人结肠癌SW620细胞上清作用下树突状细胞内皮样分化的研究

发布时间：2018-01-04 16:30

本文关键词：人结肠癌SW620细胞上清作用下树突状细胞内皮样分化的研究　出处：《郑州大学》2009年硕士论文　论文类型：学位论文

【摘要】：研究背景: 树突状细胞(dendritic cells,DCs)是目前已知的体内抗原呈递功能最强的专职抗原呈递细胞(antigen presenting cells,APCs),是启动、调控、并维持免疫反应的中心环节。在抗肿瘤免疫中发挥着重要作用。然而,许多研究证实在肿瘤患者循环血中和肿瘤周围浸润的树突状细胞的表型及其呈递功能均下降,肿瘤细胞可以分泌多种免疫抑制因子抑制DCs的功能成熟。肿瘤的无限制生长需要血管的供应。传统理论认为肿瘤血管形成是通过血管新生(angiogenesis)即从已存在的微血管上芽生出新的毛细血管的过程,目前研究发现也可以通过血管发生(vasculogenesis)——由内皮前体细胞分化形成新血管的过程。内皮细胞(endothelial cells,ECs)可以来源于骨髓内皮前体细胞(endothelialprogenitors,EPCs)和人外周血中CD34~+细胞群。研究证实多数肿瘤细胞都高表达血管内皮生长因子(vascular endothelial growth factor,VEGF),VEGF可以通过MAPK/ERK信号途径刺激内皮细胞的增殖和分化,从而促进肿瘤血管生成。MAPK/ERK途径是体内外多种细胞分化的重要信号通路之一。最新研究报道,在小鼠和人卵巢癌组织中发现了一种新的白细胞亚群,同时表达DCs和内皮细胞的标志。另有学者报道在肿瘤条件培养基浸润下的单核细胞能分化发育为肿瘤相关性树突细胞(tumor-associated dendritic cells,TADCs),此类细胞能在血管生成因子如VEGF和致瘤素M的诱导下分化发育为内皮样细胞(endothelial-like cells,ELCs),并且在基质胶上可以形成网状结构。本课题组前期研究发现结肠癌SW620细胞上清能抑制未成熟DCs(immature dendritic cells,iDCs)的生长过程及相关抗原表达,并且未成熟DCs在SW620细胞上清诱导下发生内皮样分化。这些研究揭示在肿瘤相关环境下DCs在抗肿瘤免疫方面受到抑制,部分DCs还发生内皮样分化。国内外近年来关于DCs的研究多集中于如何提高DCs抗肿瘤的免疫功能等方面,而肿瘤微环境下DCs的内皮样分化发生及其机制等还有待于进一步的研究。目的: 本实验探讨结肠癌细胞SW620分泌的可溶性细胞因子营造的微环境对人外周血单个核细胞来源的不同时期DCs发生内皮样分化的影响,以及MAPK/ERK信号途径在此过程中的激活情况。实验方法: 1.制备结肠癌SW620细胞上清液。 2.采集健康志愿者新鲜外周血,密度梯度离心法分离人外周血单个核细胞,RPMI 1640完全培养液调整细胞浓度为3×10~6/ml,接种于24孔培养板中,每孔1 ml,移入二氧化碳孵育箱(5%/CO_2,37℃)静置3 h,去除悬浮细胞,获取贴壁生长的单核细胞,加入含rhGM-CSF(100 ng/ml)、IL-4(5 ng/ml)和10%自体血清的1640培养液,第5d加入LPS(5 ng/ml)。诱导组除加入上述培养液外,分别于第2d(未成熟DCs诱导组)、第7d(成熟DCs诱导组)分组加入SW620细胞上清液。隔天半量换液,各诱导7d。分别于第9d和14d收集诱导组细胞和对照组DCs。培养过程中观察细胞形态并计数;提取各组细胞总蛋白,Western blotting检测内皮细胞特异性标志vWF、VE-cadherin的表达情况;透射电镜观察WP小体;增殖及杀伤实验观察诱导后细胞抗原呈递功能的改变;乙酰化低密度脂蛋白摄取实验(DiL-Ac-LDL uptake assay)了解诱导后细胞是否具有内皮细胞的功能;Westernblotting检测SW620细胞上清液对未成熟DCs刺激后15、30、60 min的ERK1/2水平的影响。使用ERK1/2上游激酶MEK的阻断剂PD98059,观察MAPK/ERK通路阻断后,SW620细胞上清诱导组细胞vWf、VE-cadherin的蛋白表达情况和DiL-Ac-LDL摄取功能。 3.胰蛋白酶法配合内皮细胞培养基培养原代脐静脉内皮细胞,作为阳性对照组。采用统计学软件包SPSS 12.0进行结果的统计分析,将所得计量数据以平均数±标准差((?)±s)表示,确定方差齐性后,进行t检验或方差分析,P＜0.05为显著性差异。结果: 1.在SW620细胞上清液诱导下,未成熟DCs诱导组细胞生长过程及其状态明显落后于对照组DCs,细胞密度较低,并且形成类管腔样和条索样结构;其对照组DCs则表现典型树突状细胞特征,细胞形态不规则,有粗细不等的毛刺状突起,有悬浮趋势;而成熟DCs诱导组细胞与其对照组DCs无明显差异。 2.Western blotting检测结果显示:未成熟DCs经SW620细胞上清液诱导7d后,内皮细胞的特异标记vWF、VE-cadherin与对照组DCs相比,均出现明显表达且两组间的差异有统计学意义。透射电镜结果显示:经SW620细胞上清液诱导的未成熟DCs胞内出现内皮细胞的特异性结构WP小体。DCs方面功能检测:未成熟DCs诱导组细胞的抗原提呈能力下降,而内皮细胞特异的乙酰化低密度脂蛋白摄取功能增强。SW620细胞上清诱导未成熟DCs以时间依赖性方式激活MAPK/ERK信号通路。PD98059阻断后vWF、VE-cadherin的蛋白表达均明显下降,DiL-Ac-LDL摄取功能也显著下降。结论: 1.在SW620细胞上清液诱导下,DCs的生长过程和功能受抑制,并且未成熟DCs上调内皮细胞特异性标志和功能,提示其发生内皮样分化。 2.SW620细胞上清通过MAPK/ERK信号途径诱导未成熟DCs内皮样分化。使用PD98059阻断剂后可明显阻断其内皮样分化进程。
[Abstract]:Research background:
