發(fā)布時間：2018-07-07 10:57

  本文選題：骨肉瘤 + 腫瘤轉(zhuǎn)移　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：研究目的：骨肉瘤是一種好發(fā)于兒童或青少年時期最常見的高度惡性骨腫瘤,高轉(zhuǎn)移潛能是骨肉瘤最主要的特性,也是當(dāng)前限制患者生存率進一步提高的關(guān)鍵因素。因此如何有效地控制轉(zhuǎn)移的發(fā)生一直是骨肉瘤治療研究領(lǐng)域的前沿?zé)狳c。抑制腫瘤相關(guān)巨噬細(xì)胞(TAM) M2型極化的抗腫瘤轉(zhuǎn)移策略受到廣泛關(guān)注,但在骨肉瘤的研究中尚處于起步階段,特別是TAM的M2型極化對骨肉瘤轉(zhuǎn)移的影響尚未見文獻報道。近年來研究報道全反式維甲酸(ATRA)可抑制多種腫瘤的侵襲和轉(zhuǎn)移過程,但其分子機制并不明確,也無其抗骨肉瘤轉(zhuǎn)移的相關(guān)報道。本部分研究將首先考察巨噬細(xì)胞M2型極化對骨肉瘤轉(zhuǎn)移的影響,然后以巨噬細(xì)胞極化為模型研究ATRA對巨噬細(xì)胞極化的調(diào)控作用,并深入探討ATRA對骨肉瘤轉(zhuǎn)移的實驗治療作用,最后闡明ATRA抑制骨肉瘤轉(zhuǎn)移的分子機制。本課題旨在明確巨噬細(xì)胞極化調(diào)控骨肉瘤轉(zhuǎn)移的作用,并闡明ATRA干預(yù)巨噬細(xì)胞極化和骨肉瘤轉(zhuǎn)移的分子機制,為抗骨肉瘤轉(zhuǎn)移的治療提供新思路。研究方法：本研究采用小鼠巨噬細(xì)胞和骨肉瘤細(xì)胞株作為研究對象。(1)SRB法檢測ATRA對巨噬細(xì)胞增殖以及巨噬細(xì)胞條件培養(yǎng)基對骨肉瘤細(xì)胞增殖的影響；(2)通過流式細(xì)胞術(shù)檢測巨噬細(xì)胞膜表面抗原F4/80、CD206和CD86的表達來考察ATRA對巨噬細(xì)胞極化的影響；(3)HE染色檢測骨肉瘤的肺轉(zhuǎn)移灶點；(4)免疫組化檢測腫瘤石蠟組織切片中巨噬細(xì)胞相關(guān)蛋白和MMP12蛋白的表達；(5)免疫熒光染色檢測冰凍切片樣本中巨噬細(xì)胞相關(guān)蛋白在腫瘤轉(zhuǎn)移組織里的表達；(6)Real-time PCR檢測M1和M2型兩類巨噬細(xì)胞相關(guān)基因的mRNA表達水平；(7)Transwell小室法和劃痕修復(fù)檢測骨肉瘤細(xì)胞的運動遷移及侵襲能力；(8)建立鼠源性骨肉瘤細(xì)胞K7M2 WT的balb/c小鼠尾靜脈或原位接種模型來模擬骨肉瘤肺轉(zhuǎn)移情況,考察巨噬細(xì)胞對骨肉瘤轉(zhuǎn)移的影響以及ATRA抗骨肉瘤轉(zhuǎn)移的作用；(9)表達譜芯片分析相關(guān)差異基因的表達；(10)ELISA檢測相關(guān)基質(zhì)金屬蛋白酶的分泌情況。研究結(jié)果：(1)采用尾靜脈注射的方式將鼠源性骨肉瘤細(xì)胞株K7M2 WT單獨或與巨噬細(xì)胞混合后接種于balb/c小鼠建立了骨肉瘤肺轉(zhuǎn)移模型,在此模型上研究發(fā)現(xiàn)M2型巨噬細(xì)胞可以促進骨肉瘤肺轉(zhuǎn)移的發(fā)生。