肿瘤相关巨噬细胞—肝癌细胞三维共培养体系中分泌蛋白的差异及CXCL2在肝癌转移中的作用

发布时间：2018-04-29 11:59

本文选题：肝细胞癌 + 肿瘤微环境　；参考：《广西医科大学》2017年博士论文

【摘要】：第一部分肿瘤相关巨噬细胞的诱导、鉴定及其对肝癌细胞的影响肝癌是经典的炎症相关性肿瘤,肝癌细胞所在微环境内浸润着大量肿瘤相关巨噬细胞,并以M2型肿瘤相关巨噬细胞为主。本部分研究旨在通过体外诱导获得不同活化状态巨噬细胞并明确其对肝癌细胞生物学行为的影响。我们首先通过人单核白血病细胞THP-1经佛波酯诱导为未分化巨噬细胞(UM0),再分别经脂多糖,白介素4和13诱导获得经典活化的巨噬细胞M1及选择性激活的巨噬细胞M2;然后通过形态学、荧光定量PCR和ELISA对其表型进行鉴定;最后从增殖、迁移及侵袭实验探索不同活化状态巨噬细胞条件培养基(conditioned medium,CM)对肝癌细胞生物学行为的影响,并检测了肝癌细胞中上皮-间质化转换(EMT)标志物的变化。结果显示:(1)与UM0相比,M1呈长梭形或多边形,TNF-α和CCL3 m RNA的表达量及IL-12的分泌均明显升高;M2伪足细长而杂乱、呈抱团式生长,AMAC-1、CCL22和Arg-1 m RNA的表达量及IL-10的分泌均显著增加。该检测结果与文献报道的结果一致,说明已成功诱导获得不同活化状态的巨噬细胞;(2)生物学功能实验显示经M2CM处理后的肝癌细胞与对照组相比,其增殖、迁移和侵袭能力均显著增强,而M1CM处理的肝癌细胞则无明显变化,说明M2型肿瘤相关巨噬细胞能显著促进肝癌细胞恶性变;(3)经M2CM处理后的肝癌细胞上皮标志物E-cadherin的表达明显下降,伴随间质细胞标志物N-cadherin、Vimentin和α-SMA显著上升,经M1CM处理的肝癌细胞各标志物的表达量均无明显变化,说明仅有经M2CM处理后的肝癌细胞发生了EMT。第二部分肿瘤相关巨噬细胞和肝癌细胞三维共培养体系中分泌蛋白的差异体内状态的肿瘤细胞生存于立体、复杂而开放的微环境中,分泌蛋白是该微环境中肿瘤细胞与TAMs间相互作用的重要桥梁。在本部分研究中,我们利用低熔点琼脂糖作为支架建立肿瘤相关巨噬细胞与肝癌细胞的三维立体共培养体系,通过i TRAQ标记为基础的定量蛋白质组学技术对三维培养体系中的分泌蛋白进行比较分析和筛选,并应用Western-Blot对部分差异蛋白的鉴定结果进行验证。结果显示:(1)不同三维立体培养体系中的条件培养基经i TRAQ标记、质谱分析及数据库搜索,一共鉴定到了26677条肽段及1640个蛋白(99%可信度)。将鉴定到的蛋白按比值1.3定义为上调,比值0.7定义为下调的准则进行筛选,发现在与M2比较时,有333个分泌蛋白差异性表达在M2+SMMC7721共培养体系;在与SMMC7721比较时,有357个分泌蛋白差异性表达在M2+SMMC7721共培养体系;同时与M2、SMMC7721单一立体培养体系比较时,有159个分泌蛋白差异性表达在M2+SMMC7721共培养体系中,其中上调的差异性分泌蛋白为63个,下调的为96个。(2)Western-Blot的结果显示IL-8、IL-1β、HB-EGF和IGFBP3的变化趋势与质谱结果完全一致,而MMP3与CXCL2,虽发现其在M2CM中的表达高于SMMC7721CM与质谱结果相反,但两者均在共培养条件培养基中升高,该趋势与质谱结果一致。第三部分差异分泌蛋白CXCL2对肝癌细胞转移功能的影响及机制研究CXCL2在本研究中被发现是共培养条件培养基里明显上升的差异蛋白,且在M2CM中的表达高于SMMC7721CM,说明其主要源于M2。其对肝癌转移能力的影响及作用机制未明。本部分通过采用不同浓度的外源性重组CXCL2蛋白刺激肝癌细胞,以观察其对肝癌细胞迁移、侵袭、粘附等生物学行为的影响,并通过信号通路芯片及通路抑制剂等方法探索其可能的作用机制。结果显示:(1)CXCL2在10例肝癌患者肝癌组织中的表达比相应癌旁组织明显增高;(2)不同浓度CXCL2刺激均可显著增强肝癌细胞的迁移能力,1ng/m L刺激组的肝癌细胞侵袭能力明显增加,10ng/m L及100ng/m L CXCL2刺激组的肝癌细胞粘附能力显著降低;(3)中和共培养条件培养基中的CXCL2后其促进肝癌细胞迁移及侵袭能力的作用均明显下降;(4)通过特异性抑制剂SB225002抑制肝癌细胞表达的CXCR2后,CXCL2对肝癌细胞迁移、侵袭能力的促进作用及对其粘附能力的抑制作用均消失,说明CXCL2通过与其受体CXCR2相结合发挥作用;(5)CXCL2过表达后,通过信号通路芯片检测发现肝癌细胞中HSP27、PRAS40、bad,Chk1、Ik Bα(Ser32/36)的磷酸化水平均显著上调,Ik Bα(Total)也明显升高;Western blot的结果证实过表达CXCL2后肝癌细胞中p-Ik Bα的磷酸化水平明显增高,说明NF-k B通路在CXCL2过表达的肝癌细胞中被激活;(6)进一步通过NF-k B特异性抑制剂BAY117082阻断肝癌细胞的NF-k B通路,发现CXCL2促进肝癌细胞细胞迁移及侵袭的能力减弱。上述结果表明肿瘤相关巨噬细胞源性的CXCL2主要通过激活NF-k B信号通路促进肝癌细胞的迁移及侵袭。结论1.在体外成功诱导获得具有典型表型特征的不同活化状态巨噬细胞M1和M2;并发现M2条件培养基可促进肝癌细胞增殖、迁移及侵袭并发生EMT。2.成功应用低熔点琼脂糖建立肝癌细胞及M2型肿瘤相关巨噬细胞的三维立体共培养体系,通过i TRAQ标记的定量蛋白质组学技术建立了不同三维立体培养体系中的分泌蛋白表达谱,并从共培养体系中鉴定到了159个差异分泌蛋白。3.CXCL2是共培养体系中显著升高的蛋白,可促进肝癌细胞的迁移、侵袭能力,并抑制其粘附能力;其作用的发挥是通过与肝癌细胞表达的CXCR2受体结合,从而激活NF-k B信号通路进行。
[Abstract]:The first part is the induction of tumor related macrophages, identification and its effect on hepatoma cells. Liver cancer is a classic inflammatory tumor. A large number of tumor related macrophages are infiltrated by a large number of tumor related macrophages in the microring of the hepatoma cells, and M2 type tumor related macrophages are the main factors. This part of the study is aimed at obtaining different activation states through in vitro induction. Macrophage and its effect on the biological behavior of hepatoma cells. First of all, we induced undifferentiated macrophages (UM0) through human mononuclear cells THP-1 via phorbol ester, and then induced by lipopolysaccharide, interleukin 4 and 13 to obtain the classic activated macrophage M1 and selectively activated macrophage M2; and then through morphology, fluoro The phenotypes were identified by light quantitative PCR and ELISA. Finally, the effects of conditioned medium (CM) on the biological behavior of hepatoma cells were investigated