發(fā)布時(shí)間：2018-01-04 16:30

  本文關(guān)鍵詞：人結(jié)腸癌SW620細(xì)胞上清作用下樹突狀細(xì)胞內(nèi)皮樣分化的研究　出處：《鄭州大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 樹突狀細(xì)胞 內(nèi)皮樣分化 人臍靜脈內(nèi)皮細(xì)胞 MAPK信號(hào)通路 ERK

【摘要】：研究背景: 樹突狀細(xì)胞(dendritic cells,DCs)是目前已知的體內(nèi)抗原呈遞功能最強(qiáng)的專職抗原呈遞細(xì)胞(antigen presenting cells,APCs),是啟動(dòng)、調(diào)控、并維持免疫反應(yīng)的中心環(huán)節(jié)。在抗腫瘤免疫中發(fā)揮著重要作用。然而,許多研究證實(shí)在腫瘤患者循環(huán)血中和腫瘤周圍浸潤的樹突狀細(xì)胞的表型及其呈遞功能均下降,腫瘤細(xì)胞可以分泌多種免疫抑制因子抑制DCs的功能成熟。 腫瘤的無限制生長需要血管的供應(yīng)。傳統(tǒng)理論認(rèn)為腫瘤血管形成是通過血管新生(angiogenesis)即從已存在的微血管上芽生出新的毛細(xì)血管的過程,目前研究發(fā)現(xiàn)也可以通過血管發(fā)生(vasculogenesis)——由內(nèi)皮前體細(xì)胞分化形成新血管的過程。內(nèi)皮細(xì)胞(endothelial cells,ECs)可以來源于骨髓內(nèi)皮前體細(xì)胞(endothelialprogenitors,EPCs)和人外周血中CD34~+細(xì)胞群。研究證實(shí)多數(shù)腫瘤細(xì)胞都高表達(dá)血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF),VEGF可以通過MAPK/ERK信號(hào)途徑刺激內(nèi)皮細(xì)胞的增殖和分化,從而促進(jìn)腫瘤血管生成。MAPK/ERK途徑是體內(nèi)外多種細(xì)胞分化的重要信號(hào)通路之一。 最新研究報(bào)道,在小鼠和人卵巢癌組織中發(fā)現(xiàn)了一種新的白細(xì)胞亞群,同時(shí)表達(dá)DCs和內(nèi)皮細(xì)胞的標(biāo)志。另有學(xué)者報(bào)道在腫瘤條件培養(yǎng)基浸潤下的單核細(xì)胞能分化發(fā)育為腫瘤相關(guān)性樹突細(xì)胞(tumor-associated dendritic cells,TADCs),此類細(xì)胞能在血管生成因子如VEGF和致瘤素M的誘導(dǎo)下分化發(fā)育為內(nèi)皮樣細(xì)胞(endothelial-like cells,ELCs),并且在基質(zhì)膠上可以形成網(wǎng)狀結(jié)構(gòu)。本課題組前期研究發(fā)現(xiàn)結(jié)腸癌SW620細(xì)胞上清能抑制未成熟DCs(immature dendritic cells,iDCs)的生長過程及相關(guān)抗原表達(dá),并且未成熟DCs在SW620細(xì)胞上清誘導(dǎo)下發(fā)生內(nèi)皮樣分化。這些研究揭示在腫瘤相關(guān)環(huán)境下DCs在抗腫瘤免疫方面受到抑制,部分DCs還發(fā)生內(nèi)皮樣分化。國內(nèi)外近年來關(guān)于DCs的研究多集中于如何提高DCs抗腫瘤的免疫功能等方面,而腫瘤微環(huán)境下DCs的內(nèi)皮樣分化發(fā)生及其機(jī)制等還有待于進(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)胞因子營造的微環(huán)境對(duì)人外周血單個(gè)核細(xì)胞來源的不同時(shí)期DCs發(fā)生內(nèi)皮樣分化的影響,以及MAPK/ERK信號(hào)途徑在此過程中的激活情況。 實(shí)驗(yàn)方法: 1.制備結(jié)腸癌SW620細(xì)胞上清液。 