【摘要】：巨噬細(xì)胞是腫瘤微環(huán)境中主要的細(xì)胞成分。腫瘤微環(huán)境中的巨噬細(xì)胞在微環(huán)境中細(xì)胞因子、分泌蛋白等影響下,由可以殺傷和吞噬腫瘤的M1型巨噬細(xì)胞,轉(zhuǎn)化為促腫瘤的M2型巨噬細(xì)胞。通常認(rèn)為,M1型巨噬細(xì)胞參與炎癥反應(yīng)和抗腫瘤免疫,而M2型巨噬細(xì)胞發(fā)揮抗炎和促腫瘤的作用。巨噬細(xì)胞極性的變化,受到微環(huán)境中細(xì)胞因子、趨化因子和細(xì)胞外基質(zhì)的調(diào)控。本研究主要集中于腫瘤細(xì)胞分泌的細(xì)胞因子和生物小分子,探索它們?cè)谀[瘤微環(huán)境中對(duì)巨噬細(xì)胞的極化作用。首先構(gòu)建了Src蛋白高表達(dá)的NIH3T3/Src細(xì)胞系,發(fā)現(xiàn)NIH3T3/Src細(xì)胞的培養(yǎng)上清可以誘導(dǎo)ANA-1細(xì)胞向M2型巨噬細(xì)胞極化。為了明確NIH3T3/Src細(xì)胞培養(yǎng)上清中誘導(dǎo)巨噬細(xì)胞極化的具體成分,利用細(xì)胞因子芯片檢測(cè)了NIH3T3/Src田胞分泌的細(xì)胞因子,發(fā)現(xiàn)與對(duì)照組NIH3T3/p3.1細(xì)胞培養(yǎng)上清相比,IL-6、MCP-1、sTNFR, VEGF、KC、GM-CSF和axl等因子在NIH3T3/Src細(xì)胞培養(yǎng)上清中分泌水平升高。通過NF-κB的特異性抑制劑PDTC和JAK-STAT的特異性抑制劑AG490抑制NIH3T3/Src細(xì)胞NF-κB和JAK-STAT信號(hào)通路后,ELISA檢測(cè)發(fā)現(xiàn)上清中IL-6和MCP-1等細(xì)胞因子受到不同程度的抑制。同時(shí)NIH3T3/Src細(xì)胞培養(yǎng)上清誘導(dǎo)巨噬細(xì)胞極化的能力明顯降低。這些結(jié)果提示細(xì)胞因子IL-6在誘導(dǎo)巨噬細(xì)胞極化過程中起著關(guān)鍵作用。此外,在NIH3T3/Src細(xì)胞培養(yǎng)上清中還檢測(cè)到乳酸(Lactic acid)分泌升高。當(dāng)用α-氰基-4-羥基肉桂酸CHC抑制巨噬細(xì)胞表面的單羧酸轉(zhuǎn)運(yùn)蛋白MCT,使巨噬細(xì)胞對(duì)培養(yǎng)基中的乳酸攝入減少后,NIH3T3/Src細(xì)胞培養(yǎng)上清誘導(dǎo)巨噬細(xì)胞極化的能力降低。提示乳酸在誘導(dǎo)巨噬細(xì)胞極化中發(fā)揮重要作用。隨著APCMin/+小鼠結(jié)直腸腫瘤的進(jìn)展,小鼠結(jié)直腸組織間隙液中IL-6、IL-9和1L-10等因子分泌量維持高水平。在IL-6特異性敲除的小鼠IL-6tmlKopf中,當(dāng)用AOM-DSS化學(xué)誘導(dǎo)小鼠發(fā)生結(jié)直腸腫瘤,與對(duì)照組AOM-DSS小鼠相比,IL-6tmlKopf-AOM-DSS小鼠的成瘤性明顯受到抑制,提示IL-6在腫瘤形成過程中起重要作用。綜上所述,IL-6、乳酸等因子在NIH3T3/Src田胞培養(yǎng)上清誘導(dǎo)巨噬細(xì)胞極化過程中起著重要作用。我們目前的研究提示了一個(gè)誘導(dǎo)巨噬細(xì)胞極化的可能的機(jī)制,同時(shí)也提示一個(gè)潛在的抑制巨噬細(xì)胞極化的途徑,或許能成為一種新的有前途的癌癥治療方式。
[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 偽-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 偽-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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R73-3

【共引文獻(xiàn)】

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