Dendritic cells (dendritic cells DCs) is currently the most powerful antigen-presenting in professional antigen-presenting cells (antigen, presenting, cells, APCs) is started, regulation, central link and maintain immune responses. In antitumor immunity and play an important role. However, many studies have demonstrated that the phenotype and function in presentation peripheral blood of patients with tumor and tumor infiltrating dendritic cells decreased, tumor cells can secrete a variety of immunosuppressive factors inhibit the function of DCs.
No limit the growth of tumors require blood supply. The traditional theory that tumor angiogenesis is through angiogenesis (angiogenesis) from the process of the existing micro vascular sprouting of new capillaries, the present study found that also through angiogenesis (vasculogenesis) - by the process of endothelial precursor cells to form new blood vessels endothelial. Cells (endothelial cells, ECs) can be derived from bone marrow endothelial progenitor cells (endothelialprogenitors, EPCs) and CD34~+ in peripheral blood cells. Studies have confirmed that the majority of tumor cells have high expression of vascular endothelial growth factor (vascular endothelial, growth factor, VEGF VEGF), the MAPK/ERK signaling pathways stimulated the proliferation and differentiation of endothelial cells in order to promote tumor angiogenesis,.MAPK/ERK pathway is one of the important signaling pathway on the differentiation of other cells in the body.
The latest research reports, in mouse and human ovarian carcinomas discovered a new leukocyte subsets, the expression of markers of DCs and endothelial cells. Other scholars reported monocyte culture medium under the invasion and differentiation of tumor related to dendritic cells in tumor conditions (tumor-associated dendritic cells, TADCs), such cells can induce angiogenesis factors such as VEGF and tumorigenesis of M differentiation into endothelial like cells (endothelial-like, cells, ELCs), and reticular formation in Matrigel. Ourprevious studies the node supernatant of colon cancer SW620 cells can inhibit the maturation of DCs (immature dendritic cells, iDCs) expression the growth process and related antigen, and immature DCs in SW620 cells was induced in dermoid differentiation. These studies reveal the tumor related environment DCs is inhibited in antitumor immune aspects Part of the DCs system, also occurred in dermoid differentiation. In recent years at home and abroad research on DCs mostly focus on how to improve the immune function of DCs anti-tumor and other aspects, while the endothelial like differentiation of tumor microenvironment under the occurrence of DCs and its mechanism still needs further study.
