【摘要】： 目的：樹突狀細(xì)胞(dendritic cell，DC)是目前發(fā)現(xiàn)的功能最強(qiáng)大的專職抗原提呈細(xì)胞(antigen presenting cells，APC)，能攝取提呈抗原，強(qiáng)烈刺激初始型T細(xì)胞(naive T cell)增殖，促進(jìn)細(xì)胞毒性T細(xì)胞(CTL)和T輔助細(xì)胞的生成，誘導(dǎo)高效特異的細(xì)胞免疫和體液免疫反應(yīng)，是免疫反應(yīng)的啟動因子和調(diào)節(jié)因子。基于DC強(qiáng)大抗原提呈功能的主動特異性免疫治療已逐漸成為腫瘤免疫治療研究的主要方向。雖然DC在體內(nèi)分布廣泛，但數(shù)量少且分散，，而未成熟DC主要分布在外周非淋巴組織內(nèi)，直接分離、提純十分困難，不能滿足體內(nèi)或體外研究及臨床應(yīng)用的需要。如何在體外大量擴(kuò)增DC一直是DC研究的熱點(diǎn)。目前大多數(shù)體外誘導(dǎo)DC的研究都使用了有血清培養(yǎng)基，但由于添加胎牛血清存在安全性低，不宜質(zhì)控等缺點(diǎn)，無血清培養(yǎng)基正得到越來越廣泛的應(yīng)用，無血清培養(yǎng)基的使用也是DC療法應(yīng)用于臨床的前提。本研究的目的是建立可用于臨床治療功能成熟的DC體外無血清擴(kuò)增的優(yōu)化培養(yǎng)方案，并比較無血清、替代血清和動物血清之間培養(yǎng)DC的效率。 方法：本研究通過采用X—VIV020無血清培養(yǎng)基聯(lián)合細(xì)胞因子rhGM-CSF(100ng/ml)和rhIL-4(50ng/ml)擴(kuò)增人外周血分離的單個核細(xì)胞，細(xì)胞培養(yǎng)分別按5x10~6/ml、6x10~6/ml和7x10~6/ml的密度，加入6孔培養(yǎng)板(2ml/孔)。第6d加入rhTNF-a(100ng/ml)聯(lián)合培養(yǎng)，分別于第6d，第9d和12d收獲細(xì)胞。從形態(tài)學(xué)、細(xì)胞表面標(biāo)志以及混合淋巴細(xì)胞反應(yīng)(MLR)中對同種異體未致敏T淋巴細(xì)胞刺激能力等三個方面進(jìn)行鑒定。我們對DC體外誘導(dǎo)擴(kuò)增的無血清條件下的細(xì)胞培養(yǎng)密度和培養(yǎng)時間進(jìn)行了探索和優(yōu)化。并比較無血清培養(yǎng)，替代血清培養(yǎng)和動物血清培養(yǎng)DC之間的效率差異。 本研究分兩部分進(jìn)行：1、無血清培養(yǎng)基對人外周血來源DC的體外擴(kuò)增和鑒定。2、無血清培養(yǎng)，替代血清培養(yǎng)和動物血清培養(yǎng)DC的效率比較。 結(jié)果： 1、形態(tài)學(xué)觀察：倒置顯微鏡觀察，經(jīng)過9d誘導(dǎo)后，培養(yǎng)細(xì)胞懸浮生長，體積較前增大，可見有胞體拉長、表面可見毛刺狀突起具有典型樹突細(xì)胞外形。 2、細(xì)胞表面標(biāo)記分析：流式細(xì)胞儀分析，6x10~6/ml密度的細(xì)胞培養(yǎng)組培養(yǎng)到第9天最宜。培養(yǎng)細(xì)胞其表面共刺激分子CDla的表達(dá)率為36.57％+0.92％。 3、MLR中，培養(yǎng)DC能刺激同種異體未致敏T淋巴細(xì)胞的增殖。 4、無血清培養(yǎng)，替代血清培養(yǎng)和動物血清培養(yǎng)DC的效率之間存在統(tǒng)計學(xué)差異(P值＜0.05)。其中動物血清培養(yǎng)組，獲得65.00％±5.27％CDla細(xì)胞；替代血清培養(yǎng)組，獲得47.33％±5.46％CDla細(xì)胞；無血清培養(yǎng)組，能夠獲得36.57％±0.92％CDla細(xì)胞。 結(jié)論： 1、培養(yǎng)細(xì)胞具有典型的DC的形態(tài)特征，細(xì)胞表型及功能實驗證實其DC的特性，說明本實驗建立的無血清培養(yǎng)基聯(lián)合細(xì)胞因子rhGM-CSF、rhIL-4和rhTNF-a體外誘導(dǎo)DC的方法是切實可行的。 2、無血清培養(yǎng)，替代血清培養(yǎng)和動物血清培養(yǎng)DC效率之間存在差異。說明血清對DC分化發(fā)育存在影響。動物血清或替代血清的添加可能更有利于DC的獲得。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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