免疫磁珠正负筛选在分离外周血CD8+和 CD4+T细胞亚群中的应用

为了探讨免疫磁珠分离技术及其正、负筛选策略在人外周血T淋巴细胞亚群分离提纯中的应用,应用补 体细胞毒法和正负筛选策略相结合的免疫磁珠法分离健康人外周血中CD4 T细胞、CD8 T细胞;用流式细胞术法 比较两种分离方法分离的靶细胞纯度;台盼蓝染色染色法和细胞培养后平板计数法检测免疫磁珠法分离的CD4 T 细胞、CD8 T细胞的活力和生长情况。结果表明:补体细胞毒法分离后CD4 T细胞纯度、CD8 T细胞纯度分别为 (76.0±2.8)％和(77.0±3.0)％,明显高于分离前的(34.6±3.7)％和(32.0±3.7)％(P<0.05);免疫磁殊法 分离后CD4 T细胞和CD8 T细胞纯度为(94.2±1.4)％和(93.8±3.0)％,显著高于补体细胞毒法(P<0.05)。 磁珠分离法获得的CD4 T细胞、CD8 T细胞细胞活力分别为(96.0±2.4)％和(95.0±3.6)％;细胞计数法表明 磁珠分离法获得的CD4 T细胞、CD8 T细胞对植物血凝素(PHA)的刺激保持了良好的增殖能力。结论:免疫磁珠 法较补体细胞毒法分离效率高,不影响细胞的活力和增殖能力;正负筛选策略相结合的免疫磁珠法在同时获取高 纯度的CD4 T细胞、CD8 T细胞时可获得满意效果,尤其适应于只有较少量血标本的情况,为进一步研究CD4 T 细胞、CD8 T细胞的生物学特性创造了条件。

Application of Immunomagnetic Screening Strategy for Separation of CD4 + and CD8 + T Cell Subpopulations of Peripheral Blood

To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.