cAMP-PKA信号转导通路在利多卡因上调大鼠肺泡Ⅱ型上皮细胞表面活性物质相关蛋白-A表达中的作用  

Role of cAMP-PKA signal transduction pathway in lidocaine-induced up-regulation of expression of surfactant protein-A in rat alveolar epithelial type Ⅱ cells

作　　者：戴长宗 王德明[2] 肖继[2] 

机构地区：[1]长沙市第一医院麻醉科,410005 [2]南华大学附属第二医院麻醉科

出　　处：《中华麻醉学杂志》2014年第5期620-623,共4页Chinese Journal of Anesthesiology

基　　金：湖南省自然科学基金（11JJ3120）

摘　　要：目的 评价环腺苷酸-蛋白激酶A（cAMP-PKA）信号转导通路在利多卡因上调大鼠肺泡Ⅱ型上皮细胞（AECⅡ）表面活性物质相关蛋白-A（SP-A）表达中的作用.方法 健康雄性SD大鼠,处死后分离、纯化、培养AECⅡ,接种于24孔细胞培养板中,100μl孔,细胞密度1×106个/ml,培养2h后,换无血清DMEM培养基,采用随机数字表法,将其分为4组（n=48）：正常对照组（C组）培养基中不加入任何药物处理;腺苷酸环化酶激动剂佛司可林组（F组）培养基中加入佛司可林10 μmol/ml;利多卡因组（L组）培养基中加入利多卡因200 μg/ml; PKA抑制剂H89＋利多卡因组（P＋L组）培养基中加入H89 10μmol/ml孵育AECⅡ10 min后,再加入200μg/ml利多卡因.于药物孵育6、12、24 h（T1-3）时采用ELISA法检测AECⅡ内环cAMP含量和PKA活性,采用实时荧光定量PCR法检测SP-A mRNA表达,采用Western blot法检测SP-A表达.结果 与C组比较,F组T1-3时、L组T2.3时AECⅡSP-A和SP-AmRNA表达上调,cAMP水平和PKA活性升高（P＜0.05）;与L组比较,P＋L组T1-3时AECⅡSP-A和SP-A mRNA表达下调,PKA活性降低（P＜0.05）,cAMP水平差异无统计学意义（P＞0.05）.结论 利多卡因可通过激活cAMP-PKA信号转导通路上调大鼠AECⅡ的SP-A表达.Objective To evaluate the role of cyclic adenosine monophosphate-protein kinase A （cAMP-PKA） signal transduction pathway in lidocaine-induced up-regulation of the expression of surfactant protein-A （SP-A） in rat alveolar epithelial type Ⅱ cells （AEC Ⅱ）.Methods Healthy male Sprague-Dawley rats were sacrificed and AEC Ⅱ were isolated,purified and incubated in 24-well culture plates （100μd/hole） with density of 1 × 106/ml.After being incubated for 2 h,the culture medium was replaced with serum-free medium DMEM.The cells were randomly divided into 4 groups （n =48 each）：control group （group C）,forskolin （adenylate cyclase agonist） group （group F）,lidocaine 200 μg/ml group （group L）,and PKA inhibitor H89 ＋ L group （group P＋ L）.Forskolin 10 μmol/ml was added to DMEM in group F.Lidocaine 200 μg/ml was added to DMEM in group L.H89 10μnol/ ml was added to DMEM and AEC Ⅱ were incubated for 10 min,and then lidocaine 200 μg/ml was added in group P ＋ L.At 6,12 and 24 h of incubation （T1-3）,cAMP content and PKA activity （using ELISA）,and expression of SP-A mRNA （by real-time fluorescent quantitative PCR） and SP-A （by Western blot） were measured.Results Compared with group C,the expression of SP-A mRNA and SP-A was significantly upregulated,and cAMP level and PKA activity were increased at T1-3 in group F and at T2,3 in group L.Compared with group L,the expression of SP-A and SP-A mRNA was down-regulated,PKA activity was decreased,and no significant change was found in cAMP level at T1-3 in group P ＋ L.Conclusion Lidocaine can up-regulate the expression of SP-A in AEC Ⅱ of rats through activating cAMP-PKA signal transduction pathway.

关 键 词：利多卡因 环AMP 环AMP依赖性蛋白激酶类 上皮细胞 肺泡 肺表面活性物质相关蛋白质A 

分 类 号：R614[医药卫生—麻醉学]