免疫磁珠两步法体外分离纯化小鼠脾脏CD4^+CD25^+调节性T细胞的应用研究

Application research of two-step method of immunomagnetic beads on isolation and purification of CD4+CD25+ regulatory T cells of mice spleen

目的研究免疫磁珠两步法体外分离纯化小鼠脾脏CD4+CD25+调节性T细胞(regu-latory T cells,Treg)的效果。方法将C57BL/6(H-2b)小鼠脾脏剪碎、过滤、去除红细胞后离心洗涤,获得小鼠脾细胞悬液。用Ficoll法分离获得单个核细胞后,通过免疫磁珠阴性加阳性选择两步法分离获得CD4+CD25+Treg和CD4+CD25-Treg。用流式细胞仪检测所获得CD4+CD25+Treg的纯度,并比较纯化分离前后脾脏CD4+CD25+Treg的比例,用台盼蓝染色后计算CD4+CD25+Treg的细胞存活率,并对分离后的细胞表型进行鉴定。结果免疫磁珠阴性加阳性选择两步法纯化分离后CD4+CD25+Treg的纯度为(88.7±2.9)%,分离前CD4+CD25+Treg的平均比例为11.6%,纯化分离后CD4+CD25+Treg的平均比例为93.7%。细胞存活率为(93.7±4.1)%。与CD4+CD25-Treg相比,CD4+CD25+Treg的CD44表达略有增加(P>0.05),CD45RB的表达明显下降(P<0.05),CTLA-4和Foxp3的表达明显增加(均为P<0.05)。结论使用免疫磁珠两步法分离纯化,能避免激惹CD4分子,可获得高纯度且具有天然CD4+CD25+Treg表型的细胞。

Objectives To study the effect of the two-step method of immunomagnetic beads on isolating and purifying CD4^＋CD25^＋ regulatory T cells （Treg） of mice spleen. Methods Splenocytes suspension of C57BL/6 （H-2b） mice was gathered by centrifuge washing after the spleen was cut and filtered, and red blood ceils were eliminated. Monocytes were isolated by Fieoll method, then CD4^＋CD25^＋ Treg and CD4^＋CD25^＋ Treg were isolated and collected by two-step method of immunomagnetic beads negative and positive selection. The purity of CD4^＋CD25^＋ Treg was detected by flow cytometer. And the proportion of CD4^＋CD25^＋ Treg of spleen was compared before and after isolation. Survival rate of CD4^＋CD25^＋ Treg was estimated and calculated by blue dyeing. And phenotype of isolated cells was identified. Results The purity of CD4^＋CD25^＋ Treg was （88.7 ± 2. 9） % after isolation and purification by two-step method of inununomagnetic beads negative and positive selection. The average proportion of CD4^＋CD25^＋ Treg was 11.6 % before isolation and 93.7 % after isolation. Survival rate of CD4^＋CD25^＋ Treg was （ 93.7 ± 4. 1 ） % . Compared with CD4^＋CD25^＋ Treg, the expression of CD44 slightly increased （ P 〉 0. 05） and the expression of CD44RB significantly decreased in CD4^＋CD25^＋ Treg （P 〈 0. 05 ） . The expression of CTLA- 4 and Foxp3 in CD4^＋CD25^＋ Treg also significantly increased （ all in P 〈 0. 05） . Conclusion Isolation and purification by the two-step method of immunomagnetic beads can avoid stimulating CD4 and get CD4^＋CD25^＋ Treg with high purity and nature phenotype.