CAJ | 학술논문

目的研究内皮素转换酶-1（ECE-1）反义RNA对人气道上皮细胞ECE-1表达的抑制作用。方法构建ECE-1反义RNA表达载体，用脂质体(FuGENG 6)转染人气道上皮细胞株（16HBE），G418筛选得到稳定表达反义ECE-1 RNA的细胞株（anti-16HBE）。RT-PCR检测转染细胞ECE-1 mRNA的表达，Western bloting检测细胞ECE蛋白表达情况。结果①经酶切及DNA测序鉴定证明，0.7kb的ECE-1 DNA片段反向连接到带有萤光蛋白基因为报告基因的pEGPC1表达载体；荧光倒置显微镜显示大量细胞分泌荧光蛋白并能反复传代稳定表达；② anti-16HBE细胞ECE mRNA基因表达(ECE/actin β 0.012 5±0.058)比16HBE细胞(ECE/actin β 0.134 7±0.067)明显低(P＜0.05)；而Wester bloting亦显示anti-16HBE细胞ECE表达量比16HBE细胞明显低。结论ECE反义RNA表达载体可抑制人气道上皮细胞ECE-1表达与分泌。
목적연구내피소전환매-1（ECE-1）반의RNA대인기도상피세포ECE-1표체적억제작용。방법구건ECE-1반의RNA표체재체，용지질체(FuGENG 6)전염인기도상피세포주（16HBE），G418사선득도은정표체반의ECE-1 RNA적세포주（anti-16HBE）。RT-PCR검측전염세포ECE-1 mRNA적표체，Western bloting검측세포ECE단백표체정황。결과①경매절급DNA측서감정증명，0.7kb적ECE-1 DNA편단반향련접도대유형광단백기인위보고기인적pEGPC1표체재체；형광도치현미경현시대량세포분비형광단백병능반복전대은정표체；② anti-16HBE세포ECE mRNA기인표체(ECE/actin β 0.012 5±0.058)비16HBE세포(ECE/actin β 0.134 7±0.067)명현저(P＜0.05)；이Wester bloting역현시anti-16HBE세포ECE표체량비16HBE세포명현저。결론ECE반의RNA표체재체가억제인기도상피세포ECE-1표체여분비。
Objective　To explore the effects of endothelin converting enzyme-1 (ECE-1) antisense RNA on ECE-1 expression in human bronchial epithelial cell line(16HBE). Methods ECE-1 antisense RNA expression vector with green fluorescent protein as a reporter was constructed and transfected into 16HBE with Fugene 6TM.The ECE-1 expression was measured by RT-PCT and Western bloting. Results Agrose gel electrophoresie showed that 0.717kp ECE DNA fragment was inserted into pEGFPC1 plasmid in reverse orientation. After G418 selection, anti-16HBE cell line stably expressing ECE-1 antisense RNA was developed. Both ECE-1 mRNA(ECE/actin β 0.012 5±0.058) and protein expression in anti-16HBE cells were significantly lower than those in normal cells(ECE/actin β 0.134 7±0.067) detected by RT-PCR and Western bloting respectively(P＜0.05). Conclusion　ECE-1 expression in 16HBE cells can be inhibited by ECE-1 antisense RNA in vitro.