间充质干细胞对脐血CIK/NK细胞CD69表达及培养体系中T调节细胞量比的影响

Effect of Mesenchymal Stem Cells on Expression of CD69 in Cord Blood CIK/NK Cells and Quantity Ratio of T Regulatory Cells in CIK/NK Cell Culture

本研究通过Transwell细胞隔离培养体系探讨骨髓源间充质干细胞(MSC)对脐血(CB)来源细胞因子诱导杀伤细胞(CIK)/自然杀伤细胞(NK)CD69的表达以及对培养体系中CD4+CD25+T调节(Treg)细胞量比的影响。利用聚碳酯膜孔径0.4μm的Transwell隔离细胞培养系统将实验组分为隔离培养(Transwell)组和直接混合(mix-ture)培养组;将MSC和CIK/NK细胞按1:20、1:50和1:100的比例分别在Transwell组和mixture组中进行隔离和直接混合培养,每组6个重复孔;培养48小时后通过流式细胞术检测各组CIK/NK细胞上CD69的表达以及培养体系中CD4+CD25+细胞量比的变化。结果显示:加入MSC的实验各组CB-CIK细胞和NK细胞上表达的CD69比例均显著低于对照组(p值均<0.001).Transwell组中的CIK和NK细胞上表达的CD69,在MSC的高浓度组(1:20组)均显著低于低浓度组(1:50组和1:100组),其中CIK细胞组间的p值分别为0.046、0.020;NK细胞组间的p值均为0.000。mixture组中不同比例组间CIK细胞上表达的CD69无显著性差异,而NK细胞上CD69的表达在MSC的高浓度组(1:20组)均显著低于低浓度组(1:50组和1:100组)。加入MSC的CB-CIK/NK细胞体系中的CD4+CD25+细胞的量比不论在Transwell还是mixture组中均显著高于对照组(p值均<0.01);在Transwell组中,CIK/NK细胞体系中的CD4+CD25+细胞的量比在1:20组、1:50组均显著高于1:100组;在mixture各组中,CIK/NK细胞体系中的CD4+CD25+T调节细胞的量比均有显著性差异。MSC的高浓度组(1:20组),CIK/NK细胞体系中的CD4+CD25+细胞量比在mixture组显著高于Transwell组;而在MSC的低浓度组(1:50组和1:100组),2组体系中的CD4+CD25+细胞量比无显著性差异。结论:MSC能以直接接触或间接作用的方式抑制异基因CIK/NK细胞的活化,这可能与MSC能上调培养体系中的CD4+CD25+Treg细胞量比有关,且这种作用与MSC的数量呈浓度依赖关系。

This study was purposed to explore the effect of bone marrow derived mesenchymal stem cells （MSCs） on the expression of CD69 on cytokine-induced killer（ CIK）/natural killer（NK） cells derived from cord blood and on the quantity ratio of CD4^＋ CD25^＋ T regulatory cells in CIK/NK cell culture system using Transwell non-contact cell culture system. The experiments were divided into two groups.. Transwell non-contact culture and mixture culture. The ratio of MSC to CIK/NK cells was 1 ： 20, 1 ： 50 and 1 ： 100. In mixture culture groups, MSC and CIK/ NK cells were cocultured by together contact as the same ratio of Transwell non-contact culture groups. The expression of CD69 on CIK/ NK cells, as well as the quantity ratio of CD4^＋ CD25^＋ T regulatory cells in CIK/NK cell culture were evaluated by flow cytometry. The results showed that the expression of CD69 on CIK/NK cells in experimental groups were significantly lower than that in control group（p 〈0. 001 ）. As to TransweU groups, CD69 expression on the CIK/NK cells at 1：20 ratio of MSC and CIK/NK was significantly lower than that at 1 ： 50 and 1 ： 100 ratio. There were no differences in the expression of CD69 on CIK cells in mixture groups with various MSC ratios, whereas the expression of CD69 on NK cells at 1： 20 ratio was significantly lower than that at 1：50 and 1 ： 100. The quantity ratio of CD4^＋ CD25^＋ ceils in CIK/ NK cell culture system of experimental groups with MSC co-culture was significantly higher than that in control. As to Transwell groups, the ratio of CD4^＋ CD25^＋ cells in CIK/NK cell culture system at 1 ： 20 and 1 ： 50 was significantly higher than that at 1 ： 100. The quantity ratio of CD4^＋ CD25^＋ cells in CIK/NK cell culture system showed significant differences in various mixture groups. As to 1 ： 20 ratio the amount of CD4^＋ CD25^＋ cells in CIK/NK cell culture system of mixture groups was significantly higher than that in Transwell groups, while there were no differences of the quantity ratio of CD4^＋ CD25^＋ cells in CIK/NK cell culture at 1 ： 50 and 1 ： 100. It is concluded that either by non-contact Transwell or mixed co-culture, the MSC can suppress the activation of allogeneic CB-CIK/NK cells, which maybe relate to up-regulating the ratio of CD4^＋ CD25^＋ T regulatory cells in CIK/NK cell culture system in dose-dependent manner.