摘要
目的:探讨经典瞬时受体电位通道6(TRPC6)对血小板源性生长因子(PDGF)诱导的气道平滑肌细胞(ASMCs)增殖的影响。方法:组织贴块联合酶消化法培养原代大鼠ASMCs。间接免疫荧光法鉴定平滑肌细胞及检测TRPC6在ASMCs上的表达。CCK-8法检测PDGF诱导ASMCs的增殖。Real-time PCR检测PDGF作用后TRPC6 mRNA的表达。Western blotting检测PDGF作用后TRPC6蛋白的表达。CCK-8法检测TRPC6阻断剂对PDGF诱导ASMCs增殖的作用。结果:细胞免疫荧光显示:TRPC6广泛存在于气道平滑肌细胞。CCK-8法检测细胞的增殖发现,20μg/L PDGF作用后ASMCs发生增殖(P<0.05);PDGF与TRPC6阻断剂SKF96365共同作用于ASMCs,ASMCs的增殖较单独使用PDGF组减弱(P<0.05),且减弱的程度具有剂量及时间依赖性。Real-time PCR结果显示:PDGF分别作用于ASMCs 12 h、24 h和48 h后,TRPC6 mRNA表达与相应的对照组比较明显升高(P<0.05)。Western blotting检测结果显示:PDGF分别作用ASMCs 24 h和48 h后,TRPC6蛋白表达与相应的对照组比较明显升高(P<0.05)。结论:TRPC6参与了PDGF诱导ASMCs增殖的过程,PDGF促进ASMCs增殖可能与其上调TRPC6 mRNA和蛋白表达相关。
AIM ： To investigate the role of canonical transient receptor potential channel 6 （TRPC6） in the proliferation of airway smooth muscle cells （ASMCs） induced by platelet-derived growth factor （PDGF）. METHODS ： Rat ASMCs were cultured by enzyme digestion and tissue adhesion. The method of indirect immunofluorescence was applied to identify the ASMCs and to detect the expression of TRPC6 in ASMCs. The proliferation of ASMCs was determined by CCK-8 assay. The mRNA expression of TRPC6 was tested by real-time PCR. The protein expression of TRPC6 was ana- lyzed by Western blotting. The influence of TRPC6 blocker at different concentrations on the proliferation of ASMCs was measured by CCK-8 assay. RESULTS ： The results of immunofluorescence indicated that TRPC6 expression in ASMCs was positive. PDGF at concentration of 20 p^g/L induced the proliferation of ASMCs compared with control group （P 〈 0.05）. When ASMCs were treated with both PDGF and different concentrations of TRPC6 blocker SKF96365, the proliferation of ASMCs was attenuated in dose-dependent and time-dependent manners as compared with the ceils treated with PDGF alone （P 〈 0.05）. The mRNA expression of TRPC6 in PDGF group was significantly increased （P 〈 0.05 ） after ASMCs were treated with PDGF for 12 h, 24 h and 48 h. The protein level of TRPC6 in PDGF group was significantly increased after ASMCs were treated with PDGF for 24 h and 48 h compared with control group （P 〈 0.05）. CONCLUSION ： Up-regula- tion of TRPC6 at mRNA and protein levels is most possibly related to the proliferation of ASMCs induced by PDGF. There- fore, TRPC6 is involved in the proliferation of ASMCs.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第12期2229-2234,共6页
Chinese Journal of Pathophysiology
基金
广东省科技计划(No.2012145)
广州市高校科技项目(No.10A149)
关键词
血小板源性生长因子
气道平滑肌细胞
瞬时受体电位通道6
气道重塑
Platelet-derived growth factor
Airway smooth muscle cells
Transient receptor potential channel 6
Airway remodeling
