Liu Jun-Quan, Zhu Yun, Chen Fu-Xing, Zhang Nan-Zheng, Lv Xiao-Ting, Zhou Zhong-Hai, Zhang Juan, Zhang Song, Tao Zheng-Zhong, Li Yi
Biotherapy Center, 97th Hospital of PLA, Xuzhou 221004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Apr;28(4):367-70.
To observe the costimulation of multiple activating factors effects on the proliferation and phenotype of T lymphocytes in vitro.
Peripheral blood mononuclear cells (PBMCs) were separated by fractionation on Ficoll-Hypaque gradient. According to adding different cytokines (CD3 mAb, CD28 mAb, IFN-γ, IL-1α, 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.
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-15 and IL-21 for three days (76.2%, 60.3% and 70.6%, 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.
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 cell-directed culture.
观察多种激活因子共刺激对体外T淋巴细胞增殖及表型的影响。
采用Ficoll-Hypaque密度梯度离心法分离外周血单个核细胞（PBMCs）。根据添加不同细胞因子（CD3单克隆抗体、CD28单克隆抗体、干扰素-γ、白细胞介素-1α、白细胞介素-2和白细胞介素-15），将实验分为七组。用全自动血液分析仪计数不同细胞因子对PBMC增殖的影响，分为五类。采用流式细胞术检测共刺激细胞表面表达的表型（CD3、CD4、CD8、CD28、CD16、CD56(+)；CD16、CD3(+)；CD8(+)；，CD3(+)；CD4(+)；，CD3(+)；CD56(+)；，CD45RO），并用乳酸脱氢酶释放法检测共刺激细胞对SGC-7901、SW-1990和SW-116细胞系的细胞毒性。
在PBMCs培养体系中添加不同细胞因子时，增殖有显著差异，增殖倍数最高的组是含有细胞因子CD3、CD28、干扰素-γ、白细胞介素-2、白细胞介素-1α、白细胞介素-15和白细胞介素-21的组，细胞培养第10天时增殖倍数为255.3±6.3，明显高于仅含CD3、干扰素-γ和白细胞介素-2的其他培养体系（166.6±5.5）（P<0.05）。添加不同激活因子后部分细胞表型发生改变。无白细胞介素-15时，CD16(+)；CD56(+)；（NK）细胞和CD3(+)；CD56(+)；细胞的比例高于其他组；延迟三天添加白细胞介素-15和白细胞介素-21时，CD45RO(+)；记忆细胞最为明显。用不同激活因子培养10天的PBMCs的细胞毒性有显著差异，最高的是延迟三天添加白细胞介素-15和白细胞介素-21的组（分别为76.2%、60.3%和70.6%），高于含CD3、干扰素-γ和白细胞介素-2的细胞培养组（分别为54.9%、44.6%和50.4%）（P<0.05）。培养的细胞对SGC-7901胃癌细胞的细胞毒性最强。
不同刺激因子激活后PBMCs的增殖、表型和细胞毒性有显著差异，在培养体系中添加匹配的刺激因子对细胞定向培养有重要价值。
