CX3CR1对人肝细胞癌及其肿瘤微环境的影响

发布时间：2019-02-24 09:13

【摘要】：目的:探索趋化因子受体CX3CR1对人肝细胞癌,以及通过调节巨噬细胞极化对肿瘤微环境的影响及其机制。方法:在体外实验中,用50ng/ml氟波脂(PMA)将人单核细胞THP-1诱导成巨噬细胞(M0),并用干扰RNA干扰M0细胞中的CX3CR1,然后通过RT-PCR的方法检测CX3CR1的表达水平。处理后的巨噬细胞表型通过RT-PCR检测M1和M2相关标记来确定,然后分别收集巨噬细胞M0和干扰CX3CR1后巨噬细胞的条件培养基,并将条件培养基加入到Hep G2和7721细胞中,通过流式、MTT、划痕、Transwell等试验方法观察肿瘤细胞的增殖、凋亡、迁移、侵袭等生物学效应的改变,同时用过表达质粒和干扰RNA,过表达7721细胞中的CX3CR1、抑制HepG2细胞中的CX3CR1的表达,通过MTT、流式、划痕、Transwell等方法检测肿瘤细胞增殖、迁移、侵袭等改变,最后通过Western blotting的方法检测肿瘤细胞中PI3K-AKT和ERK-MAPK相关信号通路的变化,初步探究CX3CR1发挥调节作用的机制。结果:在用干扰RNA处理诱导成功的巨噬细胞后,巨噬细胞中的CX3CR1的水平受到了明显的抑制,检测巨噬细胞相关指标发现M1型巨噬细胞相关标记升高,M2型相关标记下降,将收集的条件培养基加入Hep G2和7721细胞中观察到,HepG2和7721细胞的增殖、迁移、侵袭能力受到明显抑制,凋亡增加,过表达CX3CR1促进了7721细胞的增殖、迁移和侵袭能力,干扰CX3CR1抑制了HepG2细胞的增殖、迁移和侵袭能了。结论:抑制了CX3CR1的表达后,可促进巨噬细胞向M1型极化,并且抑制了HepG2和7721细胞的增殖、迁移、侵袭,促进了肿瘤细胞的凋亡,过表达CX3CR1促进了7721细胞的增殖、迁移和侵袭能力,干扰CX3CR1抑制了HepG2细胞的增殖、迁移和侵袭能了。
[Abstract]:Aim: to investigate the effects of chemokine receptor (CX3CR1) on human hepatocellular carcinoma (HCC) and the effect of macrophage polarization on tumor microenvironment. Methods: in vitro, human monocyte THP-1 was induced into macrophages (M0) by 50ng/ml fluroid (PMA), and CX3CR1, in M0 cells was interfered by RNA. Then the expression of CX3CR1 was detected by RT-PCR. The phenotypes of treated macrophages were determined by RT-PCR detection of M1 and M2 markers, then the macrophage M0 and conditioned medium interfering with CX3CR1 were collected, and the conditioned medium was added to Hep G2 and 7721 cells. The biological effects of proliferation, apoptosis, migration and invasion of tumor cells were observed by flow cytometry, MTT, scratch and Transwell. The expression plasmid and CX3CR1, in 7721 cells were also used to interfere with RNA, overexpression. The expression of CX3CR1 was inhibited in HepG2 cells. The changes of proliferation, migration and invasion of tumor cells were detected by MTT, flow, scratch and Transwell. Finally, the changes of PI3K-AKT and ERK-MAPK related signaling pathways in tumor cells were detected by Western blotting. To explore the mechanism of regulation of CX3CR1. Results: the level of CX3CR1 in macrophages induced by interfering RNA was significantly inhibited. The related markers of M 1 type macrophages were increased, while M 2 type related markers were decreased. It was observed that the proliferation, migration, invasion and apoptosis of HepG2 and 7721 cells were significantly inhibited and apoptosis increased. Overexpression of CX3CR1 promoted the proliferation, migration and invasion of 7721 cells. Interfering with CX3CR1 inhibits the proliferation, migration and invasion of HepG2 cells. Conclusion: inhibiting the expression of CX3CR1 can promote macrophage polarization to M1 type, inhibit the proliferation, migration and invasion of HepG2 and 7721 cells, promote the apoptosis of tumor cells, and over-express CX3CR1 can promote the proliferation of 7721 cells. The ability of migration and invasion inhibited the proliferation of HepG2 cells by interfering with CX3CR1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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