from the proliferation, migration and invasion experiments, and the changes in the epithelial mesenchymal transition (EMT) markers in the hepatoma cells were detected. The results showed: (1) M, compared with UM0, M. 1 long spindle shape or polygon, the expression of TNF- alpha and CCL3 m RNA and the secretion of IL-12 were significantly increased; M2 pseudo foot was elongated and messy and cluttered. The expression of AMAC-1, CCL22 and Arg-1 m RNA and IL-10 secretion were all increased significantly. The results of this detection were consistent with the results reported in the literature, indicating that different activation states have been successfully induced. (2) (2) biological function experiments showed that the proliferation, migration and invasion of HCC cells treated by M2CM were significantly enhanced, while the M1CM treated hepatoma cells had no significant changes, indicating that the macrophages of type M2 tumor related macrophages could significantly promote the malignant transformation of liver cancer cells; (3) the epithelial cells of liver cancer treated by M2CM The expression of the marker E-cadherin was significantly decreased, with the interstitial cell marker N-cadherin, Vimentin and alpha -SMA significantly increased, and the expression of all the hepatoma cells treated by M1CM had no significant changes, indicating that the only M2CM treated hepatoma cells had a three dimensional co culture of the tumor related macrophages and hepatoma cells of the EMT. second parts. Tumor cells secreting proteins in the nutrient system exist in a three-dimensional, complex and open microenvironment. Secretory proteins are an important bridge between the interaction of tumor cells and TAMs in this microenvironment. In this part, we use low melting point agarose as a support to establish tumor related macrophages and hepatoma cells. A three-dimensional co culture system was used to compare and screen the secretory proteins in the three-dimensional culture system by quantitative proteomics based on I TRAQ markers. The results of identification of partial differential proteins were verified by Western-Blot. The results showed that: (1) the conditioned medium in different three-dimensional culture systems was treated with I TRAQ Markers, mass spectrometry and database search, 26677 peptides and 1640 proteins (99% reliability) were identified. The identified protein was up to up by ratio 1.3, and the ratio 0.7 was defined as down regulation. It was found that when compared with M2, 333 secretory proteins were expressed differentially in the M2+SMMC7721 co culture system; and in SMMC772 1 in comparison, 357 secretory proteins were expressed differently in the M2+SMMC7721 co culture system; at the same time, when compared with M2, SMMC7721 single stereoscopic culture system, 159 secretory proteins were expressed differently in the M2+SMMC7721 co culture system, among which 63 differential secretory proteins were up-regulated and 96 were downregulated. (2) the results of Western-Blot showed IL-8, I The changes in L-1 beta, HB-EGF and IGFBP3 were in full agreement with the mass spectrum, while MMP3 and CXCL2 showed that their expression in M2CM was higher than that of SMMC7721CM and mass spectrometry, but both of them were increased in co culture medium, and the trend was in accordance with the mass spectrum. Third differential secretory protein CXCL2 has a shadow on the metastasis function of liver cancer cells. In this study, CXCL2 was found to be a distinct rising