利用CFSE标记检测CD8~+淋巴细胞增殖

目的 ]介绍一种新的检测淋巴细胞增殖的方法。 方法 ]用本室构建的重组蛋白疫苗hsp6 5-CAP3对C5 7小鼠进行 3次免疫后 ,分离小鼠的脾细胞 ,以照射灭活的CAP3转基因细胞为刺激细胞 ,在体外刺激淋巴细胞的增殖反应。淋巴细胞在与刺激细胞共培养之前用绿色荧光染料羧乙基锗倍半氧化物 (CFSE)进行标记。在共培养 72h后 ,用PE标记的抗鼠CD8分子的单抗标记淋巴细胞。收集细胞进行FACS检测 ,出现在二维点图左上象限的细胞代表CFSE含量减少的CD8 淋巴细胞 (CD8 CFSElow)即增殖的CD8 淋巴细胞 ,利用流式细胞仪记数左上象限CD8 CFSElow细胞的比例。 结果 ]经体外刺激的脾淋巴细胞中 ,实验组CD8 CFSElow的平均百分比值为 (16 .4 7± 2 .10 ) % ,对照组CD8 CFSElow 的平均百分比值为 (7.6 9± 1.6 5 ) % (P <0 .0 5 )。 结论 ]通过FACS结果判定CD8 淋巴细胞发生了特异性的增殖。CFSE标记是一种有效的检测淋巴细胞增殖的方法。

CFSE-Based proliferative assay for CD8~+ T lymphocytes

Objective] To introduce a new method of lymphocyte proliferation assay.Methods] C57 mice were immunized with hsp65-CAP3 3 times.lymphocytes were isolated from spleen and labelled with green flurescent dye CFSE. To stimulate the in vitro proliferation of CD8 + T cells, lymphocytes were incubated with CAP3 gene tranfected B16 cells. After 72 hours incubation, lymphocytes were stained with PE-labeled anti-CD8 antibody.then these cells were subjected to FACS assay to detect the cells appeared in upper left quandrant in dot plot. this group is the CD8 + cells with reduced CFSE and stand for proliferated CD8 + lymphocytes (CD8 +CFSE low ). Results] In the experimental groups,the mean percentage of CD8 +CFSE low is(16.47±2.10)%, higher than (7.69±1.65)% dected in the control group(P<0.05).Conclusion] FACS results showed that CD8 + lymphoctyes underwent specific proliferation. CFSE staining is an effective method for lymphoctye proliferative assay.