Separation and culture of dendritic cells from regional lymph nodes of patients with breast cancer
ZHOU Cai-ming, ZHANG Kong-ming, TAN Jing-jun, MO Ming-cong, HE Xin-ming, LIN Yun-en
Published 2013-04-08
Cite as Chin J Exp Surg, 2013,30(04): 687-690. DOI: 10.3760/cma.j.issn.1001-9030.2013.04.013
Abstract
Objective To investigate the separation and culture of dendritic cells (DC) from regional lymph nodes of patients with breast cancer,and the possibility of them used as immunotherapy for the tumor.Methods DC were isolated from fresh tumor-free lymph nodes of 21 cases of breast cancer undergoing surgical operations,by using mechanical crush,density gradient centrifugation and transient adherent culture.The isolated cells were divided into 3 groups,then cultured in vitro with recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) (1000 U/ml) + recombinant human interleukin (rhIL)-4 (500 U/ml),and rhGM-CSF (1000 U/ml) + rhIL-4 (500 U/ml) + tumor necrosis factor-α (TNF-α) (200 U/ml) in group B and group C,respectively.The morphology,size and changes of DC were observed under an inverted microscope,and the phenotype of DC was detected by using flow cytometry.Results (52.84 ± 20.87) × 105 DC were obtained from half of 5-12 lymph nodes of each patient with breast cancer,and the purity of isolated DC was (73.78 ± 13.31)%.These cells showed DC morphological characteristics after being cultured.After being cultured for 7 days,the purity and number of DC in three groups were greater than before,and there was significant difference between every two groups (P <0.05).The number of CD1 a + CD83 + cells and CD1 a-CD83 + cells in three groups was also greater than before,and there was significant difference between every two groups (P < 0.05).The growth rate of CD1a+ CD83 + cells and CD1a-CD83 + cells in three groups was significantly higher than that of CD1a+CD83-cells (P < 0.01).There was no significant difference in the number and growth rate of DC,CD1 a +CD83-cells,CD1a+ CD83+ cells and CD1a-CD83+ cells among three groups.Conclusion A large quantity and high purity of DC can be isolated from regional lymph nodes of patients with breast cancer.More mature DC with dendritic morphology characteristics can be obtained after these cells being cultured in vitro.
Key words:
Breast carcinoma; Lymph nodes; Dendritic cells; Cell culture
Contributor Information
ZHOU Cai-ming
Department of General Surgery, the First Affiliated Hospital of Gugangzhou Medical College, Guangzhou 510120, China
ZHANG Kong-ming
Department of General Surgery, the First Affiliated Hospital of Gugangzhou Medical College, Guangzhou 510120, China
TAN Jing-jun
Department of General Surgery, the First Affiliated Hospital of Gugangzhou Medical College, Guangzhou 510120, China
MO Ming-cong
Department of General Surgery, the First Affiliated Hospital of Gugangzhou Medical College, Guangzhou 510120, China
HE Xin-ming
Department of General Surgery, the First Affiliated Hospital of Gugangzhou Medical College, Guangzhou 510120, China
LIN Yun-en
Department of General Surgery, the First Affiliated Hospital of Gugangzhou Medical College, Guangzhou 510120, China