蛋白激酶C在支气管哮喘模型大鼠气道平滑肌细胞增殖中的信号转导机制研究

A study of protein kinase C signal pathway in regulating airway smooth muscle cell proliferation in asthmatic rats

目的 探讨蛋白激酶C(PKC)信号转导途径在支气管哮喘 (简称哮喘 )大鼠气道平滑肌细胞 (ASMC)增殖中的作用。方法 (1) 4 8只Wistar大鼠分为哮喘组 (A组 )及对照组 (B组 ) ,根据激发时间 (2、4、8周 )又分别分为A1、A2 、A3 组和B1、B2 、B3 组 ,其中A、B组大鼠各 12只 ,A2 、A3 、B2 及B3 组各 6只。用流式细胞术、四甲基偶氮唑盐 (MTT)法、增殖细胞核抗原 (PCNA)染色等方法观察每组ASMC增殖 ;(2 )用PKC激活剂 12 肉蔻酰 13 乙酸佛波酯 (PMA)及抑制剂Ro 31 82 2 0分别干预A1、B1组ASMC ,观察ASMC增殖的变化 ;(3)用逆转录 聚合酶链测定 (RT PCR)和免疫细胞化学法检测A1、A2 、A3 组和B1组ASMCPKC α的表达。结果 (1)A组ASMCS期比例、吸光度 (A)值、PCNA表达增高 ,与B组比较差异有显著性 (P <0 0 1)。 (2 )A1组ASMCS期比例、A值、PCNA阳性表达率在干预前分别为 (19± 3) %、0 4 5 9± 0 0 36、(80± 10 ) % ;10nmol/LPMA处理后分别为 (2 7± 4 ) %、0 5 99± 0 0 78、(95± 9) % ;5 0nmol/LPMA处理后为 (14± 3) %、0 346± 0 0 38、(5 3± 8) % ;Ro 31 82 2 0处理后为 (14± 3) %、0 343± 0 0 4 8、(4 9± 8) %。各干预剂处理后与处理前比较差异均有显著性 (P <0 0 1) ;5 0nmol/LPMA处?

Objective To investigate the role of protein kinase C(PKC) signal pathway in airway smooth muscle cell(ASMCs) proliferation in asthmatic rats. Methods (1) Forty-eight Wistar rats were divided into an asthmatic group(group A) and a control group(group B), and then subdivided into group A 1, A 2, A 3 and group B 1, B 2, B 3 based on the time of challenge(2, 4, 8 weeks). The proliferation of ASMCs isolated from group A and B was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen(PCNA) immunocytochemical staining. (2) ASMCs from group A 1 and B 1 were treated with PKC agonist PMA and inhibitor Ro-31-8220, and their proliferations were examined. (3) The expression of PKC-α in group A 1, A 2, A 3 and B 1 was observed by immunocytochemical staining and RT-PCR, respectively. Results (1) The percentage of S phase, absorbance (A) value and the expression rate of PCNA protein in ASMCs from group A were significantly increased compared to those of group B( P<0.01). (2) Before ASMCs from A 1 group were treated, the percentage of S phase, A value and the expression rate of PCNA were (19±3)%,0.459±0.036,and (80±10)% respectively; After treated with 10 nmol/L PMA, these values reached (27±4)%, 0.599±0.078,and (95±9)% respectively; After treated with 50 nmol/L PMA, these values were (14±3)%, 0.346±0.038, and (53±8)% respectively; After treatment with Ro-31-8220, these values were (14±3)%, 0.343±0.048, and(49±8)% respectively. Statistical analysis revealed that there were significant differences in the percentage of S phase, A value and the expression of PCNA before and after treatment( P<0.01). After treatment with 50 nmol/L PMA, there was a significant difference as compared to 10 nmol/L PMA treatment( P<0.01), whereas there was no statistical significance as compared to Ro-31-8220 treatment( P>0.05). (3) Compared with that of group B 1 (0.84±0.07), the ratios of A value of PKC-α mRNA in group A 1 (1.08±0.06), group A 2 (1.12±0.05),and group A 3 (1.10±0.08) were significantly increased( P<0.01). Compared with that of group B 1 (28±4)%, the expression rates of PKC-α protein in group A 1 (54±5)%,group A 2 (56±5)%,and group A 3 (53±6)% were significantly increased( P<0.01). Conclusions Airway smooth muscle cell proliferation increases in asthmatic rats. PKC and it′s alpha subtype may contribute to this process.