本文關(guān)鍵詞：樹(shù)突狀細(xì)胞在結(jié)腸癌細(xì)胞SW620上清液作用下內(nèi)皮樣分化傾向的研究　出處：《鄭州大學(xué)》2008年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 樹(shù)突狀細(xì)胞 內(nèi)皮樣分化 人臍靜脈內(nèi)皮細(xì)胞 流式細(xì)胞術(shù)

【摘要】：研究背景 樹(shù)突狀細(xì)胞(dendritic cell,DC)是在機(jī)體的免疫應(yīng)答過(guò)程中發(fā)揮著關(guān)鍵作用的細(xì)胞群體,其傳統(tǒng)的抗原提呈功能近年來(lái)已得到廣泛的研究。DC的APC功能即在體內(nèi)激活T細(xì)胞反應(yīng)。DC不僅提成抗原給未致敏的T細(xì)胞誘發(fā)其反應(yīng),而且還能產(chǎn)生刺激分子使靜止的T細(xì)胞進(jìn)入細(xì)胞周期并刺激它們的活化。然而,多數(shù)研究證實(shí)腫瘤浸潤(rùn)性樹(shù)突狀細(xì)胞的表型及其遞呈功能均下降,腫瘤組織分泌多種免疫因子均能抑制DC的成熟,例如IL-10,VEGF或IL-6和M-CSF等。因此,DC的數(shù)量及功能成熟與否與腫瘤的發(fā)生發(fā)展密切相關(guān)。 腫瘤血管形成是腫瘤迅速生長(zhǎng)重要標(biāo)志。傳統(tǒng)理論認(rèn)為只有通過(guò)血管新生(angiogenesis)即預(yù)存的內(nèi)皮細(xì)胞經(jīng)發(fā)芽形成毛細(xì)血管;血管發(fā)生(vasculogenesis)主要在胚胎期,近來(lái)研究表明血管發(fā)生在成年生物也可實(shí)現(xiàn),腫瘤的血管化作用也包括血管發(fā)生,即內(nèi)皮細(xì)胞從不成熟的前體細(xì)胞發(fā)育而來(lái)。目前認(rèn)為,bFGF、VEGF、HGF與腫瘤血管形成有關(guān)。 2004年《Nature Medicine》報(bào)道Coukos G等在小鼠及人的卵巢癌上發(fā)現(xiàn)了一種新型的共表達(dá)DC和血管內(nèi)皮細(xì)胞的標(biāo)志的白細(xì)胞群,接著有學(xué)者提出了腫瘤相關(guān)DC在腫瘤微環(huán)境作用下可能發(fā)生內(nèi)皮樣分化。這些研究揭示在腫瘤相關(guān)環(huán)境下DC在抗腫瘤免疫方面受到抑制,部分DC還可能發(fā)生內(nèi)皮樣分化,參與腫瘤血管的形成,促進(jìn)腫瘤生長(zhǎng)。國(guó)內(nèi)外近年來(lái)關(guān)于DC的研究多集中于如何提高DC抗腫瘤的免疫功能等方面,而腫瘤微環(huán)境下DC的內(nèi)皮樣分化傾向發(fā)生及其機(jī)制以及如何參與腫瘤血管的形成等還有待于進(jìn)一步的研究。 目的 本實(shí)驗(yàn)探討結(jié)腸癌細(xì)胞SW620分泌的可溶性細(xì)胞因子營(yíng)造的微環(huán)境對(duì)人單個(gè)核細(xì)胞來(lái)源的不同時(shí)期樹(shù)突狀細(xì)胞(Dendritic cells,DCs)分化發(fā)育的影響,以及是否發(fā)生內(nèi)皮樣分化的傾向。 實(shí)驗(yàn)方法 1.制備結(jié)腸癌細(xì)胞SW620培養(yǎng)上清液。 2.采集健康志愿者新鮮外周血,密度梯度離心法分離人外周血單個(gè)核細(xì)胞,RPMI1640完全培養(yǎng)液調(diào)整細(xì)胞濃度為3×10~6/ml,接種于24孔培養(yǎng)板中,每孔1ml,移入二氧化碳孵育箱(5%CO_2,37℃)靜置3h,去除懸浮細(xì)胞,獲取貼壁生長(zhǎng)的單核細(xì)胞,加入含GM-CSF(100 ng/ml)、IL-4(5ng/ml)和10%自體血清的1640培養(yǎng)液培養(yǎng)DC,誘導(dǎo)組除加入上述培養(yǎng)液外,分別于第2d、7d分組加入SW620培養(yǎng)上清液,隔天半量換液,各誘導(dǎo)7d。分別于第10d和14d收集誘導(dǎo)組DC和對(duì)照組DC。 3.培養(yǎng)過(guò)程中觀察細(xì)胞形態(tài)并計(jì)數(shù)。