WHBE兔骨髓、外周血和脾脏来源树突状细胞的比较

doi:10.3969/j.issn.1674-5817.2012.03.008

摘要/Abstract

摘要： 目的建立大耳白黑眼兔(WHBE兔)树突状细胞(DC)体外诱生培养的经济有效的方法,为进一步实验奠定基础。方法分离WHBE兔骨髓、外周血和脾脏三种来源单核细胞分别作为DC的前体细胞,以 GM-CSF和IL-4联合诱生未成熟DC,并经LPS刺激分化成熟。通过DC形态学和超微结构观察、表型鉴定以及同种异体混合淋巴细胞反应检测,比较三种DC的生物学特征和免疫功能。结果三种来源单核细胞经LPS刺激后其表面均显示出明显的树枝状突起,外周血和脾脏的DC分化速度较骨髓稍快,均能成功诱生出10⁶~10⁷数量级的DC,其中外周血来源获得的数量最多。CD86分子表达显著升高,脾脏来源DC的表达稍高于另外两组。具有显著刺激T细胞增殖的能力(P<0.05),但三组之间无显著性差异(P>0.05)。结论采集外周血单核细胞体外诱导扩增DC是获得WHBE兔树突状细胞最简便经济的手段。

关键词: 大耳白黑眼兔, 树突状细胞, 骨髓, 外周血, 脾脏, 体外诱导

Abstract: Objective To generate dendritic cells (DC) from different sources of WHBE rabbits (Oryctolagus cuniculusz). Methods DC were generated from mononuclear cells of bone marrow, peripheral blood and spleen by GM-CSF and IL-4. The morphology and ultrastructure were observed and the phenotypes of DC were analyzed. Immunological fuctions of the three differently derived dendritic cells were examined by allo-MLR. Results DCs generated from mononuclear cells of bone marrow, peripheral blood and spleen were matured by lipopolysaccharide (LPS). All three types of the mature DC exhibited extensive dendrites on their surfaces. DCs from peripheral blood and spleen were matured earlier than bone marrow. About 10⁶-10⁷ DCs were generated from three different sources and most of all was that from peripheral blood. All types of the DCs showed higher expression of CD86 compared with isotype control and obvious capacity to stimulate primary T cell responses in mixed lymphocyte reaction (MLR). But there were no difference among three groups (P>0.05). Conclusion Peripheral blood mononuclear cells (PBMC) were convenient and economic source to generate DC of WHBE rabbits.

Key words: WHBE rabbits, Dendritic cells(DC), Bone marrow, Peripheral blood, Spleen, Generation

中图分类号:

Q954-33

屠珏, 徐剑钦, 潘永明, 刘志华, 孔勤, 陈民利. WHBE兔骨髓、外周血和脾脏来源树突状细胞的比较[J]. 实验动物与比较医学, 2012, 32(3): 209-214.

TU Jue, XU Jian-qin, PAN Yong-ming, LIU Zhi-hua, KONG Qin, CHEN Min-li. Comparation of Dendritic Cells from Different Sources of WHBE Rabbits[J]. Laboratory Animal and Comparative Medicine, 2012, 32(3): 209-214.

