痰瘀对喉鳞癌干细胞miRNA表达异常的作用及调控机制

Laryngeal carcinoma (LC) is one of the most common malignant tumors in the head and neck, the death and recurrence rate of which is still too high after surgery, radiation or chemotherapy treatment. so it's necessary to continue to explore new treating ways. This project use the newly discovered genes of miRNA and the research results on the theory of "hoarseness originated from the phlegm and blood stasis " lasting 20 years to explore the difference about the ability of proliferation and differentiation of Laryngeal Cancer Stem Cells (LCSC) in phlegm and blood stasis related syndromes, through finding the difference about change form and degree of miRNA in three different syndrome animal models, including blood stasis, phlegm stasis and phlegm-blood stasis.Through testing the expressing difference of miRNA originated from LCSC in tumors growing on the back of mice which accepted three different types of classical decoctions by eliminating phlegm, removing blood stasis, and eliminating phlegm and blood stasis, to explore the difference of effect in preventing and inhibiting LCSC proliferation and differentiation. Through the study, the genetic changes mechanism and traditional Chinese medicine (TCM) syndrome foundation of promoting LCSC proliferation and differentiation will become revealed, that will provide the theory basis for using targeted TCM therapy to treat laryngeal cancer. This will be an important exploration in organic combination of cancer stem cells technology, micro gene technology, head and neck oncology and syndrome differentiation and treatment, it's a chance for traditional Chinese medicine in otolaryngology to fly over in scientific research.

喉癌是头颈部常见恶性肿瘤之一，手术和放化疗后其死亡、复发率仍然很高，因此有必要继续探索新的防治方法。本项目从新近发现的微基因miRNA入手，结合过去20年在"痰瘀致瘖"领域的研究成果，试图通过检测其在血瘀证、痰凝证、痰瘀互结证这三个不同证候动物模型上的变化形式及程度的差异来了解喉癌干细胞在痰瘀相关的不同证型基础上的分化与增殖能力差异，并通过检测经化痰、祛瘀、化痰祛瘀这三种不同功效经方处理后的喉癌干细胞以及由其在喉癌动物模型上形成的癌瘤中miRNA的表达来了解不同中医治法对抗喉癌干细胞分化与增殖能力上的差异。通过研究，将明晰促进喉癌干细胞分化与增殖的基因变化机制及其证型基础，优化抑制喉癌干细胞分化和正常基因突变的中药经方，为临床运用中药靶向治疗喉癌提供理论依据，这将是肿瘤干细胞技术、微基因技术、头颈肿瘤学与中医辨证论治思想有机结合的一次重要探索，是中医喉科学研究技术与水平的一次历史性飞越。

目的：本项目从微基因miRNA入手，结合课题组过去20年在“痰瘀致瘖”领域的研究进展，为探究痰瘀相关证型对喉癌细胞增殖的作用和化痰祛瘀中药方剂抑制喉癌细胞增殖的效果及其相关的miRNA异常改变机制。方法：一是将喉癌细胞接种至血瘀证、痰凝证、痰瘀证裸小鼠皮下，观察瘤体生长变化，取瘤后行MTT实验、miRNA检测；二是将喉癌细胞接种到随机分组的裸小鼠皮下，分别予化痰（二陈汤）、祛瘀（会厌逐瘀汤）、化痰祛瘀（二陈汤合会厌逐瘀汤）三种中药方剂干预，观察瘤体变化和miRNA检测。结果：血瘀证组裸鼠在种瘤后第3周起瘤体积增长比对照组和痰凝证组、痰瘀证组较快，且生长曲线逐渐出现分散，而痰凝证组、痰瘀证组、对照组三组裸鼠瘤体生长速度未出现明显差异；种瘤后第28天，血瘀证组裸鼠瘤体积及离体瘤重量较对照组、痰凝证组、痰瘀证组大，但组间比较差异不具有统计学意义；MTT实验表明血瘀证、痰凝证、痰瘀证组裸鼠移植瘤中的喉癌细胞活力较对照组增强；痰瘀证使喉癌细胞miRNA发生异常表达，hsa-miR-708-5p，hsa-miR-19a-3p，hsa-miR-30c-5p，hsa-miR-22-3p，hsa-miR-23a-3p，hsa-miR-96-5p，hsa-miR-194-5p等表达下调，而hsa-miR-27a-5p表达上调；另一方面研究发现二陈汤在早期炎症期有促进炎症吸收、抑制瘤体生长的作用，其瘤体体积空白组比较差异具有统计学意义，而会厌逐瘀汤在早期并不能抑制肿瘤增长；会厌逐瘀汤、二陈汤合会厌逐瘀汤在中后期（种瘤后第3周）对肿瘤的增长有抑制作用，但差异无统计学意义；会厌逐瘀汤治疗组裸鼠瘤重与对照组比较差异有统计学意义，其余各治疗组瘤重与对照组比较差异不具有统计学意义；化痰祛瘀中药方剂可使hsa-miR-19a-3p、hsa-miR-30c-5p、hsa-miR-22-3p、hsa-miR-23a-3p等miRNA表达上调。结论：痰瘀有促进喉癌细胞增殖的作用，而其中血瘀证对裸鼠移植瘤的生长促进作用较对照组和其他证型组更为明显，痰瘀可造成喉癌细胞中相关miRNA异常表达，可能是痰瘀促进喉癌生长的基因学机制之一；化痰祛瘀中药方剂有抑制喉癌细胞miRNA异常改变的作用，可能为化痰祛瘀法抑制喉癌生长的基因学机制之一，为临床化痰祛瘀中药用于喉癌治疗提供了理论依据。