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目的:建立人外周血自然杀伤T细胞(NKT)体外扩增及检测NKT细胞分泌胰岛素样生长因子-1(IGF-1)的方法。方法:采用IFN-γ,CD3和IL-2的方法从人外周血单个核细胞(PBMC)中扩增出NKT细胞,采用流式细胞仪检测扩增效率,并使用绝对实时定量PCR(real-time PCR)的方法检测扩增出的NKT细胞中IGF-1的 mRNA表达量。使用内源性管家基因GAPDH校正误差。结果:两周扩增后NKT细胞增加了4倍。受试者扩增的NKT细胞IGF-1 mRNA表达量为1.29±0.56拷贝数/10~6GAPDH。结论:从PBMC扩增NKT细胞可以获得良好的效果,扩增出的NKT细胞有IGF-1的表达,但是表达量相对较低。
Objective: To establish a method for the in vitro expansion of natural killer T cells (NKT) from human peripheral blood and to detect the secretion of insulin-like growth factor-1 (IGF-1) from NKT cells. Methods: NKT cells were amplified from human peripheral blood mononuclear cells (PBMCs) by IFN-γ, CD3 and IL-2 methods. The efficiency of amplification was detected by flow cytometry and the real- time PCR) was used to detect the mRNA expression of IGF-1 in the expanded NKT cells. The endogenous housekeeping gene GAPDH was used to correct the error. Results: NKT cells increased four-fold after two weeks of expansion. The amount of IGF-1 mRNA expression in NKT cells amplified by the subjects was 1.29 ± 0.56 copies / 10 ~ 6GAPDH. CONCLUSION: NKT cells can be expanded from PBMC to obtain good results. The expanded NKT cells have the expression of IGF-1, but the expression level is relatively low.