基于STAT3调控CyclinD1信号通路探讨化痰祛瘀法干预喉鳞癌细胞分化及增殖的作用机制

The previous study of the project team found that phlegm congealing and blood stasis syndrome is the most basic syndrome of laryngeal cancer, Chinese herb medicine of “resolving phlegm and eliminating stasis” method can inhibit the growth of laryngeal cancer cells. However the action pathway is still unknown. Recent findings have shown that signal transduction and activator of transcription 3 (STAT3) signaling pathway is closely related to the oncogenesis and development of laryngeal cancer. Laryngeal squamous cell carcinoma specimens in patients with phlegm congealing and blood stasis syndrome will be collected in this study, and laryngeal squamous cells will be extracted and cultured, meanwhile nude mouse model with phlegm congealing and blood stasis syndrome will be established, and then laryngeal squamous cell carcinoma will be generated on the nude mice model with phlegm congealing and blood stasis syndrome after injected with the cultured laryngeal squamous cells. The cultured laryngeal squamous cells and 100 nude mice with laryngeal squamous cell carcinoma will be randomly divided into blank control group, low dose and high dose of Chinese herb medicine of “resolving phlegm and eliminating stasis” method groups and the cisplatin group respectively, and the latter three groups will receive corresponding treatment. The expression of STAT3, p-STAT3 and Cyclin D1 in the laryngeal squamous cell carcinoma of patients, the cultured laryngeal squamous cells and the laryngeal squamous cell carcinoma of the nude mice will be detected by immunohistochemistry, Western Blot and RT-PCR technology; The flow cytometry technology, MTT assay and fluorescence microscopy will be performed for detecting and observing the cell cycle, proliferation and apoptosis of the cultured laryngeal squamous cells and the laryngeal squamous cell carcinoma of the nude mice. This study will try to explore the mechanism of Chinese herb medicine of “resolving phlegm and eliminating stasis” method for laryngeal carcinoma with STAT3 regulating Cyclin D1 signaling pathway, and it will provide scientific evidence for the Chinese herb medicine of “resolving phlegm and eliminating stasis” method in preventing and treating laryngeal cancer.

项目组前期研究发现“痰凝血瘀证”是喉癌的最基本证型，化痰祛瘀法中药能抑制喉癌细胞生长，但作用通路不明。目前研究认为STAT3信号通路与喉癌发生发展密切相关。本研究拟取痰凝血瘀证喉癌患者的喉癌组织，提取喉鳞癌细胞培养，并建立痰凝血瘀证裸小鼠动物模型。培养的喉癌细胞和100只种瘤后的裸小鼠均随机分为空白对照组、化痰祛瘀中药高、低剂量组和顺铂组，进行药物干预。采用免疫组化、Western Blot和RT-PCR检测STAT3、p-STAT3和Cyclin D1在人喉癌组织、培养的喉癌细胞和裸小鼠喉瘤中的表达；流式细胞术、MTT法和荧光显微镜检测和观察培养的喉癌细胞及裸小鼠喉瘤中的喉癌细胞周期、增殖和凋亡情况。尝试从STAT3调控Cyclin D1信号通路探讨化痰祛瘀法干预喉鳞癌细胞分化及增殖的作用机制，为化痰祛瘀法中药防治喉癌提供科学依据。

喉癌是头颈部常见恶性肿瘤之一，严重威胁人民的生命健康。项目组在过去20多年的研究中，发现并证实痰凝血瘀证是喉癌形成的最基本证型，是喉部鳞状细胞癌变的基础。化痰祛瘀法中药能抑制喉癌细胞生长，但作用通路不明。本研究收集痰凝血瘀证喉癌患者喉癌组织和安全边缘组织，免疫组化和RT-PCR分别检测 STAT3、p-STAT3 和 Cyclin D1蛋白及基因在人喉癌组织的表达。提取原代喉鳞癌细胞，培养喉鳞癌细胞、构建痰凝血瘀证喉癌异位移植瘤裸小鼠动物模型均随机分为空白对照组、化痰祛瘀中药高、低剂量组和顺铂组（顺铂组仅在动物实验），进行相应的药物干预，采用免疫组化、WB实验和RT-PCR检测STAT3、p-STAT3和CyclinD1在喉鳞癌细胞和裸小鼠喉瘤中的表达；流式细胞术、CCK8法和WB等实验检测和观察喉癌细胞株TU212和Hep-2的细胞周期、增殖和凋亡情况的变化。喉癌患者手术切除的肿瘤样本与安全边缘组织对比，STAT3、p-STAT3和CyclinD1蛋白表达均上升，差异具有统计学意义。临床喉癌组织培养的原代细胞，化痰祛瘀中药能抑制STAT3蛋白的表达，同时上调P27蛋白，对CyclinD1蛋白表达无影响，同时抑制喉癌原代细胞STAT3和CyclinD1mRNA的表达，但对P27mRNA表达无影响。通过体内实验，验证了化痰祛瘀中药能抑制STAT3、p-STAT3和CyclinD1蛋白的表达，延缓喉癌异位瘤的生长，且体内实验发现顺铂组具有明显的肾毒性，而化痰祛瘀中药组肾保护作用明显。体外细胞实验发现化痰祛瘀中药抑制喉癌细胞侵袭、迁移，调节喉癌细胞周期，促进喉癌细胞凋亡。本研究验证化痰祛瘀中药基于STAT3调控CyclinD1信号通路抑制喉鳞癌细胞增殖分化的作用机制，为化痰祛瘀法中药防治喉癌提供了科学依据，对推广中医药防治喉癌有重要意义。