脐带间充质干细胞共培养对中性粒细胞存活和免疫功能的影响  

The modulation of neutrophil survival and immune functions via coculturing with human umbilical cord mesenchymal stem cells

作　　者：范玉龙 孔鑫垚 王永荣 吕梦楠 马润芝 王彤 冯四洲 韩明哲 许元富 FAN Yulong;KONG Xinyao;WANG Yongrong;LYU Mengnan;MA Runzhi;WANG Tong;FENG Sizhou;HAN Mingzhe;XU Yuanfu(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China)

机构地区：[1]中国医学科学院血液病医院(中国医学科学院血液学研究所)实验血液学国家重点实验室国家血液病临床医学研究中心,天津300020

出　　处：《免疫学杂志》2021年第1期8-16,共9页Immunological Journal

基　　金：中国医学科学院医学科学创新基金(2016-12M-1-003);国家自然科学基金(81970107,81600083);中央级公益性科研院所基本科研业务费(2018RC31002,2018PT32034)。

摘　　要：目的本文旨在通过模拟中性粒细胞在骨髓内的发育和生存环境,在体外建立一种延长中性粒细胞寿命和维持其生物学活性的方法。方法建立间充质干细胞和人外周血中性粒细胞共培养体系,分别在共培养后的0 d、1 d、2 d、3 d收集中性粒细胞细胞,采用流式细胞术等技术检测其存活率,同时通过对中性粒细胞的趋化、吞噬、活性氧(reactive oxygen species,ROS)释放等功能进行分析,来明确共培养体系对中性粒细胞体外寿命和功能的影响;采用Western blot方法分析中性粒细胞中与细胞存活和凋亡相关的基因的表达情况。结果间充质干细胞共培养可以促进中性粒细胞中Mcl-1基因的持续表达,并抑制Caspase-9、Caspase-8和Caspase-3的剪切活化,从而影响细胞内源性和外源性凋亡途径,与对照相比,显著延长了中性粒细胞的寿命(P<0.01);共培养体系培养3 d后,中性粒细胞仍能保持较好的趋化运动能力、吞噬能力和ROS释放等功能,与对照组相比,两者之间有显著性差异(P<0.01)。结论体外通过与间充质干细胞共培养,可以显著延长中性粒细胞寿命和维持其生物学功能,这为我们今后进一步优化中性粒细胞体外保存方法提供了理论和实验基础,同时也为提高粒细胞输注的有效性提供了新的思路。This study was designed to construct a method of maintaining the survival and immune function of neutrophils in vitro by mimicking the environment of neutrophils storage pool in the bone marrow.Mesenchymal stem cells(MSCs)is known as the main component of bone marrow microenvironment,and in this study,to generate a storage milieu in vitro,we developed a co-culture system of human umbilical cord MSC(HUC-MSC)and human peripheral blood-derived neutrophils.During the experiment,neutrophils were collected at 0 d,1 d,2 d,and 3 d after co-culture with HUC-MSC,and the proportion of living cells collected from different time points were detected by flow cytometry.At the same time,chemotaxis,phagocytosis,and reactive oxygen species(ROS)release assays were carried out for functional evaluation of neutrophils.Western blot method was used to analyze the expression of genes related to neutrophil survival and apoptosis.Data showed that in co-cultured group,HUC-MSC could promote the continuous expression of Mcl-1 and inhibited the activation of Caspase-9,Caspase-8 and Caspase-3 in neutrophils,and compared with the control group,the cell death of co-culture neutrophils was significantly delayed(P<0.01).Furthermore,the neutrophils cocultured for 3 days still maintained the functions of chemotaxis,phagocytosis and the release of ROS,which was significantly different from the control group(P<0.01).Taken together,HUC-MSC co-culture could significantly prolong the life span and maintain the immune function of neutrophils in vitro.This study will provide a new strategy and operational experimental basis for further optimizing neutrophils preservation methods in vitro,and will also contribute to improving the effectiveness of granulocyte transfusion in the future.

关 键 词：中性粒细胞 间充质干细胞 细胞凋亡 粒细胞输注 

分 类 号：R392.12[医药卫生—免疫学]