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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(MAT01~06)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5×106.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 an in vitro screening method for screening mouse-sensitive novel inhibitory oligodeoxynucleotides (ODNs) by detecting the antiviral activity of the mouse spleen cell supernatants in vitro and to provide in vitro inhibitory ODNs for subsequent mouse in vivo experiments Screening method. Methods: The spleen cells of mice were divided into control group and CpG 2216 stimulation group. CpG 2216 stimulation supernatant (0,0.25,0.50,1.00,2.00,4.00,8.00,16.00 and 32.00 mg.L-1) And stimulated supernatant of mouse spleen cells treated with CpG2216 at different times (3, 6, 12, 24, 48, 72 and 96 h). The anti-viral activity of supernatant of each group was evaluated by A578 assayed by VSV virus protection assay CpG 2216 stimulation group, CpG 2216 + inhibitory ODN (A151) group, the same as above to determine the best A151 The spleen cells of mice were divided into control group, CpG 2216 stimulation group and CpG 2216 + inhibitory ODN (MAT01 ~ 06) group. After the cells were treated with the optimal experimental conditions such as ODN dose and action time, Stimulate the supernatant, the use of VSV virus protection experiments to observe the inhibitory effect of each inhibitory ODN in order to initially determine the feasibility of this method. Results: The screening method was established as follows: mouse splenocytes at a concentration of 5 x 106.mL-1 were plated on 96-well round-bottom cell culture plates and CpG 2216 was added to a final concentration of 2 mg.L-1 and / or inhibitory ODN The final concentration of 16 mg.L-1, incubated in an incubator for 24 h after the supernatant was collected, 1: 4 diluted supernatant was added to the adherent L929 cell plates were incubated 18 h after the supernatant was removed, the VSV virus solution Continue to cultivate 48 h. Discard the net supernatant crystal violet staining, bleaching and detection of A578. This screening method can distinguish between self-designed inhibitory ODN inhibitory activity. CONCLUSION: The screening method of inhibitory ODN to inhibit the antiviral activity of mouse spleen cells was successfully established, and the in vitro screening method of mouse suppressive ODN was established. The in vitro screening of the biological activity of subsequent inhibitory ODN in mice was provided platform.