protein in the co culture medium, and the expression in M2CM was higher than that of SMMC7721CM. It was mainly attributed to the effect of M2. on the metastasis ability of liver cancer and the mechanism of action was not clear. This part was used to stimulate liver cancer by using different concentrations of exogenous recombinant CXCL2 protein. Cell, to observe the effects of its biological behavior on the migration, invasion and adhesion of hepatoma cells, and to explore its possible mechanism by means of signal pathway chip and pathway inhibitor. The results showed: (1) the expression of CXCL2 in the liver cancer tissues of 10 patients with liver cancer was significantly higher than that of the corresponding para cancerous tissues; (2) different concentrations of CXCL2 stimulation could be used. Significantly enhanced migration of hepatoma cells, the invasion ability of hepatoma cells in 1ng/m L stimulation group increased significantly, and the adhesion ability of hepatoma cells in 10ng/m L and 100ng/m L CXCL2 stimulation group decreased significantly; (3) after neutralizing CXCL2 in the co culture medium, the effect of promoting the migration and invasion of hepatoma cells decreased obviously; (4) through special treatment. After the inhibition of the heterosexual inhibitor SB225002 to inhibit the expression of CXCR2 in the hepatoma cells, CXCL2 plays a role in promoting the migration of hepatoma cells, the promoting effect of invasion and the inhibition effect on its adhesion ability. It shows that CXCL2 is combined with its receptor CXCR2. (5) after CXCL2 overexpression, the detection of HSP27, PRAS40 in hepatoma cells through the signal pathway chip is found. The phosphorylation level of bad, Chk1, Ik B alpha (Ser32/36) was significantly up-regulated, and Ik B a (Total) also increased obviously. The results of Western blot confirmed that the phosphorylation level of p-Ik alpha in hepatoma cells was significantly higher after the overexpression CXCL2, indicating that the pathway was activated in the hepatocellular carcinoma cells, which were overexpressed. (6) further through the specific inhibitors 82 blocking the NF-k B pathway of hepatoma cells, and finding that the ability of CXCL2 to promote the migration and invasion of hepatoma cells weakened. The results showed that the CXCL2 of tumor related macrophage derived mainly through the activation of NF-k B signaling pathway to promote the migration and invasion of hepatoma cells. Conclusion 1. in vitro induction to obtain the typical phenotypic characteristics of the difference. Activated state macrophages M1 and M2, and M2 conditioned medium can promote the proliferation, migration and invasion of hepatoma cells, and EMT.2. successfully applied low melting point agarose to establish a three-dimensional co culture system of hepatoma cells and M2 type tumor related macrophages, and established different three-dimensional erect by quantitative proteomics technology of I TRAQ markers. The secretory protein expression profiles in the body culture system and 159 differentially secreted protein.3.CXCL2 are identified in the co culture system as a significant increase in the co culture system, which can promote the migration, invasiveness and inhibition of the adhesion of hepatoma cells, and its role is to combine with the CXCR2 receptor expressed in hepatoma cells. Activation of the NF-k B signal pathway is performed.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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