各組經(jīng)熒光素標(biāo)記抗體后,采用流式細(xì)胞技術(shù)檢測(cè)其免疫表型CD86,CD1a,CD11c;免疫熒光法檢測(cè)各組內(nèi)皮細(xì)胞特異性標(biāo)志vWF的表達(dá)情況;采用RT-PCR檢測(cè)各組特異標(biāo)記(CD1a、vWF和CD144)的基因表達(dá);顯微鏡觀察鋪有纖維粘連蛋白玻片上各組細(xì)胞條索樣結(jié)構(gòu)形成情況。 4.胰蛋白酶法配合內(nèi)皮細(xì)胞培養(yǎng)基培養(yǎng)原代臍靜脈內(nèi)皮細(xì)胞,作為陽(yáng)性對(duì)照組。采用統(tǒng)計(jì)學(xué)軟件包SPSS 12.0進(jìn)行結(jié)果的統(tǒng)計(jì)分析,將所得計(jì)量數(shù)據(jù)以平均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,確定方差齊性后,進(jìn)行t檢驗(yàn)或方差分析,P＜0.05為顯著性差異。 結(jié)果 1.正常DC在培養(yǎng)第3d開(kāi)始出現(xiàn)突起,第5d時(shí)突起更加明顯,并且第7d大量突起交織,之后細(xì)胞逐漸增大變圓,出現(xiàn)懸浮趨勢(shì)。SW620培養(yǎng)上清液的2d實(shí)驗(yàn)組細(xì)胞生長(zhǎng)過(guò)程及其狀態(tài)明顯落后于正常對(duì)照組,并且細(xì)胞密度較低;而7d誘導(dǎo)組細(xì)胞與DC對(duì)照組無(wú)明顯差別。 2.流式檢測(cè)結(jié)果:2d誘導(dǎo)組的未成熟DC經(jīng)SW620培養(yǎng)上清液誘導(dǎo)7d后,DC的特異標(biāo)記CD86、CD1a、CD11c與正常對(duì)照組DC相比,表達(dá)率均明顯下降且兩組間的差異具有統(tǒng)計(jì)學(xué)意義。7d誘導(dǎo)組的相對(duì)成熟DC誘導(dǎo)7d后,與其相對(duì)的正常組比較,DC特異標(biāo)記表達(dá)率未見(jiàn)有明顯下降,而二組間也無(wú)統(tǒng)計(jì)學(xué)差異。 3.免疫細(xì)胞化學(xué)法檢測(cè)內(nèi)皮細(xì)胞特異標(biāo)志vWF結(jié)果:用SW620培養(yǎng)上清液誘導(dǎo)的2d誘導(dǎo)組中存在胞漿著色的陽(yáng)性細(xì)胞,提示有vWF的表達(dá),而7d誘導(dǎo)組和正常對(duì)照組胞漿著色不明顯,而且2d誘導(dǎo)組的陽(yáng)性區(qū)面積百分比和陽(yáng)性區(qū)平均灰度高于其相應(yīng)正常對(duì)照組,且差異具有統(tǒng)計(jì)學(xué)意義;7d誘導(dǎo)組與其相應(yīng)正常對(duì)照組相比無(wú)統(tǒng)計(jì)學(xué)意義。 4.RT-PCR檢測(cè)各組特異標(biāo)記的基因表達(dá)結(jié)果:早期2d誘導(dǎo)組細(xì)胞目的基因條帶中有CD144、vWF,而CD1a缺失;其相應(yīng)的正常對(duì)照組有CD1a基因的表達(dá),而CD144、vWF低表達(dá)。7d誘導(dǎo)組與其相應(yīng)的正常組比較均有CD1a的表達(dá),CD144、vWF表達(dá)不明顯。 5.顯微鏡觀察條索樣結(jié)構(gòu):2d誘導(dǎo)組細(xì)胞在鋪有纖維粘連蛋白的96孔板里形成相似于HUVEC陽(yáng)性對(duì)照組的條索樣結(jié)構(gòu);而正常組DC和7d誘導(dǎo)組細(xì)胞均未有明顯的索樣結(jié)構(gòu)形成。 結(jié)論: 1.SW620上清液能抑制DC的生長(zhǎng)過(guò)程及相關(guān)抗原表達(dá),這可能是腫瘤細(xì)胞逃避機(jī)體免疫監(jiān)視的機(jī)制之一。 2.在SW620上清液誘導(dǎo)下,早期未成熟DC與晚期相對(duì)成熟DC比較,較高表達(dá)內(nèi)皮細(xì)胞特異性標(biāo)志CD144、vWF,較易形成內(nèi)皮細(xì)胞特異的條索樣結(jié)構(gòu),出現(xiàn)內(nèi)皮樣分化傾向。
[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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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