ATRA通过调控肿瘤相关巨噬细胞极化抑制骨肉瘤转移的作用研究

发布时间：2018-07-07 10:57

本文选题：骨肉瘤 + 肿瘤转移　；参考：《浙江大学》2016年博士论文

【摘要】：研究目的：骨肉瘤是一种好发于儿童或青少年时期最常见的高度恶性骨肿瘤,高转移潜能是骨肉瘤最主要的特性,也是当前限制患者生存率进一步提高的关键因素。因此如何有效地控制转移的发生一直是骨肉瘤治疗研究领域的前沿热点。抑制肿瘤相关巨噬细胞(TAM) M2型极化的抗肿瘤转移策略受到广泛关注,但在骨肉瘤的研究中尚处于起步阶段,特别是TAM的M2型极化对骨肉瘤转移的影响尚未见文献报道。近年来研究报道全反式维甲酸(ATRA)可抑制多种肿瘤的侵袭和转移过程,但其分子机制并不明确,也无其抗骨肉瘤转移的相关报道。本部分研究将首先考察巨噬细胞M2型极化对骨肉瘤转移的影响,然后以巨噬细胞极化为模型研究ATRA对巨噬细胞极化的调控作用,并深入探讨ATRA对骨肉瘤转移的实验治疗作用,最后阐明ATRA抑制骨肉瘤转移的分子机制。本课题旨在明确巨噬细胞极化调控骨肉瘤转移的作用,并阐明ATRA干预巨噬细胞极化和骨肉瘤转移的分子机制,为抗骨肉瘤转移的治疗提供新思路。研究方法：本研究采用小鼠巨噬细胞和骨肉瘤细胞株作为研究对象。(1)SRB法检测ATRA对巨噬细胞增殖以及巨噬细胞条件培养基对骨肉瘤细胞增殖的影响；(2)通过流式细胞术检测巨噬细胞膜表面抗原F4/80、CD206和CD86的表达来考察ATRA对巨噬细胞极化的影响；(3)HE染色检测骨肉瘤的肺转移灶点；(4)免疫组化检测肿瘤石蜡组织切片中巨噬细胞相关蛋白和MMP12蛋白的表达；(5)免疫荧光染色检测冰冻切片样本中巨噬细胞相关蛋白在肿瘤转移组织里的表达；(6)Real-time PCR检测M1和M2型两类巨噬细胞相关基因的mRNA表达水平；(7)Transwell小室法和划痕修复检测骨肉瘤细胞的运动迁移及侵袭能力；(8)建立鼠源性骨肉瘤细胞K7M2 WT的balb/c小鼠尾静脉或原位接种模型来模拟骨肉瘤肺转移情况,考察巨噬细胞对骨肉瘤转移的影响以及ATRA抗骨肉瘤转移的作用；(9)表达谱芯片分析相关差异基因的表达；(10)ELISA检测相关基质金属蛋白酶的分泌情况。研究结果：(1)采用尾静脉注射的方式将鼠源性骨肉瘤细胞株K7M2 WT单独或与巨噬细胞混合后接种于balb/c小鼠建立了骨肉瘤肺转移模型,在此模型上研究发现M2型巨噬细胞可以促进骨肉瘤肺转移的发生。(2)体外采用传代巨噬细胞系RAW264.7和原代巨噬细胞BMDM,应用经典的IL-13或IL-4诱导其发生M2型巨噬细胞极化,通过流式细胞术检测M2型巨噬细胞标记物CD206,结果发现ATRA可有效抑制由IL-13或IL-4诱导的CD206表达,Real time PCR结果也证实了ATRA可以明显抑制M2型巨噬细胞标记物MRCl和PPAR-γ的mRNA水平；另一方面,采用IFN-γ和LPS诱导的M1型巨噬细胞模型,发现ATRA可以一定程度上促进巨噬细胞系RAW264.7细胞的M1型极化,但对于原代巨噬细胞BMDM的M1型极化却没有影响。(3)进一步考察ATRA的体外抗骨肉瘤细胞转移作用,研究结果表明ATRA可以抑制由IL-13或IL-4刺激的M2型巨噬细胞上清诱导的骨肉瘤细胞株的运动迁移能力。(4)采用尾静脉接种和原位接种鼠源性骨肉瘤细胞K7M2WT的balb/c鼠建立了两种骨肉瘤转移模型,在这两个动物模型上深入评价了ATRA在体内抗骨肉瘤肺转移的作用。HE染色检测骨肉瘤肺转移灶点数的统计结果表明ATRA在体内的抗骨肉瘤肺转移作用十分良好,同时通过与巨噬细胞抑制剂氯膦酸盐脂质体(Clodronate liposome)共同作用后,我们发现抑制肿瘤相关巨噬细胞M2型极化可能是ATRA阻断骨肉瘤转移作用的主要途径。进一步免疫组化结果证实ATRA可以有效抑制肺转移组织中的M2型巨噬细胞,而对M1型巨噬细胞没有显著影响。(5)采用基因芯片分析探讨了ATRA调控巨噬细胞极化的潜在机制,发现MMP12可能是其潜在作用靶点。ELISA结果显示ATRA可以减少巨噬细胞中由IL-13刺激的MMP12分泌。引入MMP12抑制剂mmp408,发现mmp408可以有效抑制由IL-13和IL-4刺激的M2型巨噬细胞上清诱导的骨肉瘤细胞株K7M2 WT的运动迁移和侵袭能力。免疫组化结果发现MMP12在骨肉瘤肺转移组织中普遍高表达,而ATRA可显著抑制MMP12在肿瘤转移灶中的表达。结论：M2型巨噬细胞可促进骨肉瘤的肺转移,ATRA可以特异性调控巨噬细胞M2型极化进而有效抑制骨肉瘤的肺转移。MMP12有可能是ATRA干预巨噬细胞M2型极化和抗骨肉瘤转移作用的潜在分子靶点。
[Abstract]:Objective: osteosarcoma is the most common high malignant bone tumor in children or adolescents. High metastatic potential is the most important characteristic of osteosarcoma and is the key factor restricting the further improvement of the survival rate of the patients. Therefore, how to effectively control the metastasis of metastasis has been the forefront of the research field of osteosarcoma treatment. The anti tumor metastasis strategy of inhibiting tumor associated macrophage (TAM) M2 polarization is widely concerned, but it is still in the initial stage in the study of osteosarcoma, especially the effect of M2 polarization on osteosarcoma metastasis has not yet been reported. In recent years, it has been reported that all trans retinoic acid (ATRA) can inhibit the invasion and invasion of various tumors. The molecular mechanism of the metastasis is not clear, but there is no related report on the metastasis of osteosarcoma. This part of this study will first examine the effect of macrophage M2 polarization on osteosarcoma metastasis, and then use macrophage polarization as a model to study the use of ATRA to regulate the polarization of macrophages, and to explore the experiment of ATRA on osteosarcoma metastasis. The purpose of this study is to clarify the molecular mechanism of ATRA inhibition of osteosarcoma metastasis. The purpose of this study is to clarify the role of macrophage polarization to regulate osteosarcoma metastasis, and to clarify the molecular mechanism of ATRA intervention in macrophage polarization and osteosarcoma metastasis, and to provide a new way for the treatment of osteosarcoma metastasis. Cell and osteosarcoma cell lines were used as research objects. (1) SRB assay was used to detect the effect of ATRA on macrophage proliferation and macrophage conditioned medium on osteosarcoma cell proliferation. (2) the expression of macrophage membrane surface antigen F4/80, CD206 and CD86 was detected by flow cytometry to investigate the effect of ATRA on the polarization of macrophages; (3) HE staining detection Pulmonary