外泌体携带整合素引导结直肠癌转移的机制及健脾消癌方干预作用研究

Distant metastasis is one of the main causes of death in patients with colorectal cancer. Previous studies have found that CRC metastasis is related to the intracellular TGF- beta /Smads signaling pathway, and its targeting liver, lung and other metastatic foci are related to the exosomes. It was shown that the tumor cells can be induced to specific organ by the exosomes based on the integrins on their surfaces. Therefore, we speculated that exosomes secreted by the CRC, can be uptook by the liver, lung respectively induced by different ITG, to create a pre-transfer micro environment, leading to distant metastasis. In this study, 2 kinds of exosomes will be sorted by ITG, and then injected into nude mice, then the amount of exosomes uptook by the liver and lung will be observed , and the expressions of S100A4, A8, A9,TGF- beta in the target organs will be determined. Besides, the effects of Jianpi Xiaoai Decoction on the release, delivery and target organ uptake of exosomes from CRC were studied by vivo and vitro experiments. The aim of this study is to clarify that the exosomes are extracellular signal carriers for CRC metastasis, and the mechanism of Jianpi Xiaoai Decoction to interfere with the CRC transfer through the influence of the exosomes, and to provide scientific basis of traditional Chinese medicine (TCM) to prevent and treat CRC metastasis.

远处转移是结直肠癌（Colorectal Cancer,CRC）患者死亡的主要原因之一。前期研究发现CRC转移与胞内TGF-β/Smads信号通路相关，其靶向肝、肺等转移灶与外泌体有关。研究显示外泌体可基于其表面整合素引导肿瘤细胞发生器官特异性转移。故我们推测CRC分泌的外泌体，可在不同整合素引导下分别被肝、肺摄取，营造转移前微环境，导致CRC远处转移。本项目根据整合素分选2种外泌体，标记后注入裸鼠，观察外泌体被肝、肺摄取及引导标记人结肠癌HCT116细胞转移的情况，测定靶器官S100A4、A8、A9及TGF-β表达，证实上述推测。并经体内、外实验研究健脾消癌方对CRC源外泌体释放、输送、靶器官摄取等的影响。阐明CRC源外泌体可基于整合素作为引导CRC器官特异性转移的细胞外信号载体，及健脾消癌方通过影响外泌体行为干预CRC转移的机制，为中医药防治CRC转移提供科学依据。

结直肠癌（CRC）的转移与肿瘤微环境相关，结肠癌细胞外泌体通过影响肿瘤转移微环境中的相关细胞和因子促进CRC的转移。本项目体外用健脾消癌方干预后的HCT116细胞和CT26细胞外泌体分别与HFL1细胞、巨噬细胞及小鼠肝Kuffer细胞共培养，采用Western Blot、RT-PCR检测肿瘤微环境中CAFs活化标志α-SMA、PDGFR-β和TGF-β1、TGF-β RII、Smad2/3等因子，证实健脾消癌方通过抑制 HFL1 向CAFs活化的能力，降低巨噬细胞中TGF-β RII、Smad2/3蛋白的表达水平及Smad2/3蛋白的磷酸化水平以及下调肝Kupffer细胞TGF-β1表达改善肿瘤微环境而抑制CRC的转移。采用尾静脉注射HCT116细胞外泌体+HCT116细胞制备结直肠癌肝转移裸鼠模型，健脾消癌方分组给药后比较观察各组肝肺转移瘤成瘤情况，采用免疫组化法检测肝转移瘤中TNF-a、IL-1β、IL-6、TGF-β1 及 IL-10 的表达情况。结果表明健脾消癌方能提高裸鼠体重、减少结直肠癌裸鼠模型肝转移瘤的发生，其机制与抑制下调TNF-a、IL-1β、IL-6、TGF-β1 及 IL-10等肿瘤微环境炎症相关因子有关。本项目揭示肿瘤来源外泌体与CRC转移的相关性及健脾消癌方干预CRC转移的分子机制。