多种活化因子共刺激对人外周血单个核细胞体外增殖和表型的影响

Co-stimulation of multiple activating factors on proliferation and phenotype of T lymphocytes in peripheral blood in vitro

目的:观察多种活化因子共刺激后对人外周血淋巴细胞增殖和表型的影响。方法:用淋巴细胞分离液分离人外周血单个核细胞(PBMC),根据加入刺激因子(CD3 mAb、CD28 mAb、IFN-γ、IL-1α、IL-2、IL-15和IL-21)种类和方法不同将实验分为7组。用全自动五分类血液分析仪计数不同细胞因子诱导培养的PBMC增殖力、流式细胞术测定诱导后共刺激细胞表面CD3,CD4,CD8,CD28,CD16、CD56+CD16,CD3+CD8+,CD3+CD4+,CD3+CD56+,CD45RO等分子的变化、乳酸脱氢酶释放法测定诱导后的共刺激细胞对SGC-7901、SW-1990和SW-116细胞株的杀伤活性。结果:在PB-MC培养体系中加入不同的刺激因子其细胞增殖能力有明显的差异,以含刺激因子CD3、CD28、IFN-γ、IL-2、IL-1α、IL-15和IL-21组增殖倍数最高,在培养第10 d时该组的增殖倍数为255.3 6.3,明显高于仅含CD3、IFN-γ和IL-2培养体系组(166.6 5.5)(P<0.05)。在PBMC培养体系中加入不同的刺激因子其部分细胞表面标志有所差异,在培养体系中无IL-15时CD16+CD56+(NK细胞)细胞和CD3+CD56+细胞比例明显高于其他组;CD45RO+的记忆性T细胞以延迟3 d添加IL-15和IL-21组升高最明显。经不同活化因子刺激培养10 d的PBMC对SGC-7901、SW-1990和SW-1116细胞杀伤活性有明显差异,以延迟3 d添加IL-15和IL-21组最高(分别为76.2%、60.3%和70.6%),明显高于仅含CD3、IFN-γ和IL-2培养体系的细胞组(分别为54.9%、44.6%和50.4%)(P<0.05)。培养的细胞对胃腺癌细胞SGC-7901杀伤活性最强。结论:不同刺激因子活化的PBMC其增殖能力、表面标记和杀伤活性有明显差异,在培养体系中增加相应的刺激因子对细胞定向培养有一定价值。

AIM： To observe the costimulation of multiple activating factors effects on the proliferation and phenotype of T lymphocytes in vitro. METHODS： Peripheral blood mononuclear cells （PBMCs） were separated by fractionation on Ficoll-Hypacue gradient. According to adding different cytokines （CD3 mAb, CD28 mAb, IFN-γ, IL-lα, IL-2 and IL-15 ）, the experiments were divided into seven groups. Effects of different cytokines on the proliferation of PBMC were counted by automated hematology analyzer five categories. The phenotypes （CD3, CD4, CD8, CD28, CD16, CD56^＋ CD16, CD3^＋ CD8^＋, CD3^＋ CD4^＋ , CD3^＋ CD56^＋ , CD45RO） expressing on the surface of costimulatory cells were detected by flow cytometry, and the cytotoxicity of costimulatory cells on SGC-7901, SW-1990 and SW-116 cell lines was examined by lactate dehydrogenase release method. RESULTS： The proliferation has significant difference when adding different cytokines into PBMCs culture system, the highestest proliferation multiples group is the one contains cytokines CD3, CD28, IFN-γ, IL-2, IL-1α, IL-15 and IL-21, which proliferation multiple is 255.3 ± 6.3 at the tenth day of cell culture, obviously higher than the other culture systems which only contains CD3, IFN-γ and IL-2 （ 166.6 ＋5.5） （P〈0.05）. Part of cells＇ phenotype changed when adding different activating factors. Without IL-15, the proportion of CD16^＋ CD56^＋ （NK） cells and CD3^＋ CD56^＋ cells was higher than the other groups; CD45RO^＋ memory cells is most evident when delayed adding IL-15 and IL-21 for three days. The cytotoxicity of PBMCs cultured for ten days with different activating factors had significant difference, the highest was the one which delayed adding IL-15and IL-21 for three days （76.2%, 60.3% and ？0.6%, respectively. ）, higher than the cell culture groups containing CD3, IFN-γ and IL-2 （54. 9%, 44. 6% and 50. 4%, respectively） （P〈0.05）. The cultured cells had the strongest cytotoxicity on SGC-7901 gastric adenocarcinoma cells. CONCLUSION： The PBMCs＇ proliferation, phenotype and cytotoxicity had significant difference after being activated by different stimulating factors, adding matching stimulating factors into the culture system have great value on celldirected culture.