利用小鼠脾细胞上清抗病毒活性建立新型抑制性ODN的体外筛选方法  

Establishment of a screening method for mouse reactive suppressive oligodeoxynucleotide in vitro

作　　者：杨光[1,2] 孙然[1] 孙陆果[2] 万敏[2] 王莉[1] 吴秀丽[2] 胡大利[1] 王丽颖[2] 于永利[1] 

机构地区：[1]吉林大学基础医学院免疫学教研室,吉林长春130021 [2]吉林大学基础医学院分子生物学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2009年第5期785-789,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30771977)

摘　　要：目的：通过检测小鼠脾细胞上清抗病毒活性,建立一种体外筛选小鼠敏感的新型抑制性寡聚脱氧核苷酸（ODN）的方法,为后续小鼠体内实验提供抑制性ODN的体外筛选方法。方法：将小鼠脾细胞分为对照组、CpG 2216刺激组,收集不同浓度（0、0.25、0.50、1.00、2.00、4.00、8.00、16.00及32.00 mg.L-1）的CpG 2216刺激上清及CpG 2216作用小鼠脾细胞不同时间（3、6、12、24、48、72及96 h）的刺激上清,通过VSV病毒保护实验检测的A578评价各组上清的抗病毒活性,确定CpG 2216的最佳作用浓度、作用时间及刺激上清稀释度;再将小鼠脾细胞分为对照组、CpG 2216刺激组、CpG 2216＋抑制性ODN（A151）组,同上确定A151的最佳作用浓度;将小鼠脾细胞分为对照组、CpG 2216刺激组、CpG 2216＋抑制性ODN（MAT0106）组,应用上述优化的实验条件如各ODN剂量、作用时间等处理细胞后,收集刺激上清,采用VSV病毒保护实验,观察各抑制性ODN的抑制作用,以初步判定此方法的可行性。结果：筛选方法确定为：浓度为5×10^6·mL^-1的小鼠脾细胞铺于96孔圆底细胞培养板,加入CpG 2216至终浓度2 mg·L^-1和/或抑制性ODN至终浓度16 mg·L^-1,孵箱中培养24 h后收集上清,1∶4稀释上清后加入已贴壁的L929细胞板内,共孵育18 h后弃上清,加入VSV病毒液继续培养48 h。弃净上清后结晶紫染色、脱色并检测A578。此种筛选方法可以区别本室自行设计的抑制性ODN之间的抑制活性。结论：成功建立了抑制性ODN抑制小鼠脾细胞抗病毒活性的筛选方法,抑制性ODN的小鼠体外筛选方法的建立,为后续抑制性ODN在小鼠体内的生物学活性观察提供了体外筛选平台。Objective To establish a method to screen a mouse reactive suppressive oligodeoxynucleotide（ODN） in vitro for future research in mice.Methods Mouse splenocytes were divided into control and CpG 2216 groups.The supernatants of splenocytes induced by CpG 2216 with different concentrations（0,0.25,0.50,1.00,2.00,4.00,8.00,16.00 and 32.00 mg·L^-1） for different incubation time（3,6,12,24,48,72 and 96 h）were collected and detected by VSV protection bioassay to confirm the optimal concentration and incubation time of CpG 2216 and the dilution of the supernatant.Then the mouse splenocytes were divided into control group,CpG 2216 group,CpG 2216 ＋ different concentrations of suppressive ODN（A151） group to find an optimal concentration of A151.At last,the mouse splenocytes were divided into control group,CpG 2216 group,CpG 2216 ＋ suppressive ODN（developed by our lab） group.The anti-viral activities of the supernatants were detected by VSV protection bioassay using above conditions to practice the screening method established.Results The screening method established was as followed： mouse splenocytes（cell density 5×10^6·mL^-1） were cultivated with 2 mg·L^-1 CpG 2216 and/or 16 mg·L^-1 suppressive ODN for 24 h.The supernatants were collected and added to L929 cells by 1∶4 dilution.After 18 h incubation,the cells were cultivated with VSV for 48 h and A578 was detected to screen suppressive ODN.Conclusion Using CpG 2216 as the stimulator and A151 as the positive suppressive ODN,the conditions of screening experiment are established successfully and could be used for further development of novel suppressive ODN in mice.

关 键 词：抑制性寡聚脱氧核苷酸 CPGODN 小鼠 近交BALBC 脾细胞 抗病毒活性 

分 类 号：R373.33[医药卫生—病原生物学]