条件培养基影响巨噬细胞极性改变的机制研究

发布时间：2019-05-11 14:29

【摘要】：巨噬细胞是肿瘤微环境中主要的细胞成分。肿瘤微环境中的巨噬细胞在微环境中细胞因子、分泌蛋白等影响下,由可以杀伤和吞噬肿瘤的M1型巨噬细胞,转化为促肿瘤的M2型巨噬细胞。通常认为,M1型巨噬细胞参与炎症反应和抗肿瘤免疫,而M2型巨噬细胞发挥抗炎和促肿瘤的作用。巨噬细胞极性的变化,受到微环境中细胞因子、趋化因子和细胞外基质的调控。本研究主要集中于肿瘤细胞分泌的细胞因子和生物小分子,探索它们在肿瘤微环境中对巨噬细胞的极化作用。首先构建了Src蛋白高表达的NIH3T3/Src细胞系,发现NIH3T3/Src细胞的培养上清可以诱导ANA-1细胞向M2型巨噬细胞极化。为了明确NIH3T3/Src细胞培养上清中诱导巨噬细胞极化的具体成分,利用细胞因子芯片检测了NIH3T3/Src田胞分泌的细胞因子,发现与对照组NIH3T3/p3.1细胞培养上清相比,IL-6、MCP-1、sTNFR, VEGF、KC、GM-CSF和axl等因子在NIH3T3/Src细胞培养上清中分泌水平升高。通过NF-κB的特异性抑制剂PDTC和JAK-STAT的特异性抑制剂AG490抑制NIH3T3/Src细胞NF-κB和JAK-STAT信号通路后,ELISA检测发现上清中IL-6和MCP-1等细胞因子受到不同程度的抑制。同时NIH3T3/Src细胞培养上清诱导巨噬细胞极化的能力明显降低。这些结果提示细胞因子IL-6在诱导巨噬细胞极化过程中起着关键作用。此外,在NIH3T3/Src细胞培养上清中还检测到乳酸(Lactic acid)分泌升高。当用α-氰基-4-羟基肉桂酸CHC抑制巨噬细胞表面的单羧酸转运蛋白MCT,使巨噬细胞对培养基中的乳酸摄入减少后,NIH3T3/Src细胞培养上清诱导巨噬细胞极化的能力降低。提示乳酸在诱导巨噬细胞极化中发挥重要作用。随着APCMin/+小鼠结直肠肿瘤的进展,小鼠结直肠组织间隙液中IL-6、IL-9和1L-10等因子分泌量维持高水平。在IL-6特异性敲除的小鼠IL-6tmlKopf中,当用AOM-DSS化学诱导小鼠发生结直肠肿瘤,与对照组AOM-DSS小鼠相比,IL-6tmlKopf-AOM-DSS小鼠的成瘤性明显受到抑制,提示IL-6在肿瘤形成过程中起重要作用。综上所述,IL-6、乳酸等因子在NIH3T3/Src田胞培养上清诱导巨噬细胞极化过程中起着重要作用。我们目前的研究提示了一个诱导巨噬细胞极化的可能的机制,同时也提示一个潜在的抑制巨噬细胞极化的途径,或许能成为一种新的有前途的癌症治疗方式。
[Abstract]:Macrophages are the main cellular components in tumor microenvironment. Under the influence of cytokines and secretory proteins in tumor microenvironment, macrophages in tumor microenvironment are transformed from M1 macrophages which can kill and devour tumors to M2 macrophages that promote tumor. It is generally believed that M1 type macrophages are involved in inflammatory reaction and anti-tumor immunity, while M2-type macrophages play an anti-inflammatory and tumor-promoting role. The changes of macrophage polarity are regulated by cytokines, chemokines and extracellular matrix in microenvironment. This study focused on cytokines and biomolecules secreted by tumor cells and explored their polarization to macrophages in tumor microenvironment. First, a NIH3T3/Src cell line with high expression of Src protein was constructed. It was found that the supernatant of NIH3T3/Src cells could induce the polarization of ANA-1 cells to M2 type macrophages. In order to clarify the specific components of macrophage polarization induced by NIH3T3/Src cell culture supernatant, the cytokines secreted by NIH3T3/Src field cells were detected by cytokine microarray. Compared with the control group, IL-6, was found to be IL-6,. The secretion level of MCP-1,sTNFR, VEGF,KC,GM-CSF and axl in the culture supernatant of NIH3T3/Src cells was increased. After inhibition of NF- 魏 B and JAK-STAT signaling pathway in NIH3T3/Src cells by PDTC, a specific inhibitor of NF- 魏 B, and AG490, a specific inhibitor of JAK-STAT, ELISA assay showed that IL-6 and MCP-1 in the supernatant were inhibited to varying degrees. At the same time, the ability of NIH3T3/Src cell culture supernatant to induce macrophage polarization was significantly decreased. These results suggest that cytokine IL-6 plays a key role in inducing macrophage polarization. In addition, the secretion of lactic acid (Lactic acid) was also detected in the supernatant of NIH3T3/Src cells. When 伪-cyano-4-hydroxycinnamic acid CHC was used to inhibit the monocarboxylic acid transporter MCT, on the surface of macrophages, the ability of macrophages to induce polarization of macrophages was decreased after macrophage intake of lactic acid in culture medium was reduced by 伪-cyano-4-hydroxycinnamic acid MCT,. It is suggested that lactic acid plays an important role in inducing macrophage polarization. With the development of colorectal tumors in APCMin/ mice, the secretion of IL-6,IL-9 and 1L-10 in colorectal tissue space fluid of mice maintained a high level. In the IL-6tmlKopf of IL-6-specific knockout mice, the tumorigenicity of IL-6tmlKopf-AOM-DSS mice was significantly inhibited when the mice were induced by AOM-DSS chemistry to develop colorectal tumor, compared with the control group of AOM-DSS mice, the tumorigenicity of IL-6tmlKopf-AOM-DSS mice was significantly inhibited. It is suggested that IL-6 plays an important role in the process of tumor formation. In conclusion, IL-6, lactic acid and other factors play an important role in the polarization of macrophages induced by NIH3T3/Src field cell culture. Our current research suggests a possible mechanism for inducing macrophage polarization, as well as a potential way to inhibit macrophage polarization, which may be a new promising treatment for cancer.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-3

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