency mice model, apply the methods of gene transfection, Q-RT-PCR, western blot, flow cytometry, etc, combine the wholesome regulation of traditional Chinese medicine on the basis of lung cancer microenvironment, study Wenxia Formula's regulating effects on adhesion drug resistance mediated by lung cancer microenvironment Integrinβ1 in vivo and in vitro. Meanwhile, we intend to study Wenxia Formula's intervention on the apoptosis resistance signal pathway mediated by Integrinβ1 by developing into cellular and molecular levels. This study will have great significance in revealing Wenxia Formula's mechanism of enhancing sensitivity, attenuation and inducing apoptosis, which will broaden a new horizon for the mechanism study of the traditional medicine's function of enhancing sensitivity.

肺癌微环境是发生粘附耐药的主要场所，粘附耐药是导致肺癌疗效降低的重要原因。整合素β1作为微环境重要组分，既介导肿瘤细胞间粘附，又调节多条信号通路抑制肿瘤细胞凋亡，以维持肿瘤细胞耐药表型。前期体内外研究表明，温下方与顺铂合用，具有明显的增敏减毒、诱导凋亡作用。据此提出"抑制肺癌微环境整合素β1高表达，下调其诱导的FAK激活和PI3K-Akt信号通路是温下方干预粘附耐药的科学基础"。本研究建立三维立体培养细胞模型和整合素β1基因过表达免疫缺陷小鼠模型，运用基因转染、Q-RT-PCR、蛋白印迹、流式细胞术等方法，基于肺癌微环境，结合中医整体调节的作用特点，从体内外角度，研究温下方对整合素β1介导肺癌微环境粘附耐药的调节作用。又深入到细胞与分子水平，研究温下方对整合素β1介导凋亡抵抗信号通路的干预作用。这对于揭示温下方增敏减毒，诱导凋亡的机制有重要意义，为中医中药增敏作用机制研究开拓新视野。

摘要：体外建立琼脂糖为基底的A549三维细胞培养模型，形态学观察显示细胞粘附在一起呈团块状生长，细胞团大小不一；细胞粘附率显著升高，对DDP的敏感性降低。温下方含药血清和DDP合用干预后，与单独DDP干预比较，对肿瘤细胞增殖的抑制率提高，细胞粘附率降低，凋亡率显著提高，提示温下方能抑制细胞粘附，诱导细胞凋亡。免疫细胞化学等检测显示温下方含药血清和DDP合用，能抑制Intergrin β1的表达，下调细胞周期蛋白P21的表达；Q-RT-PCR和WB检测结果显示，温下方含药血清和DDP合用能显著下调PI3K、FAK和Cyclin D1 mRNA和蛋白表达，下调Akt mRNA表达，上调GSK-3β和BAD mRNA和蛋白的表达；建立整合素β1高表达的转染细胞株A549/D6，裸鼠右腋下接种制备整合素β1高表达裸鼠移植瘤模型。温下方和DDP联合干预，小鼠体重显著高于DDP组，肿瘤体积增长速度缓慢，瘤重减小，提示温下方能增强DDP抑制肿瘤生长的效果。Q-RT-PCR和WB检测结果显示，温下方和DDP联合，能显著下调PI3K、Akt、Integrinβ1、FAK、CyclinD1 mRNA和蛋白表达，上调GSK-3β和BAD mRNA和蛋白表达量。提示温下方可能是通过抑制整合素β1的高表达，下调FAK和PI3K-Akt信号通路，增强肿瘤组织对DDP的敏感性，抑制肿瘤生长。