(2)體外采用傳代巨噬細(xì)胞系RAW264.7和原代巨噬細(xì)胞BMDM,應(yīng)用經(jīng)典的IL-13或IL-4誘導(dǎo)其發(fā)生M2型巨噬細(xì)胞極化,通過流式細(xì)胞術(shù)檢測M2型巨噬細(xì)胞標(biāo)記物CD206,結(jié)果發(fā)現(xiàn)ATRA可有效抑制由IL-13或IL-4誘導(dǎo)的CD206表達,Real time PCR結(jié)果也證實了ATRA可以明顯抑制M2型巨噬細(xì)胞標(biāo)記物MRCl和PPAR-γ的mRNA水平；另一方面,采用IFN-γ和LPS誘導(dǎo)的M1型巨噬細(xì)胞模型,發(fā)現(xiàn)ATRA可以一定程度上促進巨噬細(xì)胞系RAW264.7細(xì)胞的M1型極化,但對于原代巨噬細(xì)胞BMDM的M1型極化卻沒有影響。(3)進一步考察ATRA的體外抗骨肉瘤細(xì)胞轉(zhuǎn)移作用,研究結(jié)果表明ATRA可以抑制由IL-13或IL-4刺激的M2型巨噬細(xì)胞上清誘導(dǎo)的骨肉瘤細(xì)胞株的運動遷移能力。(4)采用尾靜脈接種和原位接種鼠源性骨肉瘤細(xì)胞K7M2WT的balb/c鼠建立了兩種骨肉瘤轉(zhuǎn)移模型,在這兩個動物模型上深入評價了ATRA在體內(nèi)抗骨肉瘤肺轉(zhuǎn)移的作用。HE染色檢測骨肉瘤肺轉(zhuǎn)移灶點數(shù)的統(tǒng)計結(jié)果表明ATRA在體內(nèi)的抗骨肉瘤肺轉(zhuǎn)移作用十分良好,同時通過與巨噬細(xì)胞抑制劑氯膦酸鹽脂質(zhì)體(Clodronate liposome)共同作用后,我們發(fā)現(xiàn)抑制腫瘤相關(guān)巨噬細(xì)胞M2型極化可能是ATRA阻斷骨肉瘤轉(zhuǎn)移作用的主要途徑。進一步免疫組化結(jié)果證實ATRA可以有效抑制肺轉(zhuǎn)移組織中的M2型巨噬細(xì)胞,而對M1型巨噬細(xì)胞沒有顯著影響。(5)采用基因芯片分析探討了ATRA調(diào)控巨噬細(xì)胞極化的潛在機制,發(fā)現(xiàn)MMP12可能是其潛在作用靶點。ELISA結(jié)果顯示ATRA可以減少巨噬細(xì)胞中由IL-13刺激的MMP12分泌。引入MMP12抑制劑mmp408,發(fā)現(xiàn)mmp408可以有效抑制由IL-13和IL-4刺激的M2型巨噬細(xì)胞上清誘導(dǎo)的骨肉瘤細(xì)胞株K7M2 WT的運動遷移和侵襲能力。免疫組化結(jié)果發(fā)現(xiàn)MMP12在骨肉瘤肺轉(zhuǎn)移組織中普遍高表達,而ATRA可顯著抑制MMP12在腫瘤轉(zhuǎn)移灶中的表達。結(jié)論：M2型巨噬細(xì)胞可促進骨肉瘤的肺轉(zhuǎn)移,ATRA可以特異性調(diào)控巨噬細(xì)胞M2型極化進而有效抑制骨肉瘤的肺轉(zhuǎn)移。MMP12有可能是ATRA干預(yù)巨噬細(xì)胞M2型極化和抗骨肉瘤轉(zhuǎn)移作用的潛在分子靶點。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R738.1

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