本文選題：樹突狀細胞 + 細胞因子雞尾酒�。� 參考：《安徽醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：探討優(yōu)化人γhG-CSF動員的外周血采集物來源的樹突狀細胞（dendritic cell，DC）成熟誘導(dǎo)條件，以便制備更有效的DC疫苗應(yīng)用于臨床，觀察并比較不同誘導(dǎo)條件下制備的DC疫苗在體外特異性抗腫瘤免疫反應(yīng)的能力。 方法：利用密度梯度離心法從重組人粒細胞集落刺激因子（γhG-CSF）動員的外周血采集物分離出單個核細胞，以重組人粒細胞-巨噬細胞集落刺激因子(γhGM-CSF)和重組人白細胞介素-4(γhIL-4)體外誘導(dǎo)成不成熟樹突狀細胞（imDC），分別采用傳統(tǒng)細胞因子雞尾酒（IL-1β、IL-6、TNF-α、PGE2）和改良細胞因子雞尾酒（IL-1β、IL-6、TNF-α、polyI:C、CpG ODN）刺激成熟，并設(shè)有對照組（只加IL-4、GM-CSF）。3d后收獲成熟DC，觀察細胞形態(tài)、流式細胞儀檢測DC的內(nèi)吞能力通過異硫氰酸熒光素標記的葡聚糖（FITC-Dextran）、檢測DC表面CD83、CD1a、CD80、HLA-DR、CD86和CCR-7，酶聯(lián)免疫吸附法（ELISA）檢測其IL-12、IL-10的分泌，MTT法檢測其刺激淋巴細胞增殖能力。實驗中三組DC分別轉(zhuǎn)染A549細胞的總RNA，酶聯(lián)免疫吸附法（ELISA）檢測IFN-γ的分泌，乳酸脫氫酶(LDH)釋放法測定殺傷腫瘤活性。 結(jié)果：傳統(tǒng)細胞因子雞尾酒（IL-1β、IL-6、TNF-α、PGE2）和改良細胞因子雞尾酒（IL-1β、IL-6、TNF-α、polyI:C、CpG ODN）兩種方法均能誘導(dǎo)DC成熟，以改良細胞因子雞尾酒效果更佳，對照組幾乎不誘導(dǎo)DC成熟。改良雞尾酒方法誘導(dǎo)的成熟DC的FITC-Dextran內(nèi)吞能力明顯下降；DC成熟的表面標志物CD83、CD1a的表達率分別是84.44%、87.94%（P0.05）；IL-12分泌量明顯增高（P 0.05）；IL-10分泌量明顯增高（P 0.05）；成熟DC能有效的刺激T淋巴細胞增殖；轉(zhuǎn)染A549細胞的總RNA后的改良組DC的IFN-γ分泌水平明顯高于前兩組（P 0.05），并能誘導(dǎo)腫瘤特異性CTL的產(chǎn)生進而殺傷腫瘤細胞。 結(jié)論：從經(jīng)γhG-CSF動員的外周血采集物中利用密度梯度離心法分離誘導(dǎo)出具備典型形態(tài)和免疫表型的DC后，兩種方法誘導(dǎo)DC成熟，實驗證明改良細胞因子雞尾酒法是誘導(dǎo)DC成熟的最佳方法。
[Abstract]:Objective: to study the maturation induction conditions of dendritic cells derived from peripheral blood samples mobilized by human 緯 hG-CSF in order to prepare more effective DC vaccine for clinical application.To observe and compare the specific anti-tumor immune response of DC vaccine prepared under different induction conditions in vitro.Methods: mononuclear cells were isolated from peripheral blood samples mobilized by recombinant human granulocyte colony stimulating factor (緯 hG-CSF) by density gradient centrifugation.Immature dendritic cells (imDCA) were induced by recombinant human granulocyte-macrophage colony stimulating factor (緯 hGM-CSF) and recombinant human interleukin-4 (緯 hIL-4) in vitro. The mature cells were stimulated by traditional cytokine IL-1 尾 -IL-6TNF- 偽 PGE2 and modified cytokine cocktail IL-1 尾 IL-6TNF- 偽 polyCCpG ODN.The control group (IL-4 + GM-CSFN. 3 d) was used to harvest the mature DCs and observe the morphology of the cells.The endocytosis of DC was detected by flow cytometry by FITC-Dextranan labeled with fluorescein isothiocyanate, HLA-DRCD86 and CCR-7 by CD83T CD1a1 + CD80 on the surface of DC, and IL-12 IL-10 by MTT assay by enzyme linked immunosorbent assay (Elisa).In the experiment, the total RNAs of A549 cells transfected with three groups of DC were detected by enzyme linked immunosorbent assay (Elisa) to detect the secretion of IFN- 緯, and the activity of killing tumor was determined by lactate dehydrogenase (LDH) release method.The FITC-Dextran endocytosis of mature DC induced by modified cocktail method was significantly decreased. The expression rates of CD83- CD1a, the surface marker of DC maturation, were 84.444.444.44 / 87.94A and 87.94A respectively. The secretion of IL-12 was significantly increased in mature DCs, and the secretion of IL-10 was significantly increased in mature DCs, and the proliferation of T lymphocytes was effectively stimulated by mature DCs.The level of IFN- 緯 secretion in the modified DC after total RNA transfection was significantly higher than that in the former two groups (P 0.05), and it could induce the production of tumor-specific CTL and then kill the tumor cells.Conclusion: dendritic cells with typical morphology and immunophenotype were isolated from peripheral blood samples mobilized by 緯 hG-CSF by density gradient centrifugation.It is proved that the modified cytokine cocktail method is the best method to induce DC maturation.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392

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