肿瘤相关巨噬细胞M2型极化机制及其小分子化合物干预研究

发布时间：2018-09-13 10:04

【摘要】：第一部分低氧对巨噬细胞M2型极化的选择性促进作用及其机制研究目的：巨噬细胞具有很强的功能可塑性,根据诱导条件的不同,巨噬细胞可以极化成两种主要的功能表型：经典活化的巨噬细胞(M1型)和替代性活化的巨噬细胞(M2型)。肿瘤组织中的巨噬细胞被称为肿瘤相关巨噬细胞(TAM),其表型和功能与肿瘤微环境密切相关。低氧作为众多恶性肿瘤发生发展过程中所必然经历的微环境条件之一,已被证实在TAM的浸润中发挥着重要作用,但其在TAM极化表型转换中的作用则鲜有研究。本部分研究将以低氧和巨噬细胞为研究对象,系统探讨低氧对巨噬细胞表型极化的影响和相关作用机制。方法：本研究采用小鼠巨噬细胞系RAW264.7和原代巨噬细胞BMDM作为研究对象。(1)流式细胞术检测RAW264.7细胞和BMDM细胞膜表面抗原的表达；(2)免疫组化及免疫荧光检测瘤组织切片相关蛋白的表达；(3)RT-PCR检测M2型巨噬细胞相关mRNA的水平；(4)表达谱芯片分析相关差异基因的表达；(5)Western blot考察相关信号通路蛋白的表达。结果：首先,采用免疫荧光技术对Lewis肺癌细胞(LLC)移植瘤组织中M2型巨噬细胞的表达及分布进行检测,发现肿瘤中的巨噬细胞(F4/80+)大多表现为M2型(CD209+)且主要集中在低氧区域(PIMO+).这一结果提示,低氧可能对巨噬细胞的M2型极化有一定的调控作用。通过建立常压低氧动物模型,研究发现间歇性低氧处理C57BL/6小鼠能明显促进肿瘤组织中巨噬细胞的浸润,同时伴随着CD209+巨噬细胞比例的增加。进一步利用体外低氧共培养模型,研究发现低氧处理RAW264.7细胞能选择性地促进巨噬细胞极化因子(LLC-CM、IL6、IL4和IL13)诱导的M2型表面标记物CD209和CD206的表达,但对M1型表面标记物CD86的表达无明显影响。此外,RT-PCR结果显示低氧能显著增加IL6诱导的M2型特征性基因Arg1和Yml的mRNA水平。表达谱基因芯片结果显示低氧条件下IL6诱导的巨噬细胞表达高水平的M2型基因和低水平的M1型基因。在BMDM的原代巨噬细胞模型中,同样发现低氧能促进IL6诱导的CD209的表达。以上结果均表明,低氧能选择性地促进IL6诱导的巨噬细胞M2型极化。在机制研究方面,结合特异性抑制剂与分子生物学手段的应用,发现IL6/STAT3信号通路及低氧相关的HIF信号通路并不直接参与低氧对巨噬细胞M2型极化的选择性促进作用。进一步采用基因芯片技术分析不同诱导条件下获得的巨噬细胞的差异基因,结果发现多个差异基因在MAPK信号级联中富集。Western blot结果也显示低氧能显著增加巨噬细胞中p-ERK,p-p38,p-JNK的表达。通过采用特异性抑制剂,证实虽然低氧条件下MAPK的三条子信号通路都被激活,但只有抑制ERK信号后才能逆转低氧介导的巨噬细胞M2型极化。结论：低氧微环境可以选择性地促进巨噬细胞M2型极化,该选择性促进作用与ERK信号通路的激活密切相关。第二部分低氧介导的M2型巨噬细胞对肿瘤转移的影响目的：低氧是包括非小细胞肺癌(NSCLC)在内的多数实体瘤发展过程中所必然经历的微环境条件。长期以来,肿瘤低氧研究的重心是其对肿瘤细胞本身的影响,而忽视了低氧通过作用于非肿瘤细胞来推动肿瘤发生发展的可能性。TAM作为非肿瘤细胞的主要成分,被认为是肿瘤恶性行为的参与者。第一部分的研究已经证实低氧能选择性地促进巨噬细胞的M2型极化(将该条件下获得的M2型巨噬细胞定义为低氧介导的M2型巨噬细胞)。在本部分研究中,将以NSCLC为研究对象,考察低氧介导的M2型巨噬细胞对肿瘤转移的影响,进而探讨低氧对巨噬细胞M2型极化的作用与低氧介导的肿瘤恶性行为(转移)之间的相关性,有助于从新的角度完善肿瘤低氧研究领域,以期为肿瘤治疗提供新的理论基础和可能的治疗策略。方法：(1)免疫组化及免疫荧光检测临床病人样本和瘤组织切片相关蛋白的表达；(2)划痕修复和transwell小室法检测细胞的迁移运动能力；(3)管腔形成实验考察HUVEC细胞的管腔形成能力；(4)C57BL/6动物模型检测Lewis肺癌的肺转移率；(5)SRB染色法评价肿瘤细胞的增殖能力。结果：36例人肺组织样本基因芯片数据(GSE1987)分析和55例(其中20例为转移病人)临床NSCLC病人样本的免疫组化分析结果表明M2型TAM与NSCLC的转移密切相关。在此基础上,通过体内外实验评价第一部分中阐述的低氧介导的M2型巨噬细胞对NSCLC转移的影响。体外实验,采用条件培养基与LLC细胞或HUVEC细胞共孵育模型,通过细胞增殖实验、划痕修复实验、transwell实验和管腔形成实验等手段,发现低氧介导的M2型RAW264.7细胞上清可增加HUVEC细胞的管腔形成和LLC细胞的迁移运动,但对LLC细胞的增殖无影响。体内实验发现,将低氧介导的M2型RAW264.7细胞与LLC细胞的共接种可增加LLC的肺转移发生率(从22.6%增加到100%)和移植瘤肿瘤组织中的血管生成。以上结果表明,低氧介导的M2型巨噬细胞具有强有力的促进肿瘤血管生成和转移的能力。鉴于低氧介导的M2型巨噬细胞表现出与低氧类似的促进肿瘤演进的功能,进一步考察低氧对巨噬细胞M2型极化的促进作用与低氧介导的肿瘤恶性行为之间的相关性。结果显示,间歇性低氧处理C57BL/6小鼠可显著促进LLC的肺转移发生率,从20%增加到60%。免疫荧光实验结果表明低氧处理后,肿瘤组织中的M2型巨噬细胞显著增加。此外,本研究还发现ERK特异性抑制剂PD98059可通过靶向巨噬细胞,(而不是肿瘤细胞)抑制肿瘤细胞的迁移运动。结论：低氧介导的M2型巨噬细胞,在体内外均能显著促进肿瘤的转移(迁移)和血管生成。干扰ERK可通过靶向巨噬细胞,抑制肿瘤细胞的迁移运动。第三部分基于抑制TAMM2型极化的化合物M的发现及其抗转移作用研究目的：抑制TAM的M2型极化被认为是极具应用前景的抗肿瘤转移新策略,但相关小分子化合物的发现尚处于起步阶段。因此,寻找并发现特异性抑制TAMM2型极化的小分子化合物是开发以TAM为中心的治疗策略的一个关键方向。