CAJ | 학술논문

目的通过建立离体血管平滑肌细胞钙化模型,观察成纤维细胞生长因子21 (FGF21)对血管钙化的影响,并探讨其机制.方法使用氯化钙和β磷酸甘油诱导离体大鼠主动脉血管平滑肌细胞钙化.将细胞分为对照组(使用常规培养基)、钙化组(使用钙化培养基)、FGF21组(使用钙化培养基和FGF21)、PD166866组[使用钙化培养基、FGF21和成纤维细胞生长因子受体1(FGFR1)抑制剂PD166866]和GW9962组(使用钙化培养基、FGF21和过氧化物酶体增殖因子受体γ抑制剂GW9662).通过细胞内钙含量、碱性磷酸酶活性和茜素红染色检测血管平滑肌细胞钙化情况.FGFR1、β-Klotho、骨钙素和平滑肌22α的蛋白含量和mRNA表达水平分别用Western blot和Real-timePCR检测.结果 (1)与对照组比较,钙化组的FGFR1和β-Klotho蛋白水平(P＜0.05)和mRNA水平(P＜0.01)均较低.(2)与钙化组比较,FGF21组钙含量、碱性磷酸酶活性和骨钙素的表达量较少(P ＜0.05或0.01),同时平滑肌22α的蛋白和mRNA水平较高(P均＜0.05).茜素红染色显示,与钙化组比较,FGF21组的红色钙化结节较少.(3)PD166866组与钙化组之间的钙含量、碱性磷酸酶活性和茜素红染色结果差异均无统计学意义(P均＞0.05).(4) GW9662组与钙化组之间的钙含量、碱性磷酸酶活性和茜素红染色结果差异均无统计学意义(P均＞0.05).结论在大鼠血管平滑肌钙化模型中,FGFR1和β-Klotho的蛋白及mRNA水平下调,降低了FGF21抑制血管平滑肌细胞钙化的作用;FGF21通过过氧化物酶体增殖因子受体γ通路减轻血管钙化.
목적 통과건립리체혈관평활기세포개화모형,관찰성섬유세포생장인자21 (FGF21)대혈관개화적영향,병탐토기궤제.방법 사용록화개화β린산감유유도리체대서주동맥혈관평활기세포개화.장세포분위대조조(사용상규배양기)、개화조(사용개화배양기)、FGF21조(사용개화배양기화FGF21)、PD166866조[사용개화배양기、FGF21화성섬유세포생장인자수체1(FGFR1)억제제PD166866]화GW9962조(사용개화배양기、FGF21화과양화물매체증식인자수체γ억제제GW9662).통과세포내개함량、감성린산매활성화천소홍염색검측혈관평활기세포개화정황.FGFR1、β-Klotho、골개소화평활기22α적단백함량화mRNA표체수평분별용Western blot화Real-timePCR검측.결과 (1)여대조조비교,개화조적FGFR1화β-Klotho단백수평(P＜0.05)화mRNA수평(P＜0.01)균교저.(2)여개화조비교,FGF21조개함량、감성린산매활성화골개소적표체량교소(P ＜0.05혹0.01),동시평활기22α적단백화mRNA수평교고(P균＜0.05).천소홍염색현시,여개화조비교,FGF21조적홍색개화결절교소.(3)PD166866조여개화조지간적개함량、감성린산매활성화천소홍염색결과차이균무통계학의의(P균＞0.05).(4) GW9662조여개화조지간적개함량、감성린산매활성화천소홍염색결과차이균무통계학의의(P균＞0.05).결론 재대서혈관평활기개화모형중,FGFR1화β-Klotho적단백급mRNA수평하조,강저료FGF21억제혈관평활기세포개화적작용;FGF21통과과양화물매체증식인자수체γ통로감경혈관개화.
Objective To observe the effect and mechanism of fibroblast growth factor 21 (FGF21) on rat vascular smooth muscle cells (VSMCs) calcification in vitro.Methods VSMCs was treated with calcification medium containing calcium chloride and β-glycerophosphate to induce rat VSMCs calcification in vitro.VSMCs were divided into 5 groups: the control group (cultured in normal medium), the calcification group (incubated in calcified medium), the FGF21 group (cultured in calcified medium and FGF21), the PD166866 group (cultured in calcified medium and FGF21 and PD166866, inhibitor of fibroblast growth factor receptor-1 (FGFR1)), the GW9662 group (cultured in calcified medium and FGF21 and GW9662, inhibitor of peroxisome proliferators activated receptor-γ (PPAR-γ)).The calcification of VSMCs was detected by calcium content, alkaline phosphatase activity and alizarin red staining.The protein and mRNA expression of FGFR1, β-Klotho, osteocalcin and smooth muscle 22α (SM22α) were determined by western blot analysis and realtime-PCR, respectively.Results (1) The mRNA (P ＜ 0.01) and protein expressions of β-Klotho and FGFR1 were significantly downregulated in calcification group compared with control group (P ＜ 0.05 or 0.01).(2)The protein levels and mRNA expression of calcium content, alkaline phosphatase activity and osteocalcin were significantly downregulated, while the protein levels and mRNA of SM22α were significantly increased in FGF21 group compared with calcification group (all P ＜ 0.05).Moreover, alizarin red staining verified positive red nodules on calcified VSMCs was significantly reduced in FGF21 group than in calcification group.(3)Calcium content, alkaline phosphatase activity and alizarin red staining were similar between PD166866 group and calcification group (all P ＞ 0.05).(4) Calcium content, alkaline phosphatase activity and alizarin red staining were similar between GW9662 group and calcification group (all P ＞ 0.05).Conclusion The inhibition of VSMCs calcification by FGF21 is mediated by further downregulating FGFR1 and β-Klotho while activating PPAR-γpathways.