丁酸钠诱导人凝血因子Ⅷ的体外表达

In vitro expression of human factor Ⅷ gene induced by sodium butyrate

目的 观察丁酸钠对人凝血因子Ⅷ (hFⅧ )表达的作用 ,并初步探讨其机制。方法 采用B区缺失 (76 0～ 16 39氨基酸 )的hFⅧcDNA(BDD hFⅧcDNA)的重组质粒载体pRC/RSV BDD hFⅧ转化体外培养的小鼠NIH/ 3T3细胞 ,经丁酸钠作用后 ,分别采用一期法和ELISA法检测细胞培养上清中hFⅧ的活性 (hFⅧ∶C)和抗原含量 (hFⅧ∶Ag) ,并运用体外细胞核连缀反应技术 (Run onassay)观察丁酸钠对编码BDD hFⅧ全长、重链和轻链的cDNA转录的影响。结果 经丁酸钠诱导后 ,hFⅧ∶C和hFⅧ∶Ag较对照组提高了约 70 %。Run onassay显示丁酸钠通过增强BDD hFⅧ重链基因的转录进而增强BDD hFⅧcDNA全长的转录 ,因而提高hFⅧcDNA的表达水平。结论 丁酸钠通过增强编码hFⅧ重链cDNA的转录进而提高hFⅧ的表达水平 。

Objective To explore the effect and mechanism of sodium butyrate on expression of human clotting factor Ⅷ in vitro. Methods Mouse NIH/3T3 cell line was transfected with recombinant plasmid vector pRC/RSV-BDD-hFⅧ, which enclosed B-domain deleted (760aa～1 639aa) human factor Ⅷ cDNA (BDD-hFⅧ cDNA). Then cells were incubated in Dulbecco’s modification of Eagle’s medium (DMEM) containing sodium butyrate for 24 hours, hFⅧ∶C and hFⅧ∶Ag in the cell culture medium were measured by ELISA assay and one-stage method, respectively. In addition, the effect of sodium butyrate on transcription of cDNA encoding the whole hFⅧ, heavy and light chain of hFⅧ was also investigated by means of run-on assay. Results After stimulation of sodium butyrate, the levels of hFⅧ∶C and hFⅧ∶Ag increased 70% than those of control. Run-on assay showed that sodium butyrate enhanced the transcription of cDNA which encoded heavy chain of hFⅧ. Conclusion Sodium butyrate can improve the expression of hFⅧ through enhancing the transcription of hFⅧ heavy chain encoding cDNA. It demonstrated that sodium butyrate had potential utility in inducing the expression of hFⅧ in vitro.