乳腺癌区域淋巴结来源树突状细胞的分离和培养  

Separation and culture of dendritic cells from regional lymph nodes of patients with breast cancer

作　　者：周才明[1] 张孔明[1] 谭靖军[1] 莫明聪[1] 何新明[1] 林云恩[1] 

机构地区：[1]广州医学院第一附属医院外科,510120

出　　处：《中华实验外科杂志》2013年第4期687-690,共4页Chinese Journal of Experimental Surgery

基　　金：广东省自然科学基金资助项目（5300998）

摘　　要：目的探讨乳腺癌区域淋巴结来源树突状细胞（DC）的分离和培养及其用于抗肿瘤免疫治疗的可能性。方法选取21例乳腺癌患者术中新鲜无转移淋巴结组织，机械破碎制成细胞悬液，密度梯度离心和贴壁法分离DC。体外分3组培养，A组不加细胞诱导因子,B组加人人粒细胞-巨噬细胞集落刺激因子（rhGM-CSF，1000 U/ml）和重组人白细胞介素（rhIL）4（500 U/ml） ,C组加入 rhGM-CSF（1000 U/ml）、rhIL4（500 U/ml）和肿瘤坏死因子（TNF）-α（200 U/ml）。倒置显微镜下观察DC的细胞形态、大小及其变化，流式细胞术检测DC表型。结果平均每例5-12枚半个淋巴结可获得DC （52. 84 ± 20. 87） x 105个，其纯度达（73. 78 ± 13. 31）%，经培养后这些细胞具有DC的形态特征。培养7 d后，3组DC的纯度和数量均大于第2天，差异有统计学意义（P〈0. 05） ;3组€013＋0083_细胞数量与第2天比较差异均无统计学意义（p〉0.05），3组CDla＋CD83 ＋细胞和CDla_CD83 ＋细胞的数量均分别大于第2天，差异有统计学意义（P〈0. 05）＇;3组CDla＋CD83-、CDla ＋ CD83 ＋和CDla_CD83＋ 3类细胞的增长速率经方差分析差异均有统计学意义（/〉〈0.05）／两两比较，3组Cma＋CD83 ＋细胞和CDla-CD83 ＋细胞增长速率均大于CDla＋CD83_细胞，差异有统计学意义（/〉〈0.01）。7d 后,DC、CDla＋CD83-细胞、CDla＋CD83＋细胞和 CDla-CD83 ＋ 细胞数量及其增长速率在3组之间差异均无统计学意义（P〉0.05）。结论可从乳腺癌区域淋巴结中分离出较大数量和较高纯度的DC,经过体外培养可获得较多具有树突状形态特征的成熟DC。Objective To investigate the separation and culture of dendritic cells （DC） from re-gional lymph nodes of patients with breast cancer, and the possibility of them used as immunotherapy forthe tumor. Methods DC were isolated from fresh tumor-free lymph nodes of 21 cases of breast cancer un-dergoing surgical operations, by using mechanical crush, density gradient centrifugation and transient ad-herent culture. The isolated cells were divided into 3 groups, then cultured in vitro with recombinant humangranulocyte macrophage-colony stimulating factor （rhGM-CSF） （1000 U/ml） ＋ recombinant human inter-leukin （rhIL）-4 （500 U/ml）,and rhGM-CSF （1000 U/ml） ＋ rhIL-4 （500 U/ml） ＋ tumor necrosisfactor-α （TNF-a） （200 U/ml） in group B and group C, respectively. The morphology, size and changesof DC were observed under an inverted microscope,and the phenotype of DC was detected by using flow cy-tometry. Results （52. 84 ± 20. 87） x 105 DC were obtained from half of 5-12 lymph nodes of each patientwith breast cancer,and the purity of isolated DC was （73. 78 ± 13. 31） % These cells showed DC morpho-logical characteristics after being cultured. After being cultured for 7 days, the purity and number of DC inthree groups were grieater than before, and there was significant difference between every two groups （P 〈0.05）. The number of CDla ＋ CD83＋ cells and CDla- CD83 ＋ cells in three groups was also jgreater thanbefore, and there was significant difference between every two groups （P 〈 0. 05）. The growth rate ofCDla＋ CD83 ＋ cells and CDla - CD83 ＋ cells in three groups was significantly higher than that of CDla ＋CD83 - cells （P 〈0. 01）. There was no significant difference in the number and growth rate of DC, CD1 a ＋CD83~cells, CDla＋ CD83 ＋ cells and CDla- CD83 ＋ cells among three groups. Conclusion A largequantity and high purity of DC can be isolated from regional lymph nodes of patients with breast cancer.More mature DC with dendritic morphol

关 键 词：乳腺癌 淋巴结 树突状细胞 细胞培养 

分 类 号：R737[医药卫生—肿瘤]