作　　者： ; ; ; ;

机构地区： 南方医科大学南方医院

出　　处： 《中国组织工程研究与临床康复》 2007年第42期8532-8535,共4页

摘　　要： 目的:近年来,树突状细胞参与调节作用的研究较多。实验拟验证重组腺相关病毒(rAAV)介导癌胚抗原基因转染树突状细胞后其的免疫调节作用的变化。方法:实验于2006-06/2006-11在美国阿肯色大学医学院基因治疗中心完成。①实验方法:取健康人外周血(自愿捐献),采用改良Ficoll密度梯度离心的方法分离外周血单个核细胞,取贴壁细胞,分为rAAV/CEA转染组和空白对照组,均采用重组人粒细胞巨噬细胞集落刺激因子、白细胞介素4、肿瘤坏死因子-α诱导树突状细胞前体细胞成熟,将成熟树突状细胞与末梢血淋巴细胞按比例混合培养,可得到激活的细胞毒性T淋巴细胞。②实验评估:采用流式细胞仪检测树突状细胞表面标志表达,细胞毒性T淋巴细胞的免疫表型变化,细胞毒性T淋巴细胞γ-干扰素表达。采用酶联免疫吸附法(ELISA法)检测树突状细胞的白细胞介素12和白细胞介素10表达。结果:①rAAV/CEA转染树突状细胞的CD14较空白对照组降低,共刺激分子CD40、CD80、CD83、CD86表达均较空白对照组高。②成熟树突状细胞细胞因子表达中,rAAV/CEA转染组的白细胞介素12较空白对照组升高,白细胞介素10降低。③与空白对照组比较,rAAV/CEA转染组能高表达CD8+T细胞和其表型CD69,CD8/CD56的T细胞比例上调,CD25+CD4+的T细胞减少。④rAAV/CEA转染的细胞毒性T淋巴细胞表达相对较高水平的γ-干扰素,与杀伤实验结果相吻合。结论:rAAV/CEA转染树突状细胞能够激活细胞毒性T淋巴细胞的特异性杀伤作用,与树突状细胞和细胞毒性T淋巴细胞等免疫细胞表面标志变化和细胞内细胞因子水平变化相关。 AIM： In recent years, the dendritic cells （DC） therapy developed rapidly, received more and more attention. The study was aimed to explore the immune regulation of recombinant adeno-associated virus （rAAV）-mediated carcinoembryonic antigen （CEA） gene transfected dendritic cells （DC） on colorectal cancer. METHODS：From June to November 2006, the experiment was performed at the Gene Therapy Center, University of Arkansas for Medical Science. （1）The peripheral blood from the healthy donor was drawn voluntarily. The mononuclear cells （DC precursor） were isolated with modified Ficoll density gradient centrifugation. The adherent cells were divided into 2 groups, rAAV/CEA group and blank control group. Recombinant human granulocyte-macrophage colony stimulating factor （GM-CSF）, interleukin-4 （IL-4） and tumor necrosis factor-α （TNF-α） were added to induce DC precursor cells mature. Mature DC and PBL received mixed culture, then the activated cytotoxic T lymphocyte（CTL）was gotten. （2）The expression of DC surface markers, activated T lymphocytes immune phenotype and IFN-γ, expression of activated CTL were detected by flow cytometer （FACS）. The expression of IL-12 and IL-10 were detected by Enzyme-linked immunosorbent assay （ELISA） in the mature DC. RESULTS： （1）DC surface markers： The DC transfected with rAAV/CEA expressed the higher level of surface marker CD80, CD86, CD40 and CD83 than that of control group, and the lower level of CD14. （2）The expression of cytokines in mature DC： The DC transfected with rAAV/CEA expressed the higher level of IL-12 than that of control group, and the lower level of IL-10. （3）Activated T lymphocytes immune phenotype： The CTL population also got much higher ratio of CD8^＋ T and CD69, CD8/CD56, lower ratio of CD25^＋ CD4^＋.（4）The CTL population expressed higher lever of IFN-γ. CONCLUSION：Activated T cells that transfected CEA gene had specific killing effect. It was positively correlated to the DC and

关 键 词： 树突状细胞 癌胚抗原 腺相关病毒 细胞转染 细胞工程

领　　域： [哲学宗教]