前期研究中,通过筛选发现了特异性抑制巨噬细胞M2型极化的化合物M。在本部分研究中,将进一步评价化合物M对肿瘤转移的作用并初步探讨其作用机制,确证通过小分子化合物干预M2型极化实现抗肿瘤转移的可行性,以期为设计全新抗肿瘤转移化合物或干预手段提供思路及潜在靶点。方法：(1)SRB染色法评价细胞增殖能力；(2)流式细胞术检测RAW264.7细胞和BMDM细胞膜表面抗原的表达；(3)免疫荧光检测瘤组织切片中相关蛋白的表达；(4)RT-PCR检测M1、M2型巨噬细胞特征性mRNA水平；(5) Transwell小室法检测肿瘤细胞的迁移运动；(6)C57BL/6动物模型考察LLC的肺转移情况；(7)HE染色检测肺部转移灶点；(8)Western blot考察相关信号通路蛋白的表达。结果：体外采用经典的IL13诱导的M2型巨噬细胞极化模型,从众多化合物中筛选得到可抑制巨噬细胞M2型极化的化合物M。在此基础之上,进一步应用IL4和IL6诱导的M2型极化模型,也得到了同样的结果。另一方面,通过对IFNy和LPS诱导的M1型巨噬细胞极化模型的考察,发现化合物M能选择性抑制巨噬细胞M2型极化,而对M1型极化无影响。LLC移植瘤组织切片的免疫荧光结果表明化合物M在体内也能抑制巨噬细胞的M2型极化。进一步考察化合物M的体外抗转移作用,研究发现化合物M可通过抑制巨噬细胞的M2型极化来影响肿瘤细胞的迁移运动。LLC自发肺转移动物实验结果显示化合物M具有体内抗肿瘤转移作用。通过引入巨噬细胞“清除剂”脂质体氯磷酸盐,研究发现化合物M对肿瘤肺转移的抑制作用可能是通过抑制肿瘤组织中巨噬细胞的M2型极化来发挥作用的。免疫荧光结果证实化合物M能减少移植瘤肿瘤组织中新生血管的形成。机制研究证实化合物M通过激活AMPK来发挥抑制巨噬细胞M2型极化的作用。结论：小分子化合物M能选择性地抑制巨噬细胞M2型极化,并基于此发挥体内外抗转移作用。化合物M对巨噬细胞M2型极化的选择性抑制作用依赖于AMPK信号通路的激活。
[Abstract]:Part one: selective enhancement of hypoxia on macrophage M2 polarization and its mechanism
AIM: Macrophages have strong functional plasticity. According to different induction conditions, macrophages can be polarized into two major functional phenotypes: classically activated macrophages (M1) and alternatively activated macrophages (M2). Macrophages in tumor tissues are called tumor-associated macrophages (TAM), and their phenotypes and functions are known as tumor-associated macrophages (TAM). Hypoxia has been proved to play an important role in the invasion of TAM as one of the necessary microenvironment conditions in the development of many malignant tumors, but its role in the polarization phenotype transition of TAM is rarely studied. Effects of hypoxia on phenotypic polarization of macrophages and related mechanisms were discussed.
METHODS: Mouse macrophage line RAW264.7 and primary macrophage BMDM were used as the research objects. (1) The expression of membrane surface antigen in RAW264.7 and BMDM cells was detected by flow cytometry; (2) The expression of tumor slice-related protein was detected by immunohistochemistry and immunofluorescence; (3) The expression of M 2 macrophage-related mRNA was detected by RT-PCR. Ping; (4) Expression profiles were used to analyze the expression of related differentially expressed genes; (5) Western blot was used to investigate the expression of related signaling pathway proteins.
