大鼠肺微血管内皮细胞磷酸二酯酶mRNA表达与活性检测  

Detection on mRNA exepression and activity of phosphodiesterase in rat pulmonary microvascular endothelial cells

作　　者：侯显涛[1] 陈武[1] 张涛[1] 朱丽娜[1] 姜代勋[1] 陈益山[1] 李佳[1] 穆祥[1] 

机构地区：[1]北京农学院兽医学系中医药专业北京市兽医学重点实验室北京农学院动物科学技术系

出　　处：《解剖学报》2009年第4期590-593,共4页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(30671542);北京市教育委员会科技发展计划重点项目;北京市自然科学基金重点项目(B类)(KZ200810020008);北京市教委“学术创新团队”(5090245)

摘　　要：目的检测大鼠肺微血管内皮细胞（PMVECs）内磷酸二酯酶（PDE）同功酶mRNA表达及基础酶活性,明确PMVECs存在的PDE亚型及体外活性。方法采用植块法分离培养PMVECs,RT-PCR法测定PDE mRNA表达,以高效液相色谱（PHLC）检测环核苷酸在酶反应前后的含量变化,计算PDE活性。结果检测结果显示,PMVECs中含有PDE1A、1C、2A、3A、3B、4A、4B、4C、4D、5A、7A、7B、8A、8B、9A、10A、11A共17种PDE mRNA,而PDE1BmRNA未见表达,酶量在5~20μl范围内与PDE活性存在良好的线性关系。结论PMVECs中存在17种PDE基因表达,并有较高的基础酶活性。Objective To study the main subtypes messenger ribonucleic acid（mRNA） and the basal enzyme activity of phosphodiesterase （PDE） in rat pulmonary microvascular endothelial cells （PMVECs） through the examination mRNA expression and activity of PDE in vitro. The data were offered to reveal the relationship between PDE distributions, activity change and to accumulate data for the possibility of drug regulation of its functional alteration. Methods The cells were cultured with tissue- sticking method; the gene expression of PDEs was detected by reverse transcript polymerase chain reaction （RT-PCR）, and the activity of PDEs was calculated by cyclic nucleotides content change examined with high performance liquid chromatogram （HPLC） before and after the PDE reaction（ n = 3）. Results The PMVECs identified by cell immunofluorescence with polyclonal antibody of CD31 were dissociated and cultured, mRNAs of PDE1A, 1C, 2A,3A, 3B, 4A, 4D, 5A, 7A, 7B, 8A, 8B, 9A, 10A,11A were expressed in PMVECs, but there was no mRNA of PDE1B expressed in PMVECs. cAMP/cGMP-PDE in the extent of 5-20μl had a good linear correlation with its activity. Conclusion There are 17 kinds of PDE gene expression existing in PMVECs which contain of the basic enzyme with a higher activity.

关 键 词：肺微血管内皮细胞 磷酸二酯酶 反转录-聚合酶链反应 高效液相色谱 大鼠 

分 类 号：Q257[生物学—细胞生物学]