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目的 :评价转染血管紧张素 (Ang )受体 (AT1 )反义核苷酸 (AT1 A)对心肌成纤维细胞 (Fbs) Ang 受体亚型 m RNA、蛋白激酶 C(PKC)、丝裂素活化蛋白激酶 P38(P38MAPK)蛋白表达 ,及蛋白质合成的作用。方法 :RT-PCR克隆 AT1 c DNA片段 (4 76 bp) ,将克隆的 AT1 c DNA片段反向插入 Pc DNA 3.1(5 .4kb) ,构建一完整的含AT1 A的质粒 (PAT1 A)。转染培养的大鼠 Fbs,RT- PCR检测转染的 Fbs AT1 m RNA表达。 Ang 10 - 7m ol/ L 刺激 2 4h后 ,比较转染及非转染的 Fas Ang 受体 AT1 及 AT2 m RNA表达 (RT- PCR)、P38MAPK和 PKC蛋白表达(western blot)、蛋白质合成 (3H- L eu掺入 )。结果 :成功构建 PAT1 A。 RT- PCR显示转染 Fbs AT1 m RNA的水平降低 ,与对照 Fbs相比差异显著 (P<0 .0 1)。 Ang 10 - 7m ol/ L 刺激 2 4h后 ,与非转染 Fbs相比 ,转染 Fbs AT1m RNA明显减少 ,AT2 m RNA明显增加 (P<0 .0 1) ;但两组间 PKC和 P38MAPK蛋白表达、3H- L eu及 3H- Td R掺入量均无显著性差异 (P>0 .0 5 )。结论 :经 AT1 A封闭后 ,能显著地抑制 Fbs AT1 m RNA表达 ,同时上调 AT1 m RNA。单纯封闭 AT1 m RNA并不能有效阻断 Ang 介导的 Fbs蛋白质合成及 Fbs生长相关的信号转导。
Objective: To evaluate the effect of AT1 A transfection with Angiotensin receptor (AT1) on the expression of angiotensin Ⅱ receptor subunit m RNA, protein kinase C (PKC), mitosis Activation of protein kinase P38 (P38MAPK) protein expression, and protein synthesis. METHODS: The AT1 c DNA fragment (4 76 bp) was cloned by RT-PCR. The AT1 c DNA fragment was reverse inserted into PcDNA 3.1 (5.4 kb) to construct a complete AT1 A-containing plasmid (PAT1 A). Cultured rat Fbs were transfected and transfected Fbs AT1 m RNA expression was detected by RT-PCR. The expressions of AT1 and AT2 mRNA in transfected and non-transfected Fas Ang (RT-PCR), P38MAPK and PKC protein were compared with that of the protein (3H - L eu incorporation). Results: PAT1 A was successfully constructed. RT-PCR showed that the level of AT1 m RNA transfected with Fbs was decreased, which was significantly different from the control Fbs (P <0.01). Compared with untransfected Fbs, the AT1mRNA of transfected Fbs was significantly decreased and the AT2mRNA was significantly increased (P <0.01) after stimulation with Ang 10 - 7mol / L for 24 hours. However, the protein expressions of PKC and P38MAPK Expression of 3H-L eu and 3H-Td R had no significant difference (P> 0.05). Conclusion: Blocking AT1 A can significantly inhibit the expression of Fbs AT1 m RNA and up-regulate AT1 m RNA. Blocking AT1 m RNA alone did not effectively block Ang-mediated Fbs protein synthesis and Fbs-related signal transduction.