多个顺式作用元件调节血管紧张素原基因表达

血管紧张素原是最强的血管活性物质——血管紧张素Ⅱ的唯一前体，在不同的生理和病理条件下，其水平各异．为了研究血管紧张素原基因表达的调控，将人血管紧张素原基因５′端侧翼序列１．２ｋｂ同氯霉素乙酰转移酶（ＣＡＴ）基因编码序列连接，构成表达载体，并且在此基础上构建５′端系列缺失的突变表达载体，用这些表达载体转染ＨｅｐＧ２和ＣＯＳ－７，确定了正负调控元件；同时应用ＤＮＡ－蛋白质凝胶泳动检测技术，发现核蛋白质与该顺式元件的结合，从而证明多个顺式作用元件调节血管紧张素原基因的表达．

Multipe cis Acting Elements Regulate Angiotensinogen Gene Expression

Angiotensinogen is the precursor of the most potent vasoactive substances——angiotensin Ⅱ.Angiotensinogen levels in plasma are modulated under different physiological and pathological conditions.In order to understand the regulation of the human angiotensinogen gene expression,A 1220 bp fragment of the human angiotensinogen gene promoter that included 44 bp of the first exon was isolated by the polymerase chain reaction (PCR) using human genomic DNA.The fragment was directly cloned in the pGEM T vector and subsequently subcloned through SalⅠand SphⅠ restriction site in front of the chloramphenicol acetyl transferase (CAT) gene of pSVCAT Basic vector.5′ sequential deletion mutants were also obtained from the human angiotensinogen promoter attached to the CAT gene.An array of expression vectors were introduced into HepG 2 and COS 7 cells by calcium phosphate precipitation transfection technique.The CAT activity was assayed using 14 C chloramphenicol as substrate.Results of transient transfection suggested two negative regulatory fragments at -850～-580 and -420～-220 and two positive regulatory fragments at -580～-420 and -220～+1.Two synthetic oligonucleotides,homology with IL 6 responsive element and estrogen responsive element in positive regulatory fragments,were further analyzed by electrophoretic mobility shift assay and showed DNA protein binding bands using nuclear extract from COS 7 and HepG 2 cells.These observations indicate that expression of the human angiotensinogen gene is coordinately regulated by multiple cis acting elements that interact with DNA binding proteins.