树突状细胞在结肠癌细胞SW620上清液作用下内皮样分化倾向的研究

发布时间：2018-01-13 14:07

本文关键词：树突状细胞在结肠癌细胞SW620上清液作用下内皮样分化倾向的研究　出处：《郑州大学》2008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)是在机体的免疫应答过程中发挥着关键作用的细胞群体,其传统的抗原提呈功能近年来已得到广泛的研究。DC的APC功能即在体内激活T细胞反应。DC不仅提成抗原给未致敏的T细胞诱发其反应,而且还能产生刺激分子使静止的T细胞进入细胞周期并刺激它们的活化。然而,多数研究证实肿瘤浸润性树突状细胞的表型及其递呈功能均下降,肿瘤组织分泌多种免疫因子均能抑制DC的成熟,例如IL-10,VEGF或IL-6和M-CSF等。因此,DC的数量及功能成熟与否与肿瘤的发生发展密切相关。肿瘤血管形成是肿瘤迅速生长重要标志。传统理论认为只有通过血管新生(angiogenesis)即预存的内皮细胞经发芽形成毛细血管;血管发生(vasculogenesis)主要在胚胎期,近来研究表明血管发生在成年生物也可实现,肿瘤的血管化作用也包括血管发生,即内皮细胞从不成熟的前体细胞发育而来。目前认为,bFGF、VEGF、HGF与肿瘤血管形成有关。 2004年《Nature Medicine》报道Coukos G等在小鼠及人的卵巢癌上发现了一种新型的共表达DC和血管内皮细胞的标志的白细胞群,接着有学者提出了肿瘤相关DC在肿瘤微环境作用下可能发生内皮样分化。这些研究揭示在肿瘤相关环境下DC在抗肿瘤免疫方面受到抑制,部分DC还可能发生内皮样分化,参与肿瘤血管的形成,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抗肿瘤的免疫功能等方面,而肿瘤微环境下DC的内皮样分化倾向发生及其机制以及如何参与肿瘤血管的形成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单个核细胞来源的不同时期树突状细胞(Dendritic cells,DCs)分化发育的影响,以及是否发生内皮样分化的倾向。实验方法 1.制备结肠癌细胞SW620培养上清液。 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,RPMI1640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,获取贴壁生长的单核细胞,加入含GM-CSF(100 ng/ml)、IL-4(5ng/ml)和10%自体血清的1640培养液培养DC,诱导组除加入上述培养液外,分别于第2d、7d分组加入SW620培养上清液,隔天半量换液,各诱导7d。分别于第10d和14d收集诱导组DC和对照组DC。 3.培养过程中观察细胞形态并计数。各组经荧光素标记抗体后,采用流式细胞技术检测其免疫表型CD86,CD1a,CD11c;免疫荧光法检测各组内皮细胞特异性标志vWF的表达情况;采用RT-PCR检测各组特异标记(CD1a、vWF和CD144)的基因表达;显微镜观察铺有纤维粘连蛋白玻片上各组细胞条索样结构形成情况。 4.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.正常DC在培养第3d开始出现突起,第5d时突起更加明显,并且第7d大量突起交织,之后细胞逐渐增大变圆,出现悬浮趋势。SW620培养上清液的2d实验组细胞生长过程及其状态明显落后于正常对照组,并且细胞密度较低;而7d诱导组细胞与DC对照组无明显差别。 2.流式检测结果:2d诱导组的未成熟DC经SW620培养上清液诱导7d后,DC的特异标记CD86、CD1a、CD11c与正常对照组DC相比,表达率均明显下降且两组间的差异具有统计学意义。7d诱导组的相对成熟DC诱导7d后,与其相对的正常组比较,DC特异标记表达率未见有明显下降,而二组间也无统计学差异。 3.免疫细胞化学法检测内皮细胞特异标志vWF结果:用SW620培养上清液诱导的2d诱导组中存在胞浆着色的阳性细胞,提示有vWF的表达,而7d诱导组和正常对照组胞浆着色不明显,而且2d诱导组的阳性区面积百分比和阳性区平均灰度高于其相应正常对照组,且差异具有统计学意义;7d诱导组与其相应正常对照组相比无统计学意义。 4.RT-PCR检测各组特异标记的基因表达结果:早期2d诱导组细胞目的基因条带中有CD144、vWF,而CD1a缺失;其相应的正常对照组有CD1a基因的表达,而CD144、vWF低表达。7d诱导组与其相应的正常组比较均有CD1a的表达,CD144、vWF表达不明显。 5.显微镜观察条索样结构:2d诱导组细胞在铺有纤维粘连蛋白的96孔板里形成相似于HUVEC阳性对照组的条索样结构;而正常组DC和7d诱导组细胞均未有明显的索样结构形成。结论: 1.SW620上清液能抑制DC的生长过程及相关抗原表达,这可能是肿瘤细胞逃避机体免疫监视的机制之一。 2.在SW620上清液诱导下,早期未成熟DC与晚期相对成熟DC比较,较高表达内皮细胞特异性标志CD144、vWF,较易形成内皮细胞特异的条索样结构,出现内皮样分化倾向。
[Abstract]:Research background
Dendritic cells (dendritic cell DC) is a group of cells play a key role in the process of immune response, the antigen presenting function in recent years has been extensive research on.DC APC function is activated in vivo T cell reaction of.DC cells induced by T antigen sensitized to commission the reaction but also can produce stimulation molecules resting T cells into cell cycle and stimulate their activation. However, most of the studies have confirmed that the phenotype of tumor infiltrating dendritic cells and presenting function were decreased, the tumor tissue secretes a variety of immune factors could inhibit the maturation of DC, such as IL-10, VEGF or IL-6 and M-CSF. Therefore, close related to the occurrence and development of number and function of DC and the maturity of the tumor.
