PHA、ConA及CD3ε单克隆抗体对小鼠淋巴细胞迁移及分泌趋化因子的影响

Effects of PHA,ConA and CD3ε monoclonal antibody on mouse lymphocyte migration and chemokine secretion in vitro

目的探讨不同浓度的PHA、ConA及CD3ε单克隆抗体对Balb/c小鼠淋巴细胞迁移及分泌趋化因子的影响,初步了解这3种刺激剂与小鼠淋巴细胞迁移功能之间的关系。方法通过研磨法分离Balb/c小鼠脾脏淋巴细胞,用不同浓度的PHA、ConA及CD3ε单克隆抗体分别刺激培养淋巴细胞,收集细胞上清,用Traswell法测定细胞上清对淋巴细胞迁移率的影响;ELISA法检测细胞上清中趋化因子XCL1、CCL4、CCL5、CCL3、IL-16、IL-8及CCL20的水平。结果 PHA组各个浓度刺激下淋巴细胞的迁移率和对照组无明显差异;ConA组的刺激浓度为20μg/ml时,其对应的淋巴细胞迁移率明显高于对照组;CD3ε单抗组,其浓度分别为2.5、10μg/ml时,对应的淋巴细胞迁移率明显高于对照组。在各个刺激组中,ELISA测得的多种趋化因子水平在总体上存在明显差异。结论 ConA及CD3ε单克隆抗体分别刺激小鼠淋巴细胞后,其分泌趋化因子的功能发生了明显的改变,这些趋化因子影响着小鼠淋巴细胞的迁移功能。趋化因子XCL1、CCL4、CCL5、CCL3的水平明显高于对照,说明这几个因子在淋巴细胞迁移中起到重要的作用。根据实验结果初步推荐CD3ε单抗可以作为小鼠淋巴细胞迁移试验中的首选阳性对照,次选为ConA。

In order to understand the relationship between the three kinds of stimulators and mouse lymphocyte migration function preliminarily,we investigated the effects of different concentrations of PHA,ConA and CD3ε monoclonal antibody on Balb/c mouse lymphocyte migration and chemokine secretion in vitro.We used grinding method to separate Balb/c mouse lymphocytes from spleen and cultured with different concentrations of PHA,ConA and CD3ε monoclonal antibody.The lymphocyte migration rates of these collected cell culture supernatants were measured by transwell inserts,while the chemokine levels of XCL1,CCL4,CCL5,CCL3,IL-16,IL-8 and CCL20 in the cell culture supernatants were determined by ELISA.We found that the lymphocyte migration rate of PHA group had no difference compared with control group;the lymphocyte migration rate of 20 μg/ml ConA group was higher than that of control group.When the concentrations were 2.5 and 10 μg/ml,CD3ε monoclonal antibody group showed obvious higher lymphocyte migration rates when compared with the control group.The levels of multiple chemokines in the cell culture supernatants were significantly increased after lymphocytes were stimulated.In conclusion,ConA and CD3ε monoclonal antibodies could cause remarkable changes in mouse lymphocyte migration rates and chemokine secretions.The obviously elevated chemokines,such as XCL1,CCL4,CCL5 and CCL3,are responsible for the increase of lymphocyte migration function.According to the experimental results,CD3ε monoclonal antibody can be used as the best positive control in mouse lymphocyte migration test,second is ConA.