metastasis point of osteosarcoma; (4) immunohistochemical detection of the expression of macrophage related protein and MMP12 protein in the tumor paraffin tissue section; (5) immunofluorescence staining was used to detect the expression of macrophage related proteins in the tumor metastasis tissue, and (6) Real-time PCR detection of M1 and M2 type two macrophage related genes The expression level of mRNA; (7) Transwell cell method and scratch repair to detect the movement and invasion of osteosarcoma cells; (8) to establish the tail vein of balb/c mouse or in situ inoculation model of K7M2 WT in mouse derived osteosarcoma cells to simulate the pulmonary metastasis of osteosarcoma, to investigate the effect of macrophage on the metastasis of osteosarcoma and the anti osteosarcoma of ATRA Transfer function; (9) express the expression of related differential genes by spectral chip analysis; (10) ELISA detection of the secretion of related matrix metalloproteinases. The results: (1) the rat osteosarcoma cell line K7M2 WT was inoculated in the tail vein to establish the lung metastasis model of osteosarcoma after inoculation in balb/c mice. In this model, we found that type M2 macrophages could promote the occurrence of lung metastasis of osteosarcoma. (2) RAW264.7 and BMDM of primary macrophages were used in vitro, and M2 type macrophage polarization was induced by classical IL-13 or IL-4, and M2 type macrophage marker CD206 was detected by flow cytometry, and ATRA was found to be a result of ATRA. The CD206 expression induced by IL-13 or IL-4 was effectively inhibited, and Real time PCR results also confirmed that ATRA could obviously inhibit mRNA level of MRCl and PPAR- gamma of M2 type macrophage markers. On the other hand, the macrophage macrophage model could be promoted on a certain range by using IFN- gamma and induced macrophage model. Polarization, but did not affect the M1 type polarization of primary macrophage BMDM. (3) further investigate the effect of ATRA on osteosarcoma cell metastasis in vitro. The results showed that ATRA could inhibit the movement ability of osteosarcoma cell lines induced by IL-13 or IL-4 stimulated M2 macrophage supernatant. (4) the tail vein inoculation and in situ inoculation were used. Two models of osteosarcoma metastasis were established in rat K7M2WT balb/c mice. On these two animal models, the effect of ATRA on the pulmonary metastasis of osteosarcoma in vivo was evaluated by.HE staining, and the results showed that ATRA was very good for anti osteosarcoma lung metastasis in the body. After the co action of Clodronate liposome, a macrophage inhibitor, we found that inhibition of M2 polarization of tumor related macrophages may be the main pathway for ATRA to block the metastasis of osteosarcoma. Further immunohistochemical results confirm that ATRA can effectively inhibit M2 type macrophages in the pulmonary metastases, and to M1 type macrophages. (5) the potential mechanism of ATRA regulation of macrophage polarization was investigated by gene chip analysis. It was found that MMP12 may be a potential target for.ELISA. The result shows that ATRA can reduce the secretion of MMP12 stimulated by IL-13 in macrophages. MMP12 inhibitor mmp408 is introduced, and mmp408 can effectively inhibit IL-13 and IL-4 thorns. The movement and invasion ability of osteosarcoma cell line K7M2 WT induced by stimulated M2 macrophage supernatant. The immunohistochemical results showed that MMP12 was highly expressed in the lung metastases of osteosarcoma, and ATRA could significantly inhibit the expression of MMP12 in tumor metastasis. Conclusion: M2 type macrophage can promote the lung metastasis of osteosarcoma, ATRA can be special. The heterosexual regulation of M2 polarization of macrophages and the effective inhibition of pulmonary metastasis.MMP12 in osteosarcoma may be a potential molecular target for ATRA intervention in M2 type polarization and anti osteosarcoma metastasis.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

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