人外周血树突状细胞体外无血清诱导扩增的研究

发布时间：2018-10-26 20:06

【摘要】： 目的：树突状细胞(dendritic cell，DC)是目前发现的功能最强大的专职抗原提呈细胞(antigen presenting cells，APC)，能摄取提呈抗原，强烈刺激初始型T细胞(naive T cell)增殖，促进细胞毒性T细胞(CTL)和T辅助细胞的生成，诱导高效特异的细胞免疫和体液免疫反应，是免疫反应的启动因子和调节因子。基于DC强大抗原提呈功能的主动特异性免疫治疗已逐渐成为肿瘤免疫治疗研究的主要方向。虽然DC在体内分布广泛，但数量少且分散，，而未成熟DC主要分布在外周非淋巴组织内，直接分离、提纯十分困难，不能满足体内或体外研究及临床应用的需要。如何在体外大量扩增DC一直是DC研究的热点。目前大多数体外诱导DC的研究都使用了有血清培养基，但由于添加胎牛血清存在安全性低，不宜质控等缺点，无血清培养基正得到越来越广泛的应用，无血清培养基的使用也是DC疗法应用于临床的前提。本研究的目的是建立可用于临床治疗功能成熟的DC体外无血清扩增的优化培养方案，并比较无血清、替代血清和动物血清之间培养DC的效率。方法：本研究通过采用X—VIV020无血清培养基联合细胞因子rhGM-CSF(100ng/ml)和rhIL-4(50ng/ml)扩增人外周血分离的单个核细胞，细胞培养分别按5x10~6/ml、6x10~6/ml和7x10~6/ml的密度，加入6孔培养板(2ml/孔)。第6d加入rhTNF-a(100ng/ml)联合培养，分别于第6d，第9d和12d收获细胞。从形态学、细胞表面标志以及混合淋巴细胞反应(MLR)中对同种异体未致敏T淋巴细胞刺激能力等三个方面进行鉴定。我们对DC体外诱导扩增的无血清条件下的细胞培养密度和培养时间进行了探索和优化。并比较无血清培养，替代血清培养和动物血清培养DC之间的效率差异。本研究分两部分进行：1、无血清培养基对人外周血来源DC的体外扩增和鉴定。2、无血清培养，替代血清培养和动物血清培养DC的效率比较。结果： 1、形态学观察：倒置显微镜观察，经过9d诱导后，培养细胞悬浮生长，体积较前增大，可见有胞体拉长、表面可见毛刺状突起具有典型树突细胞外形。 2、细胞表面标记分析：流式细胞仪分析，6x10~6/ml密度的细胞培养组培养到第9天最宜。培养细胞其表面共刺激分子CDla的表达率为36.57％+0.92％。 3、MLR中，培养DC能刺激同种异体未致敏T淋巴细胞的增殖。 4、无血清培养，替代血清培养和动物血清培养DC的效率之间存在统计学差异(P值＜0.05)。其中动物血清培养组，获得65.00％±5.27％CDla细胞；替代血清培养组，获得47.33％±5.46％CDla细胞；无血清培养组，能够获得36.57％±0.92％CDla细胞。结论： 1、培养细胞具有典型的DC的形态特征，细胞表型及功能实验证实其DC的特性，说明本实验建立的无血清培养基联合细胞因子rhGM-CSF、rhIL-4和rhTNF-a体外诱导DC的方法是切实可行的。 2、无血清培养，替代血清培养和动物血清培养DC效率之间存在差异。说明血清对DC分化发育存在影响。动物血清或替代血清的添加可能更有利于DC的获得。
[Abstract]:Objective: Dendritic cell (DC) is the most powerful full-time antigen presenting cell (APC), which can capture antigen and stimulate initial T cell proliferation. It can promote the production of cytotoxic T cells (CTL) and T helper cells, induce high efficiency and specific cellular immunity and humoral immune response, and is the starting factor and regulating factor of immune response. Active specific immunotherapy based on DC strong antigen presenting function has gradually become the main direction of tumor immunotherapy research. Although DC is widely distributed in vivo, the quantity is small and dispersed, while immature DC is mainly distributed in peripheral non-lymphoid tissue, it is difficult to purify and purify, and can not meet the needs of in vivo or in vitro research and clinical application. How to amplify DC in vitro has been a hot spot in DC research. At present, most in vitro induction DC research uses serum culture medium, but because of the disadvantages of low safety, inappropriate quality control and so on, serum-free culture medium is becoming more and more widely applied, and the use of serum-free culture medium is also the premise of application of DC therapy in clinic. The objective of this study was to establish an optimized culture for DC in vitro non-serum amplification that could be used in clinical treatment and to compare serum-free, surrogate serum and animal serum for DC efficiency. Methods: The single nuclear cells isolated from peripheral blood of human peripheral blood were amplified by X-ray VIV020 without serum medium and rhGM-CSF (100ng/ ml) and rhIL-4 (50ng/ ml). The cells were cultured in a density of 5x10 ~ 6/ ml, 6x10 ~ 6/ ml and 7x10 ~ 6/ ml, respectively. (2ml/ well). The combined culture of rhTNF-a (100ng/ ml) was added to day 6d, respectively, at 6d, 9d and 12d. Harvest of cells from morphology, cell surface markers, and mixed lymphocyte response (MLR). Identification of aspects of cell culture density and incubation time under serum-free conditions for DC in vitro induction amplification Exploration and optimization were carried out. Serum-free culture, alternative serum culture and animal serum culture were compared. The results of this study were divided into two parts: 1, in vitro amplification and identification of DC from human peripheral blood from serum-free medium. No serum culture was used instead of serum culture. nourishing and moving The results were as follows: 1. Morphological observation: observation of the inverted microscope, after 9 d induction, the cell suspension growth was cultured, and the volume was lower. pre-increased, visible cell elongation, surface visible burr-like projections with typical dendritic cell appearance. 2, cell list Surface Labeling Analysis: Flow Cytometry Analysis, 6x10 ~ 6/ ml Density of Cell Culture Cell Culture to The expression rate of CDla on the surface of cultured cells was 36. In 57% + 0. 92%. 3, MLR, DCs were cultured to stimulate allogenic T lymphocytes. Proliferation. 4. There was a statistical difference between the serum culture and the efficiency of serum culture DC (P <0.05). Among them, 65. 00% CD5. 27% CDla cells were obtained. clear culture In group, 47. 33% CD5. 46% CDla cells were obtained; serum-free culture group was able to obtain 36. 57% CD11a cells. Conclusion: 1, cultured cells have typical DC morphological characteristics, cell phenotypes and The characteristics of DC were confirmed by functional experiments, which indicated that no serum-free medium combined with cells was established in this experiment. SubrhGM-CSF, rhIL-4 and rhTNF-a can induce DC in vitro.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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