基因转染β肾上腺素能受体激酶抑制剂对支气管哮喘小鼠β_2肾上腺素能受体和环腺苷酸的影响

Gene transfer of a β-adrenergic receptor kinase inhibitor up-regulates the level of β_2-adrenergic receptor and cyclic adenosine monophosphate in the asthmatic murine lung

目的 研究基因转染β肾上腺素能受体激酶抑制剂 (βARKct)对应用 β2 肾上腺素能受体 (β2 AR)激动剂后支气管哮喘 (简称哮喘 )小鼠肺部 β2 AR和环腺苷酸 (cAMP)的作用 ,并探讨不同基因转染途径对两者的影响。方法 (1)以 10 %卵清蛋白致敏并激发BALB/c小鼠建立哮喘模型 ,并设正常小鼠为正常对照组 (A组 )。将哮喘小鼠分为 7组 ,每组 5只 ,分别给予生理盐水 (哮喘对照组 ,B组 )肌肉注射、沙丁胺醇肌肉注射 (C、D、E、F、G组 ) ,以建立长期应用 β2 AR激动剂的哮喘动物模型。(2 )构建带有 βARKct基因表达质粒 ,通过小鼠尾静脉注射和气管内滴入的转染途径进行基因转染。D组给予静脉注射转染 βARKct、E组给予静脉注射转染空载质粒、F组给予气管滴注转染 βARKct、G组给予气管滴注转染空载质粒。(3)用Western blot检测基因的表达。放射免疫法检测小鼠肺部的 β2 AR和cAMP的水平。结果 (1)基因转染 βARKct在小鼠肺部有表达 ,且F组的基因表达量高于D组。(2 )A组 β2 AR和cAMP分别为 (10 9± 11)fmol/mg、(3 5 0± 0 5 0 )pmol/L ;B组 β2 AR和cAMP分别为 (81± 3)fmol/mg、(1 5 0± 0 2 0 )pmol/L ,与A组比较差异有显著性 (P均 <0 0 5 ) ;C组 β2 AR和cAMP分别为(6 3± 3)fmol/mg、(0 90± 0

Objective To investigate the effects of gene transfer of a β-adrenergic receptor(β 2AR) kinase inhibitor (βARKct) on pulmonary β 2-adrenergic receptor and cyclic adenosine monophosphate(cAMP) following β 2AR agonist treatment in asthmaticmice, and to analyze the relationship between the routes of gene delivery and the changes of β 2AR and cAMP. Methods (1)Thirty-five BALB/c mice were sensitized and challenged by 10% and 5% ovalbumin respectively to establish the asthmatic model and then divided into 7 groups (5 each). Sterile saline was injected intramuscularly in the asthmatic control group (group B), while β 2AR agonist (salbutamol) was injected intramuscularly in other 5 asthmatic groups (group C, D, E, F, and G) so as to establish the asthmatic model with prolonged application of β 2AR agonist. In addition, 5 healthy mice served as the normal control group(group A). (2) The plasmid with the expression of βARKct was constructed and gene transfer was performed through intravenous injection or intratracheal instillation in asthmatic mice. Intravenous injection with plasmid-βARKct was performed in group D, but with plasmid-pcDNA 3.1 in group E. Intratracheal instillation with plasmid-βARKct was performed in group F, but with plasmid-pcDNA 3.1 in group G. (3)The gene expression was measured with Western blot analysis. Radioimmunoassay was used to evaluate the changes of pulmonary β 2AR and cAMP. Results (1)The expression of transferred βARKct gene was detectable in the lungs. The level of βARKct expression was higher in the lungs of mice receiving intratracheal plasmid (group F) than those receiving intravenous plasmid (group D). (2)The levels of β 2AR and cAMP in group A were (109±11) fmol/mg and (3.50±0.50)pmol/L respectively; The levels of β 2AR and cAMP in group B were (81±3)fmol/mg and (1.50±0.20)pmol/L respectively; the differences between the two groups were significant (all P<0.05). The levels of β 2AR and cAMP in group C were (63±3)fmol/mg and (0.90±0.10)pmol/L respectively, and the differences were significant, as compared with those in group A (all P<0.01). (3)The levels of β 2AR and cAMP in group D were (86±11) fmol/mg and (2.01±0.10)pmol/L respectively after using plasmid-βARKct to the asthmatic mice treated with β 2AR agonist, and the differences were significant, as compared with those in group C (all P<0.05). The levels of β 2AR and cAMP in group F were (107±13)fmol/mg and (3.30±0.20)pmol/L respectively, and the differences were significant, as compared with those in group C (all P<0.05). The extent of up-regulation of β 2AR and cAMP was higher in group F than that in group D (all P<0.05). The levels of β 2AR and cAMP were (70±11)fmol/mg and (1.30±0.10)pmol/L in group E, (67±4)fmol/mg and (3.30±0.20)pmol/L in group G, respectively, and there were no significant differences between group C and E or G(all P<0.05). Conclusions The results indicated that there was down-regulation of β 2AR and cAMP in asthmatic mice(group B), especially in those treated with β 2AR agonist(group C). Gene transfer of βARKct could inhibit the extent of the down-regulation of β 2AR and cAMP in asthmatic mice treated with β 2AR agonist. The route of gene delivery could also affect the degree of up-regulation of β 2AR and cAMP. Gene transfer of βARKct