豚鼠哮喘模型和哮喘患者气管上皮细胞内皮素-1和内皮素转化酶mRNA表达.DOCVIP

临床医学论文-豚鼠哮喘模型和哮喘患者气管上皮细胞内皮素-1和内皮素转化酶mRNA表达 ??????????????????作者：何翔?梁永杰?汪蜀?郭忠良 【关键词】? 内皮素转换酶 【摘要】 目的 通过逆转录-DNA聚合酶链反应（RT-PCR）技术检测豚鼠哮喘模型气管上皮内皮素-1（ET-1）mRˉNA表达，检测哮喘患者气管上皮内皮素-1（ET-1）和内皮素转换酶（ECE）mRNA表达。方法 （1）动物实验部分:Hartley株豚鼠用卵蛋白腹腔注射致敏，雾化吸入激发，提取气管上皮细胞RNA，逆转录将RNA逆转为cDNA，PCR扩增DNA。（2）临床实验部分:设对照组和哮喘组。一步法提取总RNA，逆转录合成cDNA，PCR扩增β-actin片段、ET-1片段和ECE片段。PCR产物的鉴定和半定量。计算ET-1/β-actin值和ECE/β-actin值。结果 （1）动物模型检测:在电泳缓冲液内，紫外灯下可见ET-1为333bp左右条带，与标记DNA条带相对应。（2）临床实验:哮喘组ET-1mRNA表达量明显高于对照组（P0.05）。哮喘组ECEmRNA表达量与对照组比较差异无显著性（P0.05）。结论 RT-PCR技术检测出卵蛋白致敏激发的豚鼠气管上皮细胞ET-1mRNA表达。哮喘患者支气管粘膜ET-1mRNA表达明显增高，而ECEmRNA表达量与对照组比较差异无显著性。 关键词 RT-聚合酶链反应 内皮素-1 内皮素转换酶 【Abstract】 Objective To detect the mRNA expressions of endothelin-1in tracheal epithelium cell from guinea pig asthma model by reverse transcriptional polymerase chain reaction，to detect the mRNA expressions of enˉdotheline-1and endothelin converting enzyme in tracheal epithelium cell from asthma patient.Methods （1）Animal experiment department:sensitize the guinea pig by inject ovi-protein into the abdominal cavity，excitation by aspirate the ovi-protein solutions，extract RNA from tracheal epithelium cell，reverse transcribe the RNA to cDNA，amplify DNA by polymerase chain reaction.（2）Clinical department:laying the asthema group and the control group，extract RNA from tracheal epithelium cell，reverse transcribe the RNA to cDNA，amplifyβ-actin、ET-1and ECE fragment by polymerase chain reaction.evaluate and semi-quantify the PCR product，calculate ET-1/β-actin and ECE/β-actin.Results （1）Animal experiment:we can see the ET-1is about a333bp strap in the electrophoresis solution under the infrared lamp，correspond with the marking strap.（2）Clinical department:The mRNA expressions of enˉdothelin-1of the asthema group is great high than the control group（P0.05），the difference of mRNA expressions of ECE is not significant at two groups（P0.05）.Conclusion It can detect the mRNA expressions of endothelin-l in tracheal epithelium cell from guinea pig asthma model by revers