摘要
为了建立一种快速有效的体外T细胞扩增方法,本研究比较了不同培养条件对T细胞增殖及其功能和特性的影响。取外周血T细胞,用自体的树突状细胞(DC)和EB病毒转化的B细胞进行周期性刺激,用细胞增殖试验、PI和AnnexinV双标记流式细胞术、Cr释放测定及ELISPOT试验分别测定不同培养条件下细胞的增殖程度、IFN-γ分泌细胞的频率、抗原特异性T细胞的杀伤活力、细胞的凋亡状态和分化能力。结果显示:T细胞在周期性刺激后,用CD3/28微珠的短期扩增效应最好,但多轮刺激后联合应用IL-2,IL-7和IL-15对细胞的扩增最强。Cr释放试验表明,CD3/28微珠扩增的T细胞不具有杀伤活力,高浓度IL-2诱导的T细胞杀伤活性最强,其次为联合应用IL-2,IL-7和IL-15。ELISPOT显示不同培养条件诱导出的分泌IFN-γ的T细胞频率从高到低依此为高浓度IL-2>联合应用IL-2,IL-7和Il-15>低浓度的IL-2>CD3/28微珠。细胞表型分析显示高浓度IL-2和联合应用IL-2,IL-7和Il-15扩增的细胞含有较高比例的CD3+CD8+T细胞。对细胞的凋亡测定表明联合应用IL-2,IL-7和IL-15获得的T细胞的存活率最高,处于凋亡早期的细胞比例最低。细胞增殖试验显示联合应用IL-2,IL-7和IL-15扩增的细胞分化能力最强。结论:本研究建立了一种快速有效地体外扩增T细胞的方法,其中联合应用IL-2,IL-7和IL-15对T细胞的扩增最显著,获得的T细胞存活率最高,分化能力最强,该细胞同时也含有较高频率的IFN-γ分泌细胞且表现出较强的杀伤活力。
The aim of this study was to compare cell proliferation and function of the T cells acquired under various cuture conditions for establishing a simple, safe and efficient cell expansion protocol in vitro. The peripheral blood mononuclear cells (PBMNC) were isolated and stimulated with autologous dendritic cells(DC) and EBV-transformed B lymphoblastoid cell line(BLCL) weekly. The cell proliferation test, flow cytometry with PI and Annexin V double staining, Cr release test and ELISPOT test were used to detect the cell expansion level, frequency of IFN-γ producing T cells, killing activity of antigen-specific T cells, cell apoptotic status and cell differentiation potential, respectively. The results indicated that use of IL-2 combined with IL-7 and IL-15 resulted in the highest cell expansion comparing to the use of IL-2 alone and the use of CD3/28 Microbeads. Also the cells obtained under cultivating with IL-2, IL-7 and IL-15 together showed high frequency of IFN-γ producing cells, strong killing activity, high viability and high differentiation potential with large portion of CD3+CD8+ population among the T cells. It is concluded that a protocol is established in which the use of IL-2 combined with IL-7 and IL-15 induces the biggest cell expansion, expanded cells show high viability, strong differentiation potential, high frequency of IFN-γ producing cells and strong killing activity.
出处
《中国实验血液学杂志》
CAS
CSCD
2010年第6期1590-1594,共5页
Journal of Experimental Hematology