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目的采用分子生物学技术从组织和细胞水平上观察阻断15-LO/15-HETE后,缺氧对KV1.5表达的影响。方法通过酶法分离、培养Wistar大鼠肺动脉血管平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)和大鼠肺动脉,应用Western blot和RT-PCR方法分别从蛋白质水平和mRNA水平上观察在15-LO阻断剂CDC和NDGA作用下,缺氧对KV1.5表达的影响。结果从组织和细胞水平上,用CDC和NDGA阻断15-LO即阻断了内源性15-HETE的产生,KV1.5的表达量在蛋白质水平和mRNA水平与未阻断组比较都增加。在阻断了内源性15-HETE的产生以后,加入外源性15-HETE,KV1.5的表达量减低。说明不仅内源性15-HETE参与诱导缺氧对KV1.5表达的影响,外源性15-HETE也同样能影响缺氧条件下KV1.5的表达量。但在阻断了内源性15-HETE的产生以后,加入外源性15-HETE,KV1.5的表达量减低。说明不仅内源性15-HETE参与诱导缺氧对KV1.5表达的影响,外源性15-HETE也同样能影响缺氧条件下KV1.5的表达量。结论上述结果表明,从大鼠肺动脉组织和细胞水平上,内源性15-HETE介导了缺氧对KV1.5表达的抑制作用。
Objective To observe the effect of hypoxia on the expression of KV1.5 after blocking 15-LO / 15-HETE by molecular biology techniques at the tissue and cellular level. Methods Pulmonary artery smooth muscle cells (PASMCs) and pulmonary arteries of Wistar rats were isolated and cultured by enzymatic method. Western blot and RT-PCR were used to observe the effect of 15-LO on protein level and mRNA level The effects of hypoxia on the expression of KV1.5 under the action of breaking agents CDC and NDGA. Results Blockade of endogenous 15-HETE production by blocking 15-LO with CDC and NDGA both at the tissue and cellular level increased KV1.5 expression at both protein and mRNA levels compared with the un-blocked group . After blocking the production of endogenous 15-HETE, exogenous 15-HETE was added to reduce the expression level of KV1.5. It is indicated that not only the endogenous 15-HETE is involved in the induction of hypoxia on the expression of KV1.5, exogenous 15-HETE can also affect the expression of KV1.5 in hypoxia. However, after blocking the production of endogenous 15-HETE, the exogenous 15-HETE was added and the expression of KV1.5 was decreased. It is indicated that not only the endogenous 15-HETE is involved in the induction of hypoxia on the expression of KV1.5, exogenous 15-HETE can also affect the expression of KV1.5 in hypoxia. Conclusions The above results indicate that endogenous 15-HETE mediates the inhibitory effect of hypoxia on the expression of KV1.5 in rat pulmonary artery tissue and cells.