Results: Immunofluorescence technique was used to detect the expression and distribution of M2 macrophages in Lewis lung cancer cell (LLC) transplanted tumor tissues. It was found that most of the macrophages (F4/80+) in the tumor were M2 (CD209+) and mainly concentrated in the hypoxic region (PIMO+). The results suggested that hypoxia might polarize M2 of macrophages. It was found that intermittent hypoxia treatment of C57BL/6 mice could significantly promote the infiltration of macrophages in tumor tissues, accompanied by an increase in the proportion of CD209 + macrophages. In addition, RT-PCR showed that hypoxia significantly increased the mRNA levels of IL-6-induced M2 specific genes Arg1 and Yml. Gene chip analysis showed that hypoxia significantly increased the expression of IL-6-induced M2 specific genes Arg1 and Yml. In the primary macrophage model of BMDM, hypoxia can also promote the expression of CD209 induced by IL 6. These results suggest that hypoxia can selectively promote IL 6-induced M2 polarization in macrophages. It was found that IL-6/STAT3 signaling pathway and hypoxia-related HIF signaling pathway were not directly involved in the selective promotion of hypoxia on the polarization of macrophage M2. The differentially expressed genes of macrophages under different induction conditions were further analyzed by gene chip technique. Western blot also showed that hypoxia significantly increased the expression of p-ERK, p-p38, p-JNK in macrophages. Specific inhibitors were used to confirm that although all three MAPK signaling pathways were activated under hypoxia, hypoxia-mediated macrophage fineness could only be reversed by inhibiting ERK signaling. Cell type M2 polarization.
Conclusion: Hypoxic microenvironment can selectively promote the polarization of M2 in macrophages, which is closely related to the activation of ERK signaling pathway.
The second part is the effect of hypoxia mediated M2 macrophages on tumor metastasis.
OBJECTIVE: Hypoxia is an essential microenvironment in the development of most solid tumors, including non-small cell lung cancer (NSCLC). For a long time, the focus of tumor hypoxia research has been on the effect of hypoxia on tumor cells themselves, ignoring the possibility of hypoxia promoting tumor development by acting on non-tumor cells. The first part of this study has demonstrated that hypoxia selectively promotes the M2 polarization of macrophages (defined as hypoxia-mediated M2 macrophages). The effect of hypoxia-mediated M2 macrophages on tumor metastasis and the correlation between hypoxia-mediated M2 polarization and hypoxia-mediated malignant behavior (metastasis) of tumor are discussed. It is helpful to perfect the research field of tumor hypoxia from a new perspective and provide a new theoretical basis and possible therapeutic strategies for tumor therapy.
