反义寡核苷酸对气道平滑肌细胞内皮素转换酶及内皮素1表达的影响

Effect of antisense oligonucleotides on endothelin converting enzyme expression and endothlin1 release from airway smooth muscle cells

目的探讨肿瘤坏死因子α（ＴＮＦα）刺激人气道平滑肌细胞（ＡＳＭＣ）内皮素１（ＥＴ１）释放的机制及体外反义内皮素转换酶寡聚核苷酸（ＥＣＥｏＤＮＳ）是否可拮抗ＴＮＦα诱导人ＡＳＭＣ的ＥＣＥ表达及ＥＴ１分泌。方法离体培养ＡＳＭＣ并导入由阳离子脂质体：Ｌｉｐｏｆｅｃｔｉｎ携载的反义ＥＣＥｏＤＮＳ，观察其对前炎症因子ＴＮＦα诱导ＥＣＥ基因表达及ＥＴ１释放的影响，采用酶联免疫吸附（ＥＬＩＳＡ）法测定培养液ＥＴ１浓度；用逆转录多聚酶链式反应（ＲＴＰＣＲ）方法检测ＥＣＥｍＲＮＡ的表达量。结果ＴＮＦα可刺激ＡＳＭＣＥＴ１释放及ＥＣＥｍＲＮＡ表达。ＥＴ１由（７．３±０．８）ｎｇ／Ｌ升至（１０．１±０．３）ｎｇ／Ｌ，（ｔ＝５．５７６，Ｐ＜０．０１）；ＥＣＥｍＲＮＡ的表达由（０．１３２±０．０３２）升至（０．２２８±０．０１５，ｔ＝４．７２４，Ｐ＜０．０１）；导入反义ＥＣＥｏＤＮＳ后，ＥＣＥｍＲＮＡ的表达及ＥＴ１的释放均受到抑制，抑制率分别为（３５±１２）％及（２９±５）％。结论ＴＮＦα刺激人ＡＳＭＣＥＣＥ基因表达增加可能是其刺激细胞ＥＴ１释放的主要原因；反义ＥＣＥｏＤＮＳ能拮抗ＴＮＦα诱?

Objective To explore the mechanism of TNF induced ET1 release and evaluate the effects of endothelin converting enzyme (ECE) antisense oligonucleotides (oDNS) on TNF induced ET1 release from airway smooth muscle cells (ASMC). Methods ASMC transfected with the antisense oligonucleodides of ECE vectored with lipofectin were established. TNF induced ECEmRNA expression and ET1 release from above cultured cells were determined by RTPCR and ELISA. Results ET1 levels (10.10.3) ng/L and ECE expression (ECE/actin: 0.2280.015) in cultured ASMC incubated with 1 000 U/ml TNF were significantly higher than those in controls without TNF incubation (7.30.8) ng/L, (0.1320.032, all P<0.01). However, after transfecting antisense ECEoDNS in those cells, ET1 levels and ECE expressions were significantly lower than those in control cells without antisense transfection . The inhibition rates were (295)% and (3512)% respectively. Conclusions Abnormal expression of ECE mRNA may be a keypoint responsible for TNF induced ET1 release in human ASMC; TNF induced ET1 release and ECE mRNA expression from human ASMC are inhibited by antisense oligonucleotide of ECE.