細(xì)胞因子對(duì)外周血樹突狀細(xì)胞的體外誘導(dǎo)成熟及其功能影響
發(fā)布時(shí)間：2018-07-23 17:09
【摘要】：[目的] 選用幾種不同的細(xì)胞因子組合,體外培養(yǎng)人外周血單個(gè)核細(xì)胞,誘導(dǎo)樹突狀細(xì)胞(dendritic cells,DCs)產(chǎn)生,并檢測(cè)其表面分子的表達(dá)及刺激同種異體T淋巴細(xì)胞增殖的功能,從而建立誘導(dǎo)樹突狀細(xì)胞的最佳實(shí)驗(yàn)方法。并探討細(xì)胞因子對(duì)DCs體外誘導(dǎo)、成熟及其功能的影響。 [方法] 無(wú)菌采集健康人外周血,肝素鈉抗凝,采用密度梯度離心的方法獲得單個(gè)核細(xì)胞,用含10%胎牛血清的RPMI1640培養(yǎng)基在24孔板上貼壁培養(yǎng)2小時(shí),棄去未貼壁細(xì)胞,即獲得貼壁細(xì)胞,在24孔板上用含以下6組不同細(xì)胞因子的RPMI1640培養(yǎng)基進(jìn)行培養(yǎng)①對(duì)照組;不加細(xì)胞因子②rhGMCSF/rhIL-4③rhGM-CSF/rhIL-4/PHA④rhGM-CSF/rhIL-13⑤rhGM-CSF/rhIL-13/PHA⑥PHA,各利因子加入的劑量分別為(在培養(yǎng)液中的最終濃度):rhGM-CSF(50ng/ml)、rhIL-4(40ng/ml)、rhIL-13(40ng/ml)、PHA(50μg/ml),觀察DC的生成及免疫表型的表達(dá)情況。6天后,分別在各中加入LPS、IL-2、TNF-α等促成熟因子誘導(dǎo)DC成熟,加入的劑量分別為:LPS(20ng/ml)、IL-2(5ng/ml)、TNF-α(20ng/ml)鏡下觀察細(xì)胞形態(tài)。流式細(xì)胞術(shù)檢測(cè)培養(yǎng)3,6,9天細(xì)胞CD1a、CD83、CD86、CD209表達(dá):MTT法檢測(cè)誘導(dǎo)培養(yǎng)的DC體外刺激同種異體T淋巴細(xì)胞增殖的活性,比較各成熟因子對(duì)DC作用的影響。 [結(jié)果] 1、經(jīng)不同的細(xì)胞因子誘導(dǎo)培養(yǎng)6天后,倒置顯微鏡下可見多數(shù)細(xì)胞呈懸浮生長(zhǎng),細(xì)胞胞體較大,細(xì)胞表面可見有不規(guī)則突起。2、存IL-4、GM-CSF組合中加入刺激劑TNF-α、LPS或IL-2后,DC表面CD1a、CD83、CD86、CD209表達(dá)明顯升高,其刺激同種異體T淋巴細(xì)胞的能力明顯增強(qiáng),刺激細(xì)胞/反應(yīng)細(xì)胞為1:10時(shí)刺激T細(xì)胞增殖能力最明顯。在各組中,尤以TNF-α、
[Abstract]:[objective] Human peripheral blood mononuclear cells (PBMC) were cultured in vitro with several different cytokines to induce the production of dendritic cells (DCs), and to detect the expression of surface molecules and the function of stimulating the proliferation of allogeneic T lymphocytes. Thus the best experimental method for inducing dendritic cells was established. The effects of cytokines on the induction, maturation and function of DCs in vitro were investigated. [methods] Mononuclear cells were obtained by density gradient centrifugation. Mononuclear cells were collected from healthy human peripheral blood and treated with heparin sodium. The mononuclear cells were cultured on 24-well plate for 2 hours on RPMI1640 medium containing 10% fetal bovine serum, and the unadherent cells were discarded. The adherent cells were obtained and cultured on the 24-well plate in RPMI1640 medium containing the following 6 groups of cytokines. Without adding 2rhGMCSF / rhIL-43rhGM-CSF / rhIL-4 / PHA4rhGM-CSF / rhIL-135rhGM-CSF / rhGM-CSF / PHA6PHAs, the dosage of each factor was (the final concentration in the culture medium): (the final concentration of rhGM-CSF (50ng/ml), rhIL-4 (40ng/ml), rhIL-13 (40ng/ml), PHA (50 渭 g/ml), respectively), the generation of DC and the expression of immunophenotype were observed 6 days after the addition of rhGM-CSF / rhIL-43rhGM-CSF / rhIL-4rhGM-CSF / rhIL-13rhGM-CSF / rhGM-CSF / rhIL-13rhGM-CSF / PHA6PHA. The maturation of DC was induced by adding LPS-IL-2TNF- 偽, the dosage of which was 20ng/ml (20ng/ml) / IL-2 (5ng/ml) / TNF- 偽 (20ng/ml) respectively. Flow cytometry was used to detect the expression of CD83A- CD83- CD86- CD209 and the effect of mature factors on the proliferation of allogeneic T lymphocytes induced by DC in vitro. [results] 1. After 6 days of induction and culture with different cytokines, most of the cells were found to be suspended and the cell bodies were larger under inverted microscope. There were irregular