Methods: (1) Immunohistochemistry and immunofluorescence were used to detect the expression of tumor-related proteins in clinical samples and tumor tissues; (2) Scratch repair and Transwell chamber were used to detect the migration and motility of HUVEC cells; (3) lumen formation test was used to investigate the lumen formation ability of HUVEC cells; (4) C57BL/6 animal model was used to detect the lung metastasis rate of Lewis lung cancer; (5) The proliferation ability of tumor cells was evaluated by SRB staining.
RESULTS: Gene chip data of 36 human lung tissue samples (GSE1987) and immunohistochemical analysis of 55 clinical NSCLC patients (20 of whom were metastatic) showed that M2 TAM was closely related to the metastasis of NSCLC. On this basis, hypoxia-mediated M2 macrophages were evaluated in vitro and in vivo. In vitro experiments, conditioned medium was used to co-incubate with LLC cells or HUVEC cells. By means of cell proliferation, scratch repair, Transwell and lumen formation, hypoxia-mediated supernatant of M2 RAW264.7 cells was found to increase lumen formation and migration of LLC cells. In vivo experiments showed that hypoxia-mediated co-inoculation of M 2 RAW264.7 cells with LLC cells increased the incidence of lung metastasis (from 22.6% to 100%) and angiogenesis in transplanted tumor tissues. The ability of hypoxia-mediated M2 macrophages to promote tumor progression was similar to hypoxia. The relationship between hypoxia-mediated M2 polarization and hypoxia-mediated malignant behavior was further investigated. The results showed that intermittent hypoxia treatment could significantly promote LL in C57BL/6 mice. The incidence of lung metastasis of C increased from 20% to 60%. Immunofluorescence assay showed that M2 macrophages in tumor tissues were significantly increased after hypoxia treatment. In addition, this study also found that ERK-specific inhibitor PD98059 could inhibit the migration of tumor cells by targeting macrophages rather than tumor cells.
Conclusion: Hypoxia-mediated M2 macrophages can significantly promote tumor metastasis (migration) and angiogenesis in vivo and in vitro. Interference with ERK can inhibit the migration of tumor cells by targeting macrophages.
The third part is based on the discovery of compound M which inhibits TAMM2 polarization and its anti metastasis effect.
OBJECTIVE: Inhibition of TAM-2 polarization is considered to be a promising new strategy for tumor metastasis, but the discovery of related small molecule compounds is still in its infancy. In this part, we will further evaluate the effect of compound M on tumor metastasis and explore its mechanism, confirm the feasibility of anti-tumor metastasis through small molecule compounds interfering with M2 polarization, in order to design a new anti-tumor metastasis. Combination or intervention provides ideas and potential targets.
Methods: (1) SRB staining was used to evaluate cell proliferation; (2) Flow cytometry was used to detect the expression of membrane antigens on RAW264.7 cells and BMDM cells; (3) immunofluorescence was used to detect the expression of related proteins in tumor tissue slices; (4) RT-PCR was used to detect the characteristic mRNA level of M1 and M 2 macrophages; (5) Transwell chamber was used to detect the migration of tumor cells. Exercise; (6) C57BL/6 animal model to investigate the lung metastasis of LLC; (7) HE staining to detect the lung metastasis site; (8) Western blot to investigate the expression of related signal pathway proteins. Results: The classical IL 13-induced polarization model of M2 macrophages was used in vitro, and the compounds were screened from a number of compounds to inhibit the polarization of M2 macrophages. On the other hand, the polarization model of M1 macrophages induced by IFNy and LPS was investigated. It was found that compound M selectively inhibited the polarization of M2 macrophages, but had no effect on the polarization of M1 macrophages. Immunofluorescence assay showed that compound M could also inhibit the M2 polarization of macrophages in vivo. Further investigation of the anti-metastasis effect of compound M in vitro showed that compound M could affect the migration of tumor cells by inhibiting the M2 polarization of macrophages. Tumor metastasis. By introducing macrophage scavenger liposome chlorophosphate, it was found that the inhibitory effect of compound M on tumor lung metastasis may be mediated by inhibiting the M2 polarization of macrophages in tumor tissues. Immunofluorescence results showed that compound M could reduce neovascularization in tumor tissues. Mechanism study confirmed that compound M could inhibit macrophage M2 polarization by activating AMPK.
Conclusion: Small molecule compound M can selectively inhibit the polarization of macrophage M2 and exert its anti-metastasis effect in vitro and in vivo. The selective inhibition of compound M on the polarization of macrophage M2 depends on the activation of AMPK signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

【相似文献】