树突状细胞在血管内皮生长因子作用下内皮样化现象的初步探讨

发布时间：2018-02-23 18:12

本文关键词： 树突状细胞血管内皮生长因子内皮样化　出处：《郑州大学》2007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 cell，DC)是目前已知的体内抗原呈递功能最强的，而且是唯一能激活初始T细胞(naive T cell)的专职抗原呈递细胞(antigen presenting cell，APC)，是启动、调控、并维持免疫反应的中心环节。由DC激活的T细胞介导的免疫反应在机体抗肿瘤免疫中起着主导作用，DC的数量或功能的缺陷是肿瘤逃脱机体免疫监视的重要原因，因此DC的生物功能状态与肿瘤的发生发展有着重要的关系。血管内皮生长因子(vascular endothelial growth factor，VEGF)，是肿瘤细胞分泌的众多因子中的一种强力的血管生成因子，是高效的内皮细胞有丝分裂原，可直接特异地作用于内皮细胞，促进肿瘤血管新生。近年来研究已经证实，VEGF可通过抑制核因子-κB(NF-κB)的活性，使DC分化成熟障碍，并可影响DC的功能及促使其凋亡。VEGF对DC的抑制降低了机体的抗肿瘤免疫功能，从而使肿瘤细胞逃脱机体的免疫监管，促进肿瘤的生长。最新研究报道，在高分泌VEGF的荷瘤鼠和人卵巢癌肿瘤组织中发现了一种新的细胞群，能够同时表达DC和内皮细胞的标志，且体外培养的小鼠骨髓来源的DC在用高表达VEGF的肿瘤条件培养基培养时也可出现内皮样化改变，这些研究提示我们在高分泌VEGF的肿瘤环境中DC有可能出现内皮样化，但具体机制尚不清楚。这种内皮样化现象使DC在抗肿瘤免疫反应中不能发挥应有的抗原呈递作用，不但使成熟有功能的DC数量进一步减少，而且内皮样化的DC有可能参与肿瘤血管形成，反而促进肿瘤生长。国内外近年来关于DC的研究多集中于如何提高DC的抗原呈递功能及制备有效的DC瘤苗等方面，而关于DC内皮样化现象及其在人类肿瘤血管形成中的作用等则有待于进一步的研究。目的本实验拟对体外培养的人外周血单个核细胞来源的DC在VEGF诱导下能否发生内皮样化现象进行初步探讨。实验方法采集健康志愿者新鲜外周血，经Ficoll密度梯度离心法获取单个核细胞，用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1ml，移入二氧化碳孵育箱(5％CO_2，37℃)静置3h，去除悬浮细胞，获取贴壁生长的DC前体细胞，，各孔中加入1ml含rhGM-CSF(100ng／ml)和rhIL-4(5ng／ml)的RPMI 1640完全培养液培养。培养过程中观察细胞形态并计数。正常对照组在培养过程中收集第1d、3d、7d、10d、14d的细胞采用流式细胞仪动态检测细胞表型变化。实验组于培养第3d和第7d分别加入rhVEGF(10ng／ml和100ng／ml)进行诱导培养，并于隔天半量换液时半量补充因子量。分别用VEGF诱导培养7d后收集细胞，经荧光素标记抗体后，采用流式细胞技术检测DC特异性标志CD1a和内皮细胞特异性表面分子CD34、CD31的表达情况，并分别用免疫荧光法及免疫细胞化学法检测内皮细胞特异性标志vWF的表达情况。采用统计学软件包SPSS12.0对结果进行统计分析，结果用(?)±s表示，各组之间的比较采用单因素方差分析(ONE-WAY ANOVA)，组间两两比较用q检验，显著性检验水准为α=0.05。结果 1．正常对照组细胞在培养的第5d开始出现突起，第7d时突起更加明显，并且大量突起交织，之后细胞逐渐增大变圆，出现悬浮趋势。VEGF诱导的各实验组细胞生长过程明显落后于正常对照组，而且细胞密度较低。 2．正常对照组细胞在培养初期出现少量CD34、CD31表达，随培养时间的延长，其表达量逐渐降低，而DC的特异性标志CD1a的表达量逐渐升高。用VEGF诱导7d后，各实验组细胞CD1a的表达受抑制。各实验组细胞CD1a与内皮细胞表面标志CD34、CD31的共表达率均高于相应正常对照组，且差异具有统计学意义，并且其共表达情况存在如下趋势：3d大剂量组＞7d大剂量组＞3d小剂量组和7d小剂量组。 3．用VEGF诱导的实验组中存在胞浆着色的阳性细胞，提示有vWF的表达，并且各实验组之间的表达趋势和上述流式细胞仪检测到的共表达趋势相似，而且积分光密度(IOD)和阳性区面积百分比(Aera％)均高于其相应正常对照组，且差异具有统计学意义。结论： 1．在体外培养过程中，VEGF能抑制或减缓DC的生长过程，并使DC的特异性表面标志CD1a表达减少。 2．在VEGF诱导下，DC可以表达一些内皮细胞特异性标志(CD31、CD34、vWF)，出现内皮样化倾向，而且大剂量诱导比小剂量诱导更容易出现内皮样化倾向、早期诱导比晚期诱导更容易出现内皮样化倾向。
[Abstract]:Research background
Dendritic cells (dendritic cell DC) is currently the most powerful antigen-presenting body, and is the only way to activate naive T cells (naive T cell) professional antigen-presenting cells (antigen, presenting, cell, APC) is the start, control, and maintain the central link of the immune response plays a dominant role in T. Cell mediated immune response activated by DC in anti tumor immune, the number of defects or function of DC is an important cause of tumor escape from immune surveillance, so the occurrence and development of tumor and biological function of DC has an important relationship.
