基于TREM2/JAK-STAT信号通路探讨消痰散结方治疗甲状腺癌的机制研究

New cognition to the strategy of combined treatment of traditional Chinese medicine and western medicine will be generated by explaining the classic theory of Chinese medicine based on disease mechanism of western medicine. The phlegm syndrome is a classic theory of traditional Chinese medical science, especially for thyroid tumor. Based on previous studies we found: triggering receptor expressed on myeloid cells 2 (TREM2) was overexpressed in thyroid carcinoma tissue and indicated poor prognosis; overexpression of TREM2 was positively correlated with the activation of JAK-STAT signaling pathway; "Xiao Tan San Jie Decoction" could significantly inhibit TREM2 expression in the tumor tissue and induce apoptosis of tumor cells. According to these results, we expect whether there are some relations between "phlegm" in Chinese medical theory and TREM2 overexpression, and whether "phlegm-expelling" and blockage of JAK-STAT signal pathway have different approaches but equally satisfactory results. Based on the previous clinical and experimental studies, we attempt to carry out further researches: ① observing the impact of serum containing "Xiao Tan San Jie Decoction" on proliferation and apoptosis of overexpression TREM2 cells; ② comparing expressions of genes and proteins related to cell proliferation and apoptosis in subcutaneous tumor mass between nude mices received intragastric administration of decoction and the ones received JAK-STAT antagonists abdominal injection; ③ selecting candidate molecules of downstream of TREM2 signal pathway which are influenced by both decoction and TREM2 interference. By these further researches, we attempt to verify the equivalent effect between "phlegm-expelling" and interference of TREM2/JAK-STAT signal pathway and to explore the downstream molecular mechanism of "Xiao Tan San Jie Decoction" targeting to TREM2 by gene set enrichment analysis, in order to provide new ideas for Chinese and Western medicine in the treatment of thyroid cancer.

以西医致病机理解释中医经典理论可为中西医结合治疗策略带来新的认知。痰证是中医治疗甲状腺肿瘤的经典理论，我们前期研究发现：髓样细胞触发受体2（TREM2）在甲状腺癌中高表达并提示预后不良；TREM2过表达与JAK-STAT信号通路的激活正相关；消痰散结方可显著抑制肿瘤组织中TREM2的表达并诱导肿瘤细胞凋亡。故假设中医之“痰”与TREM2过表达相映射，“祛除痰浊”与阻断TREM2/JAK-STAT通路异曲同工。课题拟通过：①观察方剂含药血清对TREM2过表达细胞株增殖和凋亡的影响；②对比方剂灌胃和予JAK-STAT通路抑制剂干预的裸鼠皮下瘤荷中细胞凋亡、增殖相关基因和蛋白的变化；③通过基因富集分析筛选TREM2干扰和消痰散结方共同影响的下游信号通路的候选分子，验证祛痰与TREM2/JAK-STAT干扰的等效作用，探索消痰散结方通过靶向TREM2的下游分子机制，为中西医治疗甲状腺癌提供新思路。

甲状腺癌是内分泌系统最常见的恶性肿瘤，手术是其主要治疗手段。但术后较高的复发率及颈部遗留的手术疤痕会给患者带来困扰。中西医结合为甲状腺癌治疗提供了新思路。结合中医治疗甲状腺肿瘤的经典理论“痰症”，我们研究消痰散结方的“祛除痰浊”与阻断TREM2/JAK-STAT通路的映射关系，从机制上探索消痰散结方治疗甲状腺癌的作用靶点。本研究：1、通过对甲状腺癌患者的癌组织及癌旁组织进行PCR及Western blot检测，我们发现癌组织TREM2在基因及蛋白水平表达量均高于癌旁组织，尤其是肿瘤直径≥1cm及伴有颈部淋巴结转移的患者，TREM2表达量显著增高；2、通过对甲状腺细胞株进行CCK8、流式细胞、Transwell实验以及对增殖、凋亡及侵袭信号分子的检测，我们发现TREM2可促进甲状腺癌细胞的增殖、侵袭，抑制凋亡，其机制与激活下游信号通路Jak/STAT相关；3、配制不同浓度的消痰散结方给大鼠灌胃，提取含药血清，通过对比不同浓度的含药血清对甲状腺癌TREM2表达的影响，我们发现中等浓度及高浓度的含药血清对TREM2基因及蛋白表达的抑制作用更显著；4、以中等浓度的含药血清处理干预甲状腺癌细胞株，并进行CCK8、流式细胞、Transwell实验以及对增殖、凋亡及侵袭信号分子的检测，我们发现含有消痰散结方的血清可有效抑制甲状腺癌细胞的增殖、侵袭并促进其凋亡，含药血清与Jak抑制剂AG490联合可进一步降低STAT3的表达。该部分内容通过体外实验证明了消痰散结方可能通过TREM2/Jak-STAT信号通路抑制甲状腺癌的发生发展；5、我们将甲状腺癌细胞株皮下注射于裸鼠腋窝建立甲状腺癌瘤体模型，通过消痰散结方灌胃治疗裸鼠2周后，处死裸鼠取材瘤体组织。通过绘制瘤体生长曲线及测量肿瘤直径，TUNEL凋亡实验及对增殖、凋亡及通路信号分子的检测，发现消痰散结方可有效抑制瘤体组织的生长，促进细胞凋亡，并可以显著下调Jak/STAT信号通路的关键因子pJak、pSTAT3表达量。该部分内容通过活体实验进一步证实了消痰散结方通过调控TREM2/Jak-STAT信号通路可有效抑制甲状腺癌的发生发展。以上研究结果与我们设想基本一致，实验目标基本达成。