摘要
背景:从深低温冻存脐血中获得大量高纯度无滋养层细胞、无动物源成分的NK细胞,以便更好地开展NK细胞杀伤肿瘤的实验研究,此次研究通过使用NK扩增培养试剂盒和血清替代物,以获得安全、高效的体外扩增培养NK细胞的方法。目的:建立一种从冷冻脐血中采用纯细胞因子扩增NK细胞的技术体系并证明其体外杀瘤能力。方法:解冻液氮存储的冻存脐血,优化复苏体系,采用Ficoll密度梯度离心的方法收集脐血单个核细胞,接种于预先铺板的T175培养瓶中,采用细胞因子试剂盒+血清替代物法培养21 d后收集细胞并计数,应用流式细胞术检测细胞中CD3^(-)CD56^(+)的比例,并将所得数据与K562滋养层细胞+胎牛血清的培养方法进行对比分析。同时以髓系白血病细胞K562为靶细胞,采用CCK-8试剂盒检测不同效靶比NK细胞杀伤K562细胞的作用。最后进行了12 h内的模拟储存运输实验,采用流式检测CD3-CD56^(+)的比例变化,并使用Annexin V/PI双染法检测细胞凋亡率。结果与结论:(1)采用K562滋养层细胞+胎牛血清的培养方法扩增培养14 d,细胞扩增(51.47±4.28)倍;采用细胞因子试剂盒+血清替代物法扩增培养21 d,细胞数量5×10^(9)以上,细胞扩增(328.32±116.36)倍,细胞扩增倍数明显升高,2种方法获得的CD3^(-)CD56^(+)纯度比例均在80%以上,未见明显差异;(2)纯细胞因子方法培养后的NK细胞对K562细胞的杀伤效果(效靶比=10∶1)达80%以上;(3)在12 h的模拟储运实验中,CD3^(-)CD56^(+)的比例变化不明显,直至第12小时,NK细胞凋亡率<7%;(4)结果表明:优化复苏体系和纯细胞因子扩增方法可以高效培养出高纯度的NK细胞,同时具有有效杀伤K562肿瘤细胞的能力,且经过12 h储运后仍然具有良好的细胞纯度和较低的凋亡率。
BACKGROUND:To obtain a large number of high purity trop hoblast-free,animal-derived natural killer(NK) cells from deep cryopreserved cord blood for better experimental studies on tumor killing by NK cells,a safe and efficient method for expanding and culturing NK cells in vitro was obtained by using NK expansion culture kits and serum substitutes.OBJECTIVE:To establish a technical system for the expansion of NK cells from frozen co rd blood using pure cyto kines,and demonstrate their tumor-killing ability in vitro.METHODS:The frozen cord blood sto red in liquid nitrogen was thawed to optimize the recovery system.The individual nucleated cells were collected by Ficoll density gradient centrifugation and inoculated in pre-plated T175 culture flasks.Cells were cultured using the cytokine+serum replacement method for 21 days and counted.The ratio of CD3^(-)CD56^(+)in cells was detected by flow cytometry.The data obtained were compared with the data of K562 trophoblast cells+fetal bovine serum culture method.M eanwhile,myeloid leukemia cells K562 were used as target cells,and CCK-8 kit was used to perform different potent target ratios of NK cells to kill K562 cells.Later,simulated storage transport expe riments were performed for 12 hours.The changes in the ratio of CD3^(-)CD56^(+)were detected by flow cytometry and the apoptosis ratio was determined using Annexin V/PI double-staining method.RESULTS AND CONCLUSION:(1) The cells were expanded and cultured for 14 days using K562 trophoblast cells+fetal bovine serum,and the cells expanded(51.47±4.28) fold.After 21 days of expansion culture using cytokine+serum substitute method,the number of cells was more than 5×10^(9);the cells expanded(328.32±116.36) times;the cell expansion ploidy was significantly higher;the CD3^(-)CD56^(+)purity ratio obtained by both methods was above 80%;no significant diffe rence was seen.(2) The killing effect of NK cells cultured by pure cytokine method on K562 cells(effective target ratio=10:1) was more than 80%.(3) The ratio of CD3^(-)CD56^(+)did not change significantly during the 12-hour simulated storage expe riment until the 12 hours,when the apoptosis rate of NK cells was <7%.(4) The res ults showed that the optimized recovery system and pure factor expansion method can efficiently culture high-purity NK cells with the ability to effectively kill K562 tumor cells,and still have good cell purity and low apoptosis rate after 12-hour storage and transportation.
作者
刁小娟
石超群
李栋
王帅
肖春
刘国军
曲廷瑜
杨卫娟
贾晓鹏
Diao Xiaojuan;Shi Chaoqun;Li Dong;Wang Shuai;Xiao Chun;Liu Guojun;Qu Tingyu;Yang Weijuan;Jia Xiaopeng(Shandong Province Qilu Stem Cell Engineering Co.,Ltd.,Jinan 250100,Shandong Province,China;Cord Blood Bank of Shandong Province,Jinan 250100,Shandong Province,China;Cryomedicine Lab,Qilu Hospital,Shandong University,Jinan 250012,Shandong Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2023年第10期1572-1577,共6页
Chinese Journal of Tissue Engineering Research
基金
山东省重点研发计划(重大科技创新工程)项目(2019JZZY011012),项目负责人:曲廷瑜。
关键词
冷冻脐血
NK细胞
因子
扩增
细胞毒性
凋亡率
配型
临床应用
cryopreserved cord blood
NK cells
cytokine
expansion
cytotoxicity
apoptosis rate
matching
clinical application
