【摘要】 采用流式细胞术建立测定CD4+T细胞和B细胞相互黏附方法,探讨CD4+T细胞活化对B细胞黏附和活化状态的影响。分离正常人外周血单个核细胞(PBMC),采用磁珠分选的方法分选CD4+T细胞和B细胞后,进一步通过体外细胞黏附技术,检测并比较未经活化和经抗CD3/CD28抗体活化的CD4+T细胞与B细胞黏附能力的差异;采用流式细胞术检测培养后的B细胞中与B细胞活化相关的Pyk2分子磷酸化水平。纯化的CD4+T细胞和B细胞经体外共同培养后,可以发生直接黏附,未活化的CD4+T细胞和B细胞共同培养后,CD4+CD19+双阳性细胞占B细胞的比例为4.024%±1.078%,经不同比例(1∶8和1∶1)抗CD3/CD28抗体活化的CD4+T细胞与B细胞作用后的CD4+CD19+双阳性细胞的比例分别为5.560%±0.863%和8.358%±1.917%(P<0.05),显著高于未活化组,表明活化的CD4+T细胞与B细胞之间的黏附能力显著提高,并随着CD4+T细胞活化程度的升高而增加;同时,活化的CD4+T细胞与B细胞发生黏附后,可以促进B细胞在短时间内发生活化,B细胞内Pyk2磷酸化的水平显著升高。本研究初步建立了用于分析CD4+T细胞和B细胞黏附水平的体外细胞学检测方法,研究结果显示活化的CD4+T细胞与B细胞发生黏附的能力显著增强,并且可直接促进B细胞的活化。更多还原

【Abstract】 To investigate the impacts of CD4+T cell activation on B cells adhesion and activation through flow cytometry based T-B cell adhesion assay,peripheral blood mononuclear cells(PBMCs)from healthy donors were isolated by Ficoll-Paque density centrifugation.CD4+T cells and B cells were isolated by MACS separation.Adhesion between CD4+T cells(unstimulated CD4+T cells and CD3/CD28 activated CD4+T cells respectively)and B cells were analyzed and compared by in vitro cell adhesion assay.B cell activation was analyzed in parallel through the detection of Pyk2 phosphorylation in B cells.Sorted CD4+T cells can directly adhere to B cells after in vitro coculture.The percentage of CD4+CD19+cell population in B cells cocultured with unstimulated CD4+T cells was approximately 4.024%±1.078%.The adhesion between CD4+T cells activated by CD3/CD28stimulation(1∶8and 1∶1)and B cells was significantly increased with 5.560%±0.863% and 8.358%±1.917% of CD4+CD19+cells after coculture respectively(P<0.05),which was strongly correlated with T cell activation.Meanwhile,the direct contact of CD4+T cells with B cells triggered B cell activation since the phosphorylation of Pyk2 in B cells was significantly amplified after coculture.In the present study we have established a feasible method to analyze the adhesion between human CD4+T cells and B cells in vitro.Further study demonstrated that the activation of CD4+T cells enhanced both the adhesion to B cells and the activation of B cells,which strongly implied the critical role of T cells activation on B cell activation and differentiation.更多还原