小鼠脾脏来源树突状细胞的体外扩增培养

1、小鼠脾脏来源树突状细胞的体外扩增培养         11-03-25 09:39:00     编辑：studa20                作者：黎辉，曹宇皎 ，黄军华 ，刘俊峰【摘要】  目的：对体外诱导小鼠脾脏单个核细胞分化成的树突状细胞(DC)的生物学表型及功能进行检测，并与骨髓来源的DC进行比较。方法

2、：分离小鼠脾脏单个核细胞，在体外培养的条件下通过添加25 ng·ml-1的粒单核细胞集落刺激因子和25 ng·ml-1的白细胞介素4(IL4)将其诱导为DC，分别从细胞形态、表型以及功能3个方面对其进行检测，并与骨髓来源的DC进行比较。结果：小鼠脾脏来源的DC经磷酸脂多糖(LPS)刺激成熟后其表面显示出明显的树枝状突起，高表达CD11c、CD86及MHC类分子，并具有极强的刺激同种异基因淋巴细胞增殖的能力，其特征与骨髓来源的DC相差无几。结论：小鼠脾脏单个核细胞能够被诱导为具有正常形态、表型、功能的DC，为DC的功能研究以及进一步制备相应的肿瘤疫苗提供又一可靠来源。 【关键

3、词】  脾脏; 骨髓; 树突状细胞; 诱导; 小鼠Dendritic cells(DC) are the most potent antigen presenting cells that are responsible for priming native T cells in the immune response. Immunotherapy of DC/tumor vaccine has become an important therapeutic strategy against tumors12. However, the basic and clinical appl

4、ications of DCs have been limited by the low yield and purity of DCs generated by traditional bone marrow derivation. In this study, we induced mouse spleen mononuclear cells into dendritic cells in vitro, and then compared the biological characteristics of them with those of bone marrow mononuclear

5、 cells derived DCs.1 Materials and methods1.1 MaterialsMain reagents: recombinant mouse granulocytemacrophage colonystimulating factor(GMCSF), recombinant mouse interleukin4(IL4), recombinant mouse leukemia inhibitory factor(LIF,Peprotech), lipopolysaccharide(LPS,Sigma); mitomycin C(MMC) and methabe

6、nzthiazuron(MTT,Duchfo). DCs culture medium: high glucose DMEM plus 15% fetal calf serum plus 25 mg·L-1 rmGMCSF and rmIL4. Mice: six to eight weeks old female BALB/c and 129 mice were purchased from Animal Center of Guiyang Medical College.1.2 Methods1.2.1 Induction of DC from mouse spleen Cell

7、s were flushed out of spleen of 129 mice.Mononuclear cells at the interface were collected by centrifugation over a lympholyte gradient. They were washed and then cultured in 6well plate for 2 h with the density of 5×104 ml-1.The suspended cells were removed and the adherent cells were cultured

8、 with DC culture medium. LPS was added on day 7 and the suspended cells were harvested 24 h later, while at the same time mouse bone mononuclear cells were induced into DCs.1.2.2 Morphology observation Cells were observed under microscope daily and suspended cells were identified by electron microsc

9、opy.1.2.3 Cell surface marker analysis Cells were collected and added into centrifuge tube. Mixed with FITCCD11c and MHCPE, FITCCD86 and MHC IIPE, respectively, the cells were kept at 4 for 30 min. After being washed twice by PBS,cells were assayed by flow cytometry.1.2.4 Mixed lymphocyte reactions(

10、MLR) Collected spleen derived DC were added with 25 g·ml-1 MMC as a stimulator for 45 min and washed twice. Nylon wool purified Tcel1s as responders were added in 96well round bottom plates at various ratios for 4 d.At the same time, we set control group that didnt include stimulators. 4 h befo

11、re harvesting, cells were pulsed with 20 l MTT(5 mg·ml-1), then added with 100 l DMSO and its absorbency was measured. The stimulation index was calculated according to the following formula: SI= absorbency of stimulation groupabsorbency of contrast/the absorbency of contrast.1.3 Statistical an

12、alysisData are expressed as ±s deviation.The results of the analysis was shown by t test.A value of P<0.05 was considered statistically significant.2 Results2.1 Morphological characteristics of mouse spleen derived DCsAt the first 2-4 d, the mouse spleen mononuclear cells grew slowly and rou

13、nd in shape, the bulk was small, the population expanded rapidly with time and then the bulk became larger gradually.Immature spleen derived DCs already had irregular dendrites after being cultured for 5-7 d. After adding 1 g·ml-1 of LPS for 24 h, the dramatic veils of cytoplasm and extensive d

14、endrites were more distinct(Fig 1), just as the mature BMDCs. Besides, 5-10 cell conglomerates could be seen(Fig 2). By electromicroscopy the dramatic veils of cytoplasm and extensive dendrites were seen more clearly(Fig 3).2.2 Analysis of surface phenotypeDCs derived from mouse spleen mononuclear c

15、ells were induced to mature through exposure to LPS and high expression of surface molecules MHCand CD86 was associated with antigen presenting.The expression of peculiar surface molecule CD11c(Fig 4) in spleenDCs was just as that in BMDCs.2.3 MLRFollowing exposure to LPS,mouse spleen derived DCs we

16、re induced and became mature, which demonstrated their capacity to stimulate potent primary T cell responses in mixed lymphocyte cultures(Tab 1).Tab 1 Stimulation index for primary T cell of twosource derived DCs(±s)3 DiscussionDCs are unique among populations of antigen presenting cells by the

17、 virtue of their capacity to direct the outcome of antigen recognition by naive T cells3. DCs in the periphery capture and process antigens, express lymphocyte costimulatory molecules,migrate to lymphoid organs and secrete cytokines to initiate immune responses4.Numerous studies have shown that DCs

18、play crucial roles in controlling tumor growth, graft rejection and autoimmunity.At present, we have already induced DCs from bone marrow, etc.57, and will produce antitumor vaccine by gene transfection, tumorassociated antigen impaction and cell fusion810. The effects of research in experimental animal were exciting. Furthermore, DC immunotherapy has