大鼠骨髓来源树突状细胞的体外培养与鉴定.doc

中 文 摘 要

骨髓来源的树突状细胞(dendritic cells DC) 是已知的体内抗原提呈功能最强的抗原提呈细胞( antigenpresenting cell，APC ) ，能摄取各类抗原, 体内、外均能激发T 细胞增殖, 诱导特异性细胞杀伤性T 淋巴细胞( cy to to xic T lymphocy tes, CT L) 生成, 产生抗肿瘤效应。近些年来越来越多的人认识到DC 在肿瘤免疫中的作用, DC 的研究也已经进入了体内研究阶段。但既往体内试验主要是以裸鼠为动物模型，由于裸鼠为细胞免疫缺陷性动物, 很难反应出卵巢癌DC 疫苗的真正免疫调节作用，因此有必要对正常免疫状态的实验动物进行DC 培养。大鼠是体内实验最常用的动物模型, 而目前国内外对大鼠来源DC 培养方法有限。本实验利用大鼠骨髓细胞进行DC 的分离培养, 进一步探讨了大鼠骨髓DC 的培养方法, 为DC 体内研究奠定基础。

目的：建立体外培养、诱导和扩增大鼠骨髓来源的树突状细胞的方法。

方法：实验通过运用大鼠淋巴细胞分离液和培养过程的手法筛选相结合,培养、诱导和扩增大鼠骨髓来源的树突状细胞,通过形态学和免疫表型进行鉴定。

结果： 成功建立了体外大量培养、诱导及扩增大鼠骨髓来源树突状细胞的方法;经倒置相差显微镜、透射电镜及流式细胞仪鉴定,证实所培养细胞为树突状细胞。 

结论：通过运用大鼠淋巴细胞分离液和培养过程的手法筛选相结合能培养、诱导、扩增大量的树突状细胞。

关键词: 树突状细胞; 免疫耐受; 大鼠

Astract 

Bone marrow derived dendritic cells (dendritic cells DC) is known in vivo antigen presenting function is the strongest antigen-presenting cells(antigenpresenting, cell, APC), intake of various antigens, body, can stimulate the proliferation of T cells, induce specific cytotoxic T lymphocyte (CY to to Xic T Lymphocy tes, CTL) generation, producing antitumor effect. In recent years, more and more people realize that the role of DC in tumor immunity, DC research has entered a phase of the study in vivo. But previous in vivo test is mainly to nude mice as animal model, the nude mice for cell immunodeficiency animal, it is difficult to reflect the real immune regulation effect of ovarian cancer DC vaccine, it is necessary to experimental animal of normal immune status were cultured in DC system. The rat is the most commonly used experimental animal models, while at home and abroad on the rat DC culture co.. Isolation and culture of rat bone marrow cells in this experiment using DC, to further explore the cultivation of rat bone marrow DC, lay the foundation for DC in vivo.

Objective: to establish a method of induction and proliferation of rat bone marrow-derived dendritic cells, in vitro culture.

Methods: the experiment by using the separation of rat lymphocytes and screening combined training techniques, training,induction and proliferation of rat bone marrow-derived dendritic cells, were identified by morphology and immunophenotype.

Results: successfully established the method of in vitro, induction and proliferation of rat bone marrow-derived dendritic cells; by the inverted microscope, transmission electron microscope and flow cytometry, confirmed that the cultured cells into dendritic cells.

Conclusion: through the use of liquid separation of rat lymphocytes and training process is manual screening combining culture, induction, amplification of dendritic cells.

Keywords: dendritic cells; immune tolerance; rats

目  录

中 文 摘 要 I

Astract II

目  录 III

第一章  前  言 1

第二章  实验部分 3

2.1  实验动物 3

2.2  主要仪器和试剂 3

2.3试剂配制 4

2.4  实验方法 5

第三章  实验结果分析及讨论 7

3.1 骨髓树突状细胞的形态观察 7

3.2 讨论 7

第四章  结论 10

附  图 11

参考文献 12

致  谢 13

外文文献 14

文献翻译 17