血液储存时间对NK细胞功能影响

Effects of blood storage time on NK cell functions

目的:观察储存血中自然杀伤细胞(NK)细胞在不同储存时间功能变化。方法:留取10份健康自愿献血者外周静脉血各20ml,ACD-B抗凝,4℃保存。分别于储存第0、1、2、3、4、6天各取血2ml,用淋巴细胞分离液分离单个核细胞(PBMC);用博纳无血清选择性培养液培养NK细胞;乳酸脱氢酶释放法检测不同储存时间培养的NK细胞对K562细胞杀伤活性;流式细胞术测定不同储存时间培养的NK细胞穿孔素、颗粒酶B和CD107a的表达率。结果:我们在预试验中对储存血NK细胞进行长达30d的对比观察,发现血液储存第6天之后所有的NK细胞已经不能被激活。因此本研究仅观察储存≤6d的NK细胞功能变化。储存2d后的NK细胞激活率和功能有明显下降趋势,随着储存时间延长,NK细胞激活的百分率逐渐减低,至第6天时细胞激活率只达第0天的11.2%(P<0.01)。储存6d后诱导培养的NK细胞杀伤活性(28.73%)明显低于诱导前(杀伤活性44.45%)(P<0.05);血液储存时间对NK细胞穿孔素、颗粒酶B和CD107a的表达有明显影响,储存前穿孔素、颗粒酶B和CD107a分别为:(33.94±2.92)%、(77.11±6.87)%和(80.97±7.76)%,显著高于储存6d后[分别为:(24.53±2.83)%、(63.93±6.56)%和(73.84±7.21)%](P<0.05)。结论:储存血中能保持NK细胞功能和活性最长时间为6d;在≤6d时间内NK细胞功能随着存放时间延长而变弱。

Objective：To investigate the changes of NK cell functions at different storage time.Method：20 ml ACD-B anticoagulant peripheral blood from 10 human healthy volunteers were stored at 4℃.Peripheral blood mononuclear cells（PBMC）were separated by lymphocyte separation medium at 0,1 st,2 nd,3 rd,4 th and 6 th day respectively and cultured with Bona serum-free NK medium.Cytotoxicity to K562 cell was tested by LDH.Expressions of Perforin,Granzyme B and CD107 awere detected by flow cytometry（FCM）.Result：In our preliminary experiments,we compared NK cells in stored blood for 30 days,and found that all NK cells could not be activated after 6 days of the blood storage.Therefore,we would only observe the functional changes of NK cells with storage time less than or equal to 6 days in this study.NK cell viability and function were significantly decreased after2 days.The percentage of activated NK cell decreased gradually with the prolongation of storage time.The activation of NK cell at 6 th day only reached 11.2%.The cytotoxicity of NK cells significantly decreased to 28.73%,lower than that at 0 day,there was significant difference（P〈0.01）.The expressions of perforin,Granzyme B and CD107 aalso decreased to（24.53±2.83）%,（63.93±6.56）% and（73.84±7.21）% at 6 th day,lower than thoseat 0 day[（33.94±2.92）%,（77.11±6.87）% and（80.97±7.76）%].Conclusion：The function and activity of NK cells could maintain for a maximum of 6 days in stored blood.The function of NK cells decreased with prolonged storage time in less than 6 days.