小鼠β-防御素-3基因表达调控的初步研究

Primary study of the expression of the mouse β-defensin-3 gene and its regulatory factor

目的 研究小鼠 β 防御素 3(mBD 3)基因的表达及其调控因子。方法 通过腹腔注射内毒素 (LPS)建立小鼠急性时相反应 ,经Northern blot检测mBD3mRNA表达的组织特异性并分析在肝脏中表达的剂 效和时 效关系 ,EMSA和South westernblot分析细胞核蛋白中的转录因子。结果 内毒素注射后仅在肝脏组织中检测到mBD3mRNA并存在最小诱导剂量 ,本实验确定的最小诱导剂量为 1 .5 μg/ g(剂量 /体重比 )。以 1 .5 μg/g为诱导剂量 ,在注射后 6h才出现mBD3mRNA的表达 ,8h、1 0h达峰值 ,1 2h后表达水平下降。肝脏细胞核蛋白中有与mBD3基因调节区特异DNA片段结合的转录因子 ,分子量在 4 3.0～ 6 6 .2kD之间。结论 小鼠急性时相反应时mBD3基因在肝脏中表达上调 ,具有剂量和时间依赖性 ;NF κB参与β 防御素 3基因在肝脏中表达的转录调节。

Objective To study the expression of the mouse β defensin 3 gene and its regulatory factor.Methods Mouse systemic acute phase responsive model was established by intraperitoneal injection of LPS.The expression of the mouse β defensin3 mRNA in different tissues and the time effect and dose effect dependent expression caused by LPS were detected by Northern blot.The transcription factors binding to the 5′ flanking sequence of mBD3 gene were analyzed by electrophoretic mobility shift assay (EMSA) and South western blot.Results The expression of mBD3 gene was only detected in mouse liver after LPS injection and not in heart,lung,spleen and kidney by Northern blot.The minimum dose of LPS inducing mBD3 expression in liver was 1.5μg/g.It was found that mBD3 mRNA was not expressed until 6 hours later and the mRNA level reached maximum at 8 or 10 hours,then decreased 12 hours later after LPS injection at the dose of 1.5μ g/g.The results of EMSA showed the obvious retardant bands,which suggest that some proteins in nuclei can bind to the specific DNA sequence of mBD3 gene.The molecular mass of this DNA binding protein was assessed between 43.0 66.2kD by South Western blot.Conclusion The expression of the mouseβ defensin3 gene were up regulated in liver in a time result and dose result dependent fasion and NF κB involvement in the transcriptional regulation of the mouseβ defensin 3 gene during the systemic acute phase response.