Tumor angiogenesis is an important hallmark of rapidly growing tumors. The traditional theory thinks that only through neovascularization (angiogenesis) is stored in endothelial cells to form capillary angiogenesis by germination; (vasculogenesis) mainly in the embryo, recent studies have shown that angiogenesis in adult organisms can also be achieved, vascularization of tumors including angiogenesis. The endothelial cells of immature precursor cells developed. Now that bFGF, VEGF, HGF and angiogenesis.
In 2004, reported Coukos G in mice and human ovarian cancer is found on the white cell group marks a new co expression of DC and vascular endothelial cells, then some scholars put forward the tumor related DC in tumor microenvironment may occur under the action of endothelial differentiation. These studies reveal the tumor related environment DC inhibited in anti tumor immunity, DC may also occur in the differentiation of dermoid, participating in tumor angiogenesis and promote tumor growth. In recent years at home and abroad research on DC mostly focus on how to improve the immune function of DC anti-tumor and other aspects, and the tendency of endothelial like differentiation of tumor micro environment and the occurrence of DC and its mechanism how to participate in the formation of tumor vessels and needs to be further studied.
objective
The effects of different periods of dendritic cells in human mononuclear cells derived with micro environment soluble cytokines secreted by SW620 cells (Dendritic, cells, DCs) differentiation and development, and whether the tendency to differentiate into endothelial like cells.
Experimental method
1. SW620 culture supernatant was prepared from colon cancer cells.
2. healthy volunteers were collected fresh peripheral blood from human peripheral blood mononuclear cells by density gradient centrifugation, RPMI1640 medium cell concentration was adjusted to 3 * 10~6/ml, inoculated in 24 well plates, each hole 1ml, into carbon dioxide incubator (5%CO_2,37 C) static 3h, in addition to suspension cells. To obtain adherent mononuclear cells growth, containing GM-CSF (100 ng/ml), IL-4 (5ng/ml) 1640 and 10% autologous serum cultured DC induced group in addition to adding the cultured liquid, respectively in 2D, 7d group added to SW620 culture supernatant, the next day was half changed, the induction of 7d. respectively the 10d and 14d collection by group DC and control group DC.
3. in the training process of cell morphology was observed and counted. Groups by fluorescein labeled antibody, flow cytometry was used to determine the immune phenotype of CD86, CD1a, CD11c; immunofluorescence method to detect endothelial cell specific marker vWF was detected by RT-PCR; specific markers (CD1a, vWF and CD144) gene expression; microscope slides covered with fibronectin cell cord like structure formation.
4. trypsin method with endothelial cell cultured primary human umbilical vein endothelial cells as the positive control group. The analysis results of statistical package SPSS 12 by using statistical software, the measurement data to mean + standard deviation ((?) + s) said, to determine the homogeneity of variance, t test or analysis variance, P < 0.05 for significant differences.
Result
1. normal DC at the start of training the 3D projection, projection is more obvious in 5D, and the 7d number of protrusions intertwined, after the cells gradually became round, suspension of trend of.SW620 cultured 2D cells in the experimental group was the growth process and state was significantly lower than that in normal control group, and the cell density is low; and 7d induced group DC cells and the control group had no significant difference.
2. flow cytometry results: immature DC 2D induced group by SW620 culture supernatant after 7d induction, DC CD1a specific markers CD86, CD11c, DC compared with the normal control group, the expression rate decreased significantly and the differences between the two groups was statistically significant.7d induction group was relatively mature DC induced by 7d, and the relative comparison the normal group, no specific DC marker expression rate decreased significantly, but there was no statistically significant difference between the two groups.
3. immunocytochemical detection of endothelial cell specific markers of vWF results: SW620 culture supernatant induced 2D positive cells existed in cytoplasm in the induced group, suggesting that the expression of vWF and 7d induced group and normal control group in cytoplasm is not obvious, but the average gray positive area percentage of 2D induced group and positive area is higher than that of the normal control group, and the difference was statistically significant; 7d induced group and its corresponding normal control group showed no statistical significance.
Results the expression of 4.RT-PCR was detected by specific marker genes: early gene 2D induced cells have CD144, with vWF, and the deletion of CD1a; the corresponding normal control group the expression of CD1a gene and CD144, low expression of vWF induced by.7d group normal group were compared with the corresponding expression of CD1a and CD144. The expression of vWF was not obvious.
5. microscope cordlike structure: 2D cells in 96 well plates covered with fibronectin form similar to HUVEC positive control group cordlike structure; and the DC normal group and 7d induced group cells had no obvious cable like structure formation.
Conclusion:
1.SW620 supernatant can inhibit the growth of DC and the expression of related antigens, which may be one of the mechanisms of tumor cells to escape the immune surveillance of the body.
2. in SW620 induced by the supernatant, early and late immature DC relatively mature DC, higher expression of endothelial cell specific markers CD144, vWF, is easy to form specific endothelial cells of cord like structure, internal dermoid differentiation tendency.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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