C57BL／6小鼠树突状细胞的培养及其诱导细胞毒性T淋巴细胞对FBL-3细胞杀伤效应的研究

目的探讨C57BL／6小鼠树突状细胞（DC）的培养及其诱导细胞毒性T淋巴细胞（CTL）对FBL-3细胞的杀伤效应。方法应用GM—CSF和114培养C57BL／6小鼠骨髓DC，用冻融法制备FBL-3细胞抗原致敏DC，用3H—TdR法检测T细胞增殖反应的能力，标准的4h51cr释放测定法检测CTL杀伤活性。结果用IL4和GM—CSF联合培养小鼠骨髓细胞3天后，可见细胞形态发生改变，细胞形态不规则。培养第6～8天，细胞表面出现较多毛刺样突起，拉长，为典型的DC特征。流式细胞仪检测DC表面分子有CD80、CD86、H-2Kd及I-Ad表达。FBL-3细胞冻融组和PBS组的DC在体外均可以诱导T细胞增殖，冻融组的DC体外诱导T细胞增殖能力明显高于PBS组。冻融组DC体外诱导的CTL对FBL-3细胞有明显的细胞毒作用。结论应用IL-4联合GM—CSF培养C57BL／6小鼠骨髓细胞，可大量扩增成熟DC，培养的DC符合其自身的特性；用冻融法制备FBL-3细胞抗原致敏DC，可以诱导T细胞细胞大量增殖，并能诱导出杀伤效应较强的CTL。

Culturing of dendritic cells from bone marrow of C57BL/6 mice and its induction CTL to kill FBL-3 cells in vitro

Objective： To explore a method of expanding dendritic cells from bone marrow of C57BL/6 mice in vitro and its induction CTL to kill FBL-3 cells in vitro. Methods： The mouse marrow DC was cultured using GM-CSF and IL-d, and DC loaded with FBL-5 cell＇ s antigen was prepared by freezing melting, and the T cell multiplication ability was examined with the 3H-TdR, and the killing effect of CTL on FBL-3 cell was examined by 4 h the 51Cr release measuring method. Re- suits： With IL-4 and GM-CSF, morphological changes were found and dendritic protrusion appeared on the 3rd day after culture; dendritic protrusion prolonged on the 5th day after culture; flow-cytometric analysis showed that expressions of CD83 and CD86, H-2Kd and I-Ad. The freezing melting and PBS group＇s DC might induce the T cell multiplication in vitro, but the freezing melting group＇s DC induced the T cell multiplication ability and was be significantly higher than that of the PBS group. The freezing melting group g DC induced CTL cytotoxin effect on the FBL-3 cell and was significantly higher than that of the PBS group. Conclusion： Dendritic cells from bone marrow of C57BL/6 mice can be cultured. Dendritic cells of mice have typical morphology and characteristic phenotype. DC loaded with tumour antigen prepared by freezing melting can induce the T cell massive multiplication and CTL with stronger killing effect on FBL-3 cell .