protrusions on the surface of the cells. The expression of CD1a- CD83, CD86, CD209, and the ability of stimulating allogeneic T lymphocytes were obviously increased after the addition of TNF- 偽 LPS or IL-2 to the combination of IL-4 and GM-CSF, and the expression of CD863 and CD86 + CD209 on the surface of the DC was significantly increased after the addition of the stimulator TNF- 偽 or IL-2. The T cell proliferation was the most obvious at 1:10. In each group, especially TNF- 偽,
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392
本文編號(hào)：2140077
[Abstract]:[objective] Human peripheral blood mononuclear cells (PBMC) were cultured in vitro with several different cytokines to induce the production of dendritic cells (DCs), and to detect the expression of surface molecules and the function of stimulating the proliferation of allogeneic T lymphocytes. Thus the best experimental method for inducing dendritic cells was established. The effects of cytokines on the induction, maturation and function of DCs in vitro were investigated. [methods] Mononuclear cells were obtained by density gradient centrifugation. Mononuclear cells were collected from healthy human peripheral blood and treated with heparin sodium. The mononuclear cells were cultured on 24-well plate for 2 hours on RPMI1640 medium containing 10% fetal bovine serum, and the unadherent cells were discarded. The adherent cells were obtained and cultured on the 24-well plate in RPMI1640 medium containing the following 6 groups of cytokines. Without adding 2rhGMCSF / rhIL-43rhGM-CSF / rhIL-4 / PHA4rhGM-CSF / rhIL-135rhGM-CSF / rhGM-CSF / PHA6PHAs, the dosage of each factor was (the final concentration in the culture medium): (the final concentration of rhGM-CSF (50ng/ml), rhIL-4 (40ng/ml), rhIL-13 (40ng/ml), PHA (50 渭 g/ml), respectively), the generation of DC and the expression of immunophenotype were observed 6 days after the addition of rhGM-CSF / rhIL-43rhGM-CSF / rhIL-4rhGM-CSF / rhIL-13rhGM-CSF / rhGM-CSF / rhIL-13rhGM-CSF / PHA6PHA. The maturation of DC was induced by adding LPS-IL-2TNF- 偽, the dosage of which was 20ng/ml (20ng/ml) / IL-2 (5ng/ml) / TNF- 偽 (20ng/ml) respectively. Flow cytometry was used to detect the expression of CD83A- CD83- CD86- CD209 and the effect of mature factors on the proliferation of allogeneic T lymphocytes induced by DC in vitro. [results] 1. After 6 days of induction and culture with different cytokines, most of the cells were found to be suspended and the cell bodies were larger under inverted microscope. There were irregular protrusions on the surface of the cells. The expression of CD1a- CD83, CD86, CD209, and the ability of stimulating allogeneic T lymphocytes were obviously increased after the addition of TNF- 偽 LPS or IL-2 to the combination of IL-4 and GM-CSF, and the expression of CD863 and CD86 + CD209 on the surface of the DC was significantly increased after the addition of the stimulator TNF- 偽 or IL-2. The T cell proliferation was the most obvious at 1:10. In each group, especially TNF- 偽,
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392
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，本文編號(hào)：2140077
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