Objective:
This experiment to investigate the influence of node micro environment soluble cytokine secretion of SW620 cells of human peripheral blood mononuclear cells from different periods of DCs epidermoid differentiation, and activation of MAPK/ERK signaling pathway in the process.
Experimental methods:
1. the supernatant of colon cancer SW620 cells was prepared.
2. healthy volunteers were collected fresh peripheral blood from human peripheral blood mononuclear cells by density gradient centrifugation. RPMI 1640 medium cell concentration was adjusted to 3 * 10~6/ml, inoculated in 24 well plates, each hole 1 ml into carbon dioxide incubator (5%/CO_2,37 C) standing for 3 h, the removal of suspended cells obtain mononuclear cells adherent growth, containing rhGM-CSF (100 ng/ml), IL-4 (5 ng/ml) 1640 and 10% autologous serum culture medium, the 5D LPS (5 ng/ml). In addition to adding the culture medium induced group, respectively in 2D (immature DCs induction group), 7d (mature DCs induced group) divided into SW620 cell supernatant. The next day was half changed, the induction of 7d. and 14d were in the 9D group and the control group were collected by DCs. in the training process observation of cell morphology and cell counting; extraction of total protein, Western blotting detection of endothelial cell specific markers vWF, VE-cadheri The expression of N was observed by transmission electron microscope; WP bodies; the change of cell proliferation induced by antigen presenting function and killing experiment observation; acetylated low density lipoprotein uptake experiment (DiL-Ac-LDL uptake assay) to know whether the induced cells have the function of endothelial cells; Westernblotting detection of SW620 cell supernatant on Min effect of 15,30,60 ERK1/2 levels of the immature DCs after the stimulation. The use of ERK1/2 upstream MEK kinase inhibitor PD98059, observe the MAPK/ERK pathway after inhibition of SW620 cells induced by supernatant of vWf cells, the expression of VE-cadherin protein and DiL-Ac-LDL uptake.
The primary umbilical vein endothelial cells were cultured with 3. trypsin method and endothelial cell culture medium as the positive control group.
Statistical software package SPSS 12 was used to conduct statistical analysis of the results. The measured data were expressed by mean + standard deviation ((?) s). After determining the homogeneity of variance, t test or variance analysis were performed. P < 0.05 was a significant difference.
Result:
1. in the supernatant of SW620 cells induced by immature DCs cells growth and the state is obviously behind the control group DCs, the cell density is low, and the formation of tube like and cord like structure; the control group DCs showed typical characteristics of dendritic cells, irregular cell morphology, burr like protrusions with unequal thickness. Suspended trend; mature DCs induced cells with the control group DCs showed no significant difference.
2.Western blotting test results show that: the immature DCs cells with SW620 induced by the supernatant after 7d, vWF specific markers of endothelial cells, compared with the control group VE-cadherin DCs, showed obvious expression and the differences between the two groups was statistically significant. TEM results showed that the SW620 cells induced by the supernatant of immature DCs intracellular specific structure WP body.DCs function detection of endothelial cells decreased in DCs induced group cells of immature antigen-presenting ability, and endothelial cell specific acetylated low density lipoprotein uptake enhanced.SW620 cell supernatant induced by immature DCs activation of MAPK/ERK signaling pathway after occlusion of.PD98059 vWF in a time dependent manner, the expression of VE-cadherin protein was significantly decreased, DiL-Ac-LDL uptake also decreased significantly.
Conclusion:
1. in the supernatant of SW620 cells induced by growth process and the function of DCs was inhibited, and immature DCs up regulate endothelial cell specific markers and function, suggesting the occurrence of endothelial differentiation.
2.SW620 cell supernatant induced by immature DCs differentiate into endothelial like cells through MAPK/ERK signaling pathway. PD98059 blocker can obviously block the endothelial differentiation process.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392;R735.35

【参考文献】