目的:比较人外周血γδ T细胞不同的制备方法，摸索适于临床应用的培养和保存条件。方法：分离人外周血单个核细胞，分别以固相化和可溶性antiTCRγδ抗体进行刺激，以添加小牛血清或人AB血浆的RPMI 1640培养液或AIM V无血清培养液进行培养，采用锥虫蓝染色法检测γδ T细胞存活率，采用免疫荧光染色法考察γδ T细胞的纯度和亚型，采用乳酸脱氢酶（lacate dehydrogenase，LDH）法检测γδ T细胞对不同肿瘤细胞系的杀伤作用。结果：添加15%小牛血清和添加5%人AB血浆的RPMI 1640培养液对γδ T细胞的培养效果接近，均优于AIM V无血清培养液。可溶性抗体与固相化抗体扩增外周血γδ T细胞的效果相似；可溶性抗体扩增的γδ T细胞受体（T cell receptor，TCR）库容完整，同时具有TCR Vδ1和Vδ2两种亚型；对肺癌、肝癌和卵巢癌细胞系均有体外杀伤活性。这些γδ T细胞在含1%人AB血浆或0.25%人血白蛋白的生理盐水中、4 ℃条件下保存12 h,细胞存活率仍达95%以上。结论：可溶性antiTCR γδ抗体和含有人AB血浆的RPMI 1640培养液可以用于人外周血γδ T细胞的体外扩增，扩增得到的γδ T细胞在含有血浆或白蛋白的生理盐水中稳定性较好。

Objective: To compare the different protocols for amplifying human peripheral blood γδ T cells and establish the suitable expansion and storage conditions for clinical application.Methods: Human peripheral blood mononuclear cells were isolated and stimulated by immobilized or soluble antiTCR γδ, and further cultured in RPMI 1640 supplemented with newborn bovine serum, RPMI 1640 supplemented with human AB plasma, or serum free AIM V medium separately. Cell numbers and survival rates were detected by trypan blue staining; purities and phenotypes of the expanded γδ T cells were measured by immunofluorescence staining; cytotoxicities of γδ T cells against different tumor cell lines were detected by lactate dehydrogenase (LDH) method. Results: RPMI 1640 supplemented with 15% newborn bovine serum or 5% human AB plasma had similar effects on the proliferation of γδ T cells but better than that of AIM V serum free medium. Soluble antibody stimulated the expansion of peripheral blood γδ T cells as effectively as immobilized antibody did. The expanded γδ T cells had complete T cell receptor (TCR) pool, containing TCR Vδ1 and Vδ2 subtypes, and displayed cytotoxic activities aganist lung cancer, hepatocellular carcinoma, and ovarian carcinoma cells in vitro. These γδ T cells, when suspended in normal saline supplemented with 0.25% human albumin or 1% human AB plasma, could maintain 95% survival for 12 hours at 4 ℃. Conclusion:Human peripheral blood γδ T cells can be effectively expanded in vitro by soluble antiTCR γδ in RPMI 1640 culture medium containing human AB plasma, and these expanded γδ T cells can be stably preserved in normal saline supplemented with plasma or albumin.

国家高技术研究发展计划(863计划)资助项目(No. 2007AA021109)