小鼠脾脏B淋巴细胞纯化培养方法的建立

发布时间：2018-04-14 20:34

本文选题：组织工程 + B淋巴细胞　；参考：《中国组织工程研究》2015年02期

【摘要】：背景:B淋巴细胞是机体免疫系统重要的参与者,目前的研究主要采用磁珠、补体等方法进行B淋巴细胞的分离纯化,但是这些方法费用高或者细胞损伤大、纯度低,体外分离、培养B淋巴细胞的方法还有待进一步改进。目的:探讨从小鼠脾脏细胞中对B淋巴细胞同时进行分离、培养的方法。采用加入白细胞介素4、脂多糖或者CD3单克隆抗体及其组合,探讨体外分离、培养小鼠脾脏B淋巴细胞的适宜条件。方法:体外分离培养小鼠脾脏细胞,随机分为7组,分别用白细胞介素4,CD3,脂多糖,白细胞介素4+CD3,白细胞介素4+脂多糖,CD3+脂多糖组进行干预、将未给予刺激的脾细胞作为对照组。用流式细胞仪检测在不同培养条件下小鼠脾脏细胞T、B淋巴细胞及其亚群的变化。结果与结论:与对照组比较,白细胞介素4组淋巴细胞在培养后第3-5天数量达到高峰;脂多糖组在培养初期无明显作用,第3天开始淋巴细胞数量有明显的增加,第5天达到高峰;培养体系中加入CD3单克隆抗体,可导致T淋巴细胞消失,培养2 d后,可得到较为单一的B淋巴细胞,其细胞数量在第3天达到高峰。其中B220+IgD+成熟B淋巴细胞亚群数量显著增加。体外培养24 h后,各组B220+CD93+Transitional B淋巴细胞亚群均完全消失。结果说明,体外培养的小鼠脾脏细胞加入CD3单克隆抗体和白细胞介素4可以去除T淋巴细胞,并维持成熟B淋巴细胞的生存和增殖。
[Abstract]:Background: B lymphocytes are important participants in the body's immune system. Currently, the methods of separation and purification of B lymphocytes, such as magnetic beads and complements, are mainly used, but these methods are expensive or have high cell damage, low purity and in vitro isolation.The method of B lymphocyte culture needs further improvement.Objective: to study the method of simultaneous isolation and culture of B lymphocytes from mouse spleen cells.The suitable conditions for isolation and culture of mouse splenic B lymphocytes in vitro were studied by adding interleukin 4, lipopolysaccharide or CD3 monoclonal antibodies and their combinations.Methods: mouse spleen cells were isolated and cultured in vitro and randomly divided into 7 groups. The mice were treated with interleukin-4 CD3, lipopolysaccharide, interleukin-4 CD3, interleukin-4 lipopolysaccharide CD3 respectively.Splenocytes without stimulation were used as control group.The changes of T _ (B) lymphocytes and their subsets of spleen cells in different culture conditions were detected by flow cytometry.Results and conclusion: compared with the control group, the number of lymphocytes in the IL-4 group reached the peak on the 3-5 days after culture, but in the lipopolysaccharide group there was no obvious effect at the initial stage of culture, but the lymphocyte number increased significantly at the 3rd day.On the 5th day, the T lymphocytes disappeared when CD3 monoclonal antibody was added into the culture system. After 2 days of culture, a single B lymphocyte was obtained, and the number of B lymphocytes reached its peak on the third day.The number of mature B lymphocyte subsets of B 220 IgD increased significantly.After cultured in vitro for 24 h, B lymphocyte subsets of B 220 CD93 Transitional disappeared completely in each group.The results showed that murine spleen cells cultured in vitro with CD3 monoclonal antibody and interleukin 4 could remove T lymphocytes and maintain the survival and proliferation of mature B lymphocytes.
【作者单位】：天津医科大学基础医学院生物化学与分子生物学系;
【基金】：国家自然科学基金资助项目(81070271)~~
【分类号】：R392

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