小鼠脾脏来源树突状细胞的体外扩增培养

　　　　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：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(IL4)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Ⅱ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 tumors[12]. However， the basic and clinical applications 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 cells derived DCs.

　　1 Materials and methods

　　1.1 Materials

　　Main reagents: recombinant mouse granulocytemacrophage colonystimulating factor(GMCSF)， recombinant mouse interleukin4(IL4)， recombinant mouse leukemia inhibitory factor(LIF，Peprotech)， lipopolysaccharide(LPS，Sigma); mitomycin C(MMC) and methabenzthiazuron(MTT，Duchfo). DCs culture medium: high glucose DMEM plus 15% fetal calf serum plus 25 mg·L-1 rmGMCSF and rmIL4. Mice: six to eight weeks old female BALB/c and 129 mice were purchased from Animal Center of Guiyang Medical College.

　　1.2 Methods

　　1.2.1 Induction of DC from mouse spleen Cells 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 6well plate for 2 h with the density of 5×104 ml-1.The suspended cells were removed and the adherent cells were cultured 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 microscopy.

　　1.2.3 Cell surface marker analysis Cells were collected and added into centrifuge tube. Mixed with FITCCD11c and MHCⅡPE， FITCCD86 and MHC IIPE， 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(MLR) Collected spleen derived DC were added with 25 μg·ml-1 MMC as a stimulator for 45 min and washed twice. Nylon wool purified Tcel1s as responders were added in 96well round bottom plates at various ratios for 4 d.At the same time， we set control group that didnt include stimulators. 4 h before harvesting， cells were pulsed with 20 μl MTT(5 mg·ml-1)， then added with 100 μl DMSO and its absorbency was measured. The stimulation index was calculated according to the following formula: SI= absorbency of stimulation groupabsorbency of contrast/the absorbency of contrast.

　　1.3 Statistical analysis

　　Data 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 Results

　　2.1 Morphological characteristics of mouse spleen derived DCs

　　At the first 2-4 d， the mouse spleen mononuclear cells grew slowly and round 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 μg·ml-1 of LPS for 24 h， the dramatic veils of cytoplasm and extensive dendrites were more distinct(Fig 1)， just as the mature BMDCs. Besides， 5-10 cell conglomerates could be seen(Fig 2). By electromicroscopy the dramatic veils of cytoplasm and extensive dendrites were seen more clearly(Fig 3).

　　2.2 Analysis of surface phenotype

　　DCs derived from mouse spleen mononuclear cells were induced to mature through exposure to LPS and high expression of surface molecules MHCⅡand CD86 was associated with antigen presenting.The expression of peculiar surface molecule CD11c(Fig 4) in spleenDCs was just as that in BMDCs.

　　2.3 MLR

　　Following exposure to LPS，mouse spleen derived DCs were 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 twosource derived DCs(±s)

　　3 Discussion

　　DCs are unique among populations of antigen presenting cells by the virtue of their capacity to direct the outcome of antigen recognition by naive T cells[3]. DCs in the periphery capture and process antigens， express lymphocyte costimulatory molecules，migrate to lymphoid organs and secrete cytokines to initiate immune responses[4].Numerous studies have shown that DCs play crucial roles in controlling tumor growth， graft rejection and autoimmunity.At present， we have already induced DCs from bone marrow， etc.[57]， and will produce antitumor vaccine by gene transfection， tumorassociated antigen impaction and cell fusion[810]. The effects of research in experimental animal were exciting. Furthermore， DC immunotherapy has been introduced in the clinic， and has been proved to be feasible， nontoxic and effective in some cancer patients， and particularly so when the DCs are completely mature and activated[11]. The results of the first clinical trial have been published， and unequivocally demonstrated that only mature DCs are capable of inducing potent antiKLHspecific Tcell and Bcell response[12]. The life cycle of DC includes several stages characterized by distinct functions and mechanisms of regulation[13]. The DCs derived from different sources or at different life stages may have different biological characteristics[1418].

　　In the present study， we induced DCs differentiated from mouse spleen mononuclear cell.The results showed that it could be induced into DCs with normal surface phenotype in vitro by additional appropriate signals in the form of growth factors and cytokines.The dramatic veils of cytoplasm and extensive dendrites were seen in their surface when they were induced and matured by adding LPS，and they expressed the myeloid peculiar surface molecule CD11c， MHCⅡand CD86 which were associated with antigen presenting. Compared with BMDC， the surface morphological characteristics of them were similar. They have already had a few irregular dendrites before maturity by plus LPS，and expressed lower levels of CD11c， MHCⅡ， CD86. Induced into mature by LPS， the characteristics were more evident. Mouse spleen mononuclear cell derived mature DCs demonstrates their capacity to stimulate potent primary T cell responses in mixed lymphocyte reactions. There was no statistical difference compared with BMDC. From this research， we can conclude that mouse spleen derived DCs have analogical biological characteristics to those in BMDC which has been applied in basic and clinical research. Therefore it provides a reliable source for the research of DCs and the preparation of antitumor vaccine in the next step.

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