人外周血γδ T细胞的可溶性anti-TCR γδ抗体扩增及其培养和保存的条件

Soluble anti-TCR γδ stimulates expansion of human peripheral blood γδ T cells and its culture and storage

目的:比较人外周血γδT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RPMI1640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γδT细胞存活率,采用免疫荧光染色法考察γδT细胞的纯度和亚型,采用乳酸脱氢酶(lacate dehydrogenase,LDH)法检测γδT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RPMI1640培养液对γδT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γδT细胞的效果相似;可溶性抗体扩增的γδT细胞受体(Tcell 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γδT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12h,细胞存活率仍达95%以上。结论:可溶性anti-TCRγδ抗体和含有人AB血浆的RPMI1640培养液可以用于人外周血γδT细胞的体外扩增,扩增得到的γδT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 estab- lish 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 supplemen- ted with newborn bovine serum, RPMI 1640 supplemented with human AB plasma, or serum free AIM V medium sepa- rately. Cell numbers and survival rates were detected by trypan blue staining; purities and phenotypes of the expanded γδT cells were measured by immunofluorescenee staining; cytotoxicities of γδT 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 γδT 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 VS1 and δ2 subtypes, and dis- played cytotoxic activities aganist lung cancer, hepatocellolar 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 γδT cells can be stably preserved in normal saline supplemented with plasma or albumin.