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,RPMI 1640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1 ml,移入二氧化碳孵育箱(5%/CO_2,37℃)靜置3 h,去除懸浮細(xì)胞,獲取貼壁生長的單核細(xì)胞,加入含rhGM-CSF(100 ng/ml)、IL-4(5 ng/ml)和10%自體血清的1640培養(yǎng)液,第5d加入LPS(5 ng/ml)。誘導(dǎo)組除加入上述培養(yǎng)液外,分別于第2d(未成熟DCs誘導(dǎo)組)、第7d(成熟DCs誘導(dǎo)組)分組加入SW620細(xì)胞上清液。隔天半量換液,各誘導(dǎo)7d。分別于第9d和14d收集誘導(dǎo)組細(xì)胞和對(duì)照組DCs。培養(yǎng)過程中觀察細(xì)胞形態(tài)并計(jì)數(shù);提取各組細(xì)胞總蛋白,Western blotting檢測(cè)內(nèi)皮細(xì)胞特異性標(biāo)志vWF、VE-cadherin的表達(dá)情況;透射電鏡觀察WP小體;增殖及殺傷實(shí)驗(yàn)觀察誘導(dǎo)后細(xì)胞抗原呈遞功能的改變;乙�；兔芏戎鞍讛z取實(shí)驗(yàn)(DiL-Ac-LDL uptake assay)了解誘導(dǎo)后細(xì)胞是否具有內(nèi)皮細(xì)胞的功能;Westernblotting檢測(cè)SW620細(xì)胞上清液對(duì)未成熟DCs刺激后15、30、60 min的ERK1/2水平的影響。使用ERK1/2上游激酶MEK的阻斷劑PD98059,觀察MAPK/ERK通路阻斷后,SW620細(xì)胞上清誘導(dǎo)組細(xì)胞vWf、VE-cadherin的蛋白表達(dá)情況和DiL-Ac-LDL攝取功能。 3.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.在SW620細(xì)胞上清液誘導(dǎo)下,未成熟DCs誘導(dǎo)組細(xì)胞生長過程及其狀態(tài)明顯落后于對(duì)照組DCs,細(xì)胞密度較低,并且形成類管腔樣和條索樣結(jié)構(gòu);其對(duì)照組DCs則表現(xiàn)典型樹突狀細(xì)胞特征,細(xì)胞形態(tài)不規(guī)則,有粗細(xì)不等的毛刺狀突起,有懸浮趨勢(shì);而成熟DCs誘導(dǎo)組細(xì)胞與其對(duì)照組DCs無明顯差異。 2.Western blotting檢測(cè)結(jié)果顯示:未成熟DCs經(jīng)SW620細(xì)胞上清液誘導(dǎo)7d后,內(nèi)皮細(xì)胞的特異標(biāo)記vWF、VE-cadherin與對(duì)照組DCs相比,均出現(xiàn)明顯表達(dá)且兩組間的差異有統(tǒng)計(jì)學(xué)意義。透射電鏡結(jié)果顯示:經(jīng)SW620細(xì)胞上清液誘導(dǎo)的未成熟DCs胞內(nèi)出現(xiàn)內(nèi)皮細(xì)胞的特異性結(jié)構(gòu)WP小體。DCs方面功能檢測(cè):未成熟DCs誘導(dǎo)組細(xì)胞的抗原提呈能力下降,而內(nèi)皮細(xì)胞特異的乙�；兔芏戎鞍讛z取功能增強(qiáng)。SW620細(xì)胞上清誘導(dǎo)未成熟DCs以時(shí)間依賴性方式激活MAPK/ERK信號(hào)通路。PD98059阻斷后vWF、VE-cadherin的蛋白表達(dá)均明顯下降,DiL-Ac-LDL攝取功能也顯著下降。 結(jié)論: 1.在SW620細(xì)胞上清液誘導(dǎo)下,DCs的生長過程和功能受抑制,并且未成熟DCs上調(diào)內(nèi)皮細(xì)胞特異性標(biāo)志和功能,提示其發(fā)生內(nèi)皮樣分化。 2.SW620細(xì)胞上清通過MAPK/ERK信號(hào)途徑誘導(dǎo)未成熟DCs內(nèi)皮樣分化。使用PD98059阻斷劑后可明顯阻斷其內(nèi)皮樣分化進(jìn)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.

【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】：2009
【分類號(hào)】：R392;R735.35

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本文編號(hào)：1379199