Vascular endothelial growth factor (vascular endothelial, factor growth, VEGF) is a potent angiogenic factor secreted by tumor cells in many factors, is an effective mitogen of endothelial cells, can direct specific effect on endothelial cells, promote tumor angiogenesis. Recent studies have confirmed that VEGF can inhibit nuclear factor kappa B (NF- K B) activity, the DC differentiation and maturation disorder, and can inhibit the effect of the function of DC and induce the apoptosis of DC.VEGF reduced the anti tumor immune function, so that the tumor cells escape the immune regulation, promoting tumor growth.
The latest research reports, the secretion of VEGF murine and human ovarian cancer tissues from the discovery of a new group of cells, can express the markers of DC and endothelial cells, and cultured mouse bone marrow-derived DC with high expression of VEGF tumor conditioned medium also can appear when the change of endothelial like, these studies suggest that we in the secretion of VEGF in tumor environment DC likely endothelialisation, but the mechanism is still unclear. The endothelialisation phenomenon that DC can not play its due role in antigen presentation in the anti-tumor immune response, not only makes the mature quantity function of DC decreased further, and endothelial like the DC may be involved in tumor angiogenesis, but to promote tumor growth. DC tumor vaccine and other aspects of domestic and foreign research in recent years on the DC is focused on how to improve the antigen-presenting function of DC and the effective preparation, and to DC endothelia and its role in human tumor angiogenesis need to be further studied.
objective
This experiment is intended to investigate the possibility of endothelia in human peripheral blood mononuclear cells derived from human peripheral blood mononuclear cells (DC) induced by VEGF in vitro.
Experimental method
Healthy volunteers collected fresh peripheral blood mononuclear cells, obtained by Ficoll density gradient centrifugation method, using RPMI 1640 complete medium cell concentration was adjusted to 3 * 10~6 / ml were seeded in 24 well plates, each hole 1ml, into carbon dioxide incubator (5%CO_2,37 C) static 3h, remove the suspension cells. To obtain adherent DC precursor cells, into the holes in the 1ml containing rhGM-CSF (100ng / ml) and rhIL-4 (5ng / ml) RPMI 1640 were cultured. During the culture cells were observed and counted. The normal control group were collected at 1D, in the training process of 3D, 7d, 10d, 14d the cells by flow cytometry dynamic changes in phenotype. In the experimental group training section 3D and 7d were added to rhVEGF (10NG / ml and 100ng / ml) were cultured, and the next day when the quantity of liquid exchange half half amount of complement factor respectively. Induced by VEGF after cultured 7d cells were collected by fluorescein standard After antibody, the expression of DC specific marker CD1a and endothelial specific surface molecule CD34 and CD31 were detected by flow cytometry. The expression of vWF was detected by immunofluorescence and immunocytochemistry.
Statistical software package SPSS12.0 was used to analyze the results. The results were expressed by (+) s. The comparison between the groups was performed by one-way ANOVA (ONE-WAY ANOVA). Q comparison was used in the comparison between 22 groups, with a significant test level of alpha =0.05..
Result
1. normal control group cells began to appear in the processes of 5D culture, the 7d projection is more obvious, and a lot of protrusions intertwined, after cells gradually became round, the growth process was significantly lower than that in normal control group in each experimental group cell suspension trend induced by.VEGF, and the cell density is low.
2. normal control group cells occurred at the beginning of a small amount of CD34, the expression of CD31, with prolonged incubation time, the expression decreased gradually, while the DC specific marker CD1a expression gradually increased by VEGF. After 7d induction, the expression of each experimental group CD1a cells was inhibited. The experimental group of CD1a and endothelial cells cell surface marker CD34 was better than that of the normal control group co expression of CD31, and the difference was statistically significant, and the co expression has the following trends: high dose group 3D > 7d > 3D in high dose group and small dose group 7d small dose group.
Positive cells existed in cytoplasm of experimental group induced by VEGF in 3., suggesting that the expression of vWF, and the expression trend between the experimental group and the flow cytometry detected the co expression of a similar trend, and the integral optical density (IOD) and the percentage of positive area (Aera%) were higher than those of the normal the control group, and the difference was statistically significant.
Conclusion:
1. in the process of culture in vitro, VEGF can inhibit or slow down the growth of DC, and reduce the expression of the specific surface of DC on the expression of CD1a.
2., under the guidance of VEGF, DC can express some endothelial specific markers (CD31, CD34, vWF), showing endothelia like tendency. Moreover, high-dose induction is more prone to endothelia like tendency than low dose induction. Early induction is more prone to endothelial like tilting than late induction.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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