参考文献

[1] Steinman RM, Banchereau J.Dendritic cells and the control of immunity[J]. Nature, 1998, 392:245-252.
[2] Ingulli E, Mondino A, Khoruts A, et al.In vivo detection of dendritic cell antigen presentation to CD4(+) T cells[J]. J Exp Med, 1997, 185(12):2133-2141.
[3] 陈民利, 蔡月琴, 陶涛, 等. 微卫星标记对WHBE兔封闭群、日本大耳白和新西兰兔的遗传分析[J]. 中国比较医学杂志, 2008, 18(9):21-27.
[4] 朱亮, 应华忠, 陈民利, 等. 微卫星标记对封闭群和近交第五代WHBE兔的遗传分析[J]. 实验动物与比较医学, 2009, 29(5):334-340.
[5] 陈民利, 吕建敏, 徐孝平, 等. WHBE免血气一电解质分析及与日本大耳白兔和新西兰白兔比较研究[J]. 实验动物与比较医学, 2009, 29(5):331-333.
[6] 徐剑钦, 陈民利, 张利棕, 等. WHBE兔主要脏器重量及脏器系数的测定[J]. 实验动物与比较医学, 2009, 29(4): 266-268.
[7] 王晓, 裴雪涛, 李梁, 等. 脐血CD34+细胞和外周血单核也细胞两种来源的树突状细胞特性的比较研究[J]. 中华血液学杂志, 1999, 11:583-585.
[8] Cody V, Shen H, Shlyankevich M, et al.Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and IL-4[J]. Vet Immunol Immunopathol, 2005, 103(3-4):163-172.
[9] 岳嘉宁, 斯东锋, 李济宇, 等. 大鼠脾脏来源树突状细胞的分离培养及生物学特性[J]. 上海交通大学学报, 2007, 27: 311-314.
[10] 周谦君, 范慧敏, 刘中民. IL-4\GM-CSF体外诱导猪单核来源树突状细胞和功能鉴定[J]. 实验动物与比较医学, 2010; 30(3):174-179.
[11] Wamcke M, Dodero A, Dierbach H, et al.Murine dendritic cells generated under serum-free conditions have a mature phenotype and efficiently induce primary immune responses[J]. J Immunol Methods, 2006, 310(1-2):1-11.
[12] 成建平, 黄君华, 王杰, 等.小鼠脾脏树突状细胞的体外扩增与培养. 中国组织工程研究与临床康复. 2009, 13(5): 873-876.
[13] 郭佳, 黄建华. 小鼠树突状细胞亚群与T细胞关系的研究进展[J]. 中国实验动物学报, 2006, 14:236-240.
[14] 李沛寰. 树突状细胞与自身免疫性疾病关系的研究进展[J]. 医学综述, 2007, 13:411-413.
[15] 仲琳, 张运, 张梅, 等. 兔动脉粥样硬化易损斑块模型的建立[J]. 基础医学与临床, 2005, 25(4):370-374.
[16] 雷霄, 李志梁, 傅强, 等. 在兔外周血源性及骨髓源性单核细胞向树突状细胞转化过程中人粒-巨噬细胞集落刺激因子与人白细胞介素4的作用[J]. 中国组织工程研究与临床康复, 2009, 13:1913-1917.
[17] 李新田, 邱财荣, 杨喜民, 等. 乌藤镇痛胶囊对类风湿关节炎家兔树突状细胞免疫活性的影响[J]. 中国医药导报, 2007, 4:115-116.
[18] 傅继东, 钟翠平, 王国强, 等.肝树突状细胞与骨髓树突状细胞体外生物免疫特性的比较研究[J]. 解剖学报, 2000, 31:81-84.
[19] Komi J, mottonen M, Luukkainen R, et al. Non-steroidal anti-oestrogens inhibit the differentiation of synovial macrophages into dendritic cells[J]. Rheumatology (oxford). 2001, 40(2):185-191.
[20] 陈静琦, 曾波航. 不同来源树突状细胞的研究进展[J]. 肿瘤防治杂志, 2003, 10:878-880.
[21] 吴萍萍, 杜晓英, 刘书逊, 等. 不同来源人树突状细胞的培养诱导及鉴定[J]. 浙江医学, 2007, 29:928-930.
[22] Liu E, Tu W, Law HK, et al.Phenotypic expression and function of immature monocyte-derived dendritic cells in cord blood[J]. Br J Haematol, 2001, l13(1):240-246.
[23] Cody V, Shen H, Shlyankevich M.et al.Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and hIL-4[J]. Vet Immunol Immunopathol, 2005, 103(3-4):163-172.
[24] Fanger NA, Maliszewski CR, Schooley K, et al.Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)[J]. J Exp, 1999, 190(8):l155-l164.
[25] Johansson E, Domeika K, Berg M, et al.Characterisation of porcine monocyte-derived dendritic cells according to their cytokine profile[J]. Vet Immunol Immunopathol, 2003, 91(3-4):183-197.
[26] Rau H, Revets H, Cornelis P, et al.Efficacy and functionality of lipoprotein OprI from Pseudomonas aeruginosa as adjuvant for a subunit vaccine against classical swine fever[J]. Vaccine, 2006, 24(22):4757-4768.