肺癌发病率逐年上升，严重威胁人类的健康。目前临床使用的化疗药物大多存在毒副作用强及耐受性差等问题。因此，寻找安全有效的治疗肺癌的天然药物至关重要。研究表明IL-35具有促进肿瘤生长及抑制肿瘤免疫调节等作用。我们前期研究发现三叶青黄酮明显抑制人非小细胞肺癌A549细胞的增殖和促进凋亡，并明显降低IL-35，且非小细胞肺癌患者外周血和癌组织中IL-35表达明显升高，IL-35中和抗体明显抑制肿瘤的生长。提示三叶青黄酮可通过负调控IL-35，抑制肿瘤生长及增强肿瘤免疫调节。本项目拟在前期工作基础上通过观察三叶青黄酮对A549细胞和IL-35基因敲除的A549细胞的增殖、凋亡等情况，深入探究三叶青黄酮对IL-35的负调控作用，并明确介导三叶青黄酮负调控IL-35抑制肿瘤细胞生长的靶向因子，深入研究其功能，阐明三叶青黄酮对抑制肿瘤细胞增殖及肿瘤微环境的调节作用，为提高肺癌治疗效果提供新思路新方法。

Lung cancer has been one of the most serious diseases threatening human health, because of the high morbidity. Currently, most chemotherapeutic agents have strong side effects and poor tolerance. Therefore, to find a safe and effective natural medicine for lung cancer is essential. Recent investigation determining the role of tumor-derived IL-35 on tumor growth and immunity has revealed novel functions of IL-35 in promoting tumor growth and inhibition of tumor immune regulation. We have previously shown that Radix Tetrastigma Hemsleyani Flavone (RTHF) exert anti-growth and apoptosis activity against lung cancer A549 cells in dose-dependent manner, and inhibit IL-35 activity. IL-35 of patients with lung cancer in peripheral blood and tissue is abnormal expression, and IL-35 neutralizing antibody significantly inhibited the growth of tumor. We think that RTHF may regulate the IL-35 and its signaling pathway, result in changing the expression of the key functional protein, inhibiting tumor growth and enhancing immune ability. Therefore, this project aims to observe the effect of RTHF on the wild type and IL-35 gene knock out cell of A549, and regulate the IL-35 and its related cell proliferation and immune function. Clarify RTHF regulate the key functional protein in the IL-35 signaling pathway, and study its function to improve that RTHF play an important role in the cancer cell proliferation and tumor micro environment, to provide the therapeutic effects new ideas and methods for lung cancer.

肺癌发病率逐年上升，严重威胁人类的健康。目前临床使用的化疗药物大多存在毒副作用强及耐受性差等问题。因此，寻找安全有效的治疗肺癌的天然药物至关重要。研究表明IL-35具有促进肿瘤生长及抑制肿瘤免疫调节等作用。我们前期研究发现三叶青黄酮明显抑制人非小细胞肺癌A549细胞的增殖和促进凋亡，并明显降低IL-35，提示三叶青黄酮可能通过调控IL-35，抑制肿瘤生长及增强肿瘤免疫调节。本研究主要观察三叶青黄酮处理IL-35 基因敲除的A549、A549、IL-35 高表达A549各组肿瘤细胞增殖、凋亡、侵袭，并通过microarray分析三叶青黄酮负调控IL-35的靶向因子并对其进行验证。结果显示：三叶青黄酮对A549 细胞、IL-35 基因敲除的A549 细胞、IL-35高表达的A549 细胞抑制率分别为25.37±1.02%、21.25±0.86%、33.57±1.41%；与对照组比较，P＜0.05，IL-35高表达的A549 细胞组抑制最明显。细胞凋亡率较对照组分别升高了42.35±1.56%、39.76±1.39%、51.29±2.73%；与对照组比较，P＜0.05，IL-35高表达的A549 细胞组凋亡最明显。细胞侵袭率分别下降了52.79±2.81%、41.29%±2.28、60.95±3.14%；与对照组比较，P＜0.05，IL-35高表达的A549 细胞组抑制侵袭最明显。microarray芯片总共分析了49294个基因；与对照组比较，有4368个基因上调，1672个基因下调（FC≥2,P＜0.01）；其中与肺癌密切相关的基因有Bax、TRAILR1、ERK、P27上调，Bcl-2、Survivin、Cyclin D1、CDK2、MMP-2、VEGF下调。采用Real Time PCR和Western blot 检测上述基因的mRNA 和蛋白表达水平，显示与对照组比较差异有统计学意义。本项目深入探究三叶青黄酮对IL-35的调控作用，并初步明确三叶青黄酮调控IL-35抑制肿瘤细胞生长的关键因子，阐明三叶青黄酮对抑制肿瘤细胞增殖及肿瘤微环境的调节作用，为提高肺癌治疗效果提供新思路新方法。