利用CAL-1-L929细胞建立一种抑制性寡聚脱氧核苷酸筛选体系

Establishment of an inhibitory oligodeoxynucleotides screening system using CAL - 1 - L929 cells

目的利用树突状细胞CAL-1的肿瘤坏死因子-α（TNF—α）检测平台，以进一步应用于抑制性寡聚脱氧核苷酸的筛选。方法以含有未甲基化CpG基序的寡聚脱氧核苷酸CpG（CpGODN）0800（3μg／ml）作为刺激剂，将其作用CAL-1细胞（2×10^5个细胞／孔），48h后收集上清，作用于L929细胞（2×10^4个细胞／孔））。培养18h，加入水疱性口炎病毒（VSV），培养72h，检测570nm处的吸光度值。结果筛选条件确定为CpGODN0800终质量浓度为3μg／ml，CAL-1上清液1：5倍稀释。L929细胞数在2×10^4个细胞／孔），反应时间为24h。应用此条件成功筛选小自行设计的抑制性ODN—SAT05f。结论建立实验体系，成功检测了自行设计的抑制ODN和CpGODN诱导的CAL-1细胞的TNF—α的生物学活性。

Objective To establish a rapid screening method for the development of novel oligode- oxynucleotides （ODN） that antagonized for the development of tumor necrosis factor - α （TNF - α）. Methods The CAL - 1 ceils were stimulated by unmethy｜ated CpG motif containing oligonucleotides （ CpG ODN） 0800 （ 3 dg/ml）. After 48 h, the culture supernatant was collected and TNF -αsecretion in the culture supernatant was assessed by an in vitro cytotoxicity assay using L929 ceils （2×10^4/well）. Ater 24 h, vesicular stomatitis virus （VSV） was added into the wells, and after 72 h, the A values were detected. The experimental conditions were also well optimized. Results Screening conditions were identified as final concentration of CpG ODN 0800 of 3 μg/m], and dilution of CAL - 1 supernatant of 1/5. The A values of L929 cell was 2×10^4/well, and the experiment time on L929 was 24 h. SAT05f was successful screened by application of the methods above. Conclusion TNF - α can be suppressed by the ODNs with special sequence. This experimental method was successfully established for development of novel Inhibitory ODNs for targeting TNF -α.