Notch1高表达可使钙化的人心脏瓣膜间质细胞骨形态发生蛋白-2、4表达增加

Upregulated Notch1 expression promotes bone morphogenetic protein-2/4 expression of calcified human heart valve interstitial cells

目的 观察钙化的人心脏瓣膜间质细胞（hVIC） Notch1蛋白表达的变化及其对细胞钙化的影响,并探讨其机制.方法 体外培养hVIC,将其分为2组,分别为正常对照组和诱导钙化组,前者常规培养,后者在常规培养的基础上加用β-甘油磷酸（500μl）、抗坏血酸（200μl）和地塞米松（100μl）以诱导细胞钙化,2组细胞均培养7d.另取上述方法诱导钙化的hVIC为钙化对照组,再设钙化抑制组,即为在钙化对照组的基础上加入Notch1特异性抑制剂DAPT 50 μmol/L（4μl/孔）,2组细胞均培养7d.4组均用脂多糖（LPS）诱导细胞的炎症反应.采用蛋白印迹法检测各组细胞内Notch1、磷酸化核转录因子κB （p-NF-κB）、骨形态发生蛋白（BMP）-2和BMP4的蛋白表达水平,ELISA法测定各组细胞培养液上清中BMP-2蛋白的含量,Von Kossa染色法观察各组细胞钙化的情况.结果 （1）诱导钙化组和正常对照组细胞Notch1、p-NF-κB、BMP-2、BMP4蛋白表达和细胞培养液上清中BMP-2蛋白的含量以及Von Kossa染色结果：诱导钙化组细胞Notch1、p-NF-κB、BMP-2和BMP-4蛋白表达水平均明显高于正常对照组（P均＜0.05）.诱导钙化组培养液上清中BMP-2蛋白含量为（88.23±3.28） pg/ml明显高于正常对照组的（25.41 ±3.68） pg/ml（P=0.02）.Von Kossa染色可见诱导钙化组细胞形成大小不等的结节,胞质内的沉积物与细胞结节被染成黑色,提示有钙质沉积,而正常对照组细胞形态结构清晰,细胞周围无沉积物形成.（2）钙化对照组和钙化抑制组细胞Notch1、p-NF-κB、BMP-2、BMP-4蛋白表达和细胞培养液上清中BMP-2蛋白的含量以及Von Kossa染色结果：钙化抑制组细胞Notch1、p-NF-κB、BMP-2和BMP4蛋白的表达水平均明显低于钙化对照组（P均＜0.05）,钙化抑制组培养液上清中BMP-2蛋白的含量为（26.74±4.62） pg/ml明显低于钙化对照组的（80.41 ±2.96） pg/ml （P=0.02）.Von Kossa染色可见钙化抑制组细胞钙结节明显少于钙化对照组.结论 钙化的hVIC Notch1蛋白高表达,促进了BMP-2和BMP-4蛋白表达增加,从而使hVIC发生成骨样改变,诱导细胞钙化,这一机制可能与NF-κB的活化有关.

Objective To observe the protein expression of Notch 1 in the cultured calcified human heart valve interstitial cells （hVICs） in vitro and related mechanisms.Methods hVICs were divided into two groups：control hVICs were cultured in conventional media for 14 days and calcified hVICs were cultured with calcification inducers：[3-glycerophosphate （500 μl）,ascorbic acid （200 μl）,dexamethasone （100 μl） for 7 days.The calcified hVICs were further divided into calcified hVICs group and inhibited calcified hVICs by adding specific Notch1 inhibitor DAPT （50 μmol/L（4 μl/hole））groups and cultured for another 7 days.Inflammatory response of all groups were induced by lipopolysaccharide （LPS） for 8 to 12 hours.Western blot was used to detect the protein expression of Notch1,phosphorylation nuclear transcription factor κB （p-NF-κB）,bone morphogenetic protein-2/4（BMP-2/4）.ELISA was applied to detect the content of BMP-2 secretion of the groups.Von Kossa staining was used to observe of cellular calcification.Results （1）Von Kossa staining is positive in the induced calcification group,the expression of Notch1,p-NF-κB,BMP-2 and BMP-4 is significantly higher in the induced calcification group than in the control group （all P 〈 0.05）.The expression of BMP-2 is significantly higher in the induced calcification group than in control group （（88.23 ± 3.28） pg/ml vs.（25.41 ± 3.68） pg/ml,P =0.02）.（2） After treatment with DAPT,the calcification and the expression of Notch1,p-NF-κB,BMP-2 and BMP-4 were significantly decreased compared to calcification group （all P 〈 0.05）.The expression of BMP-2 is （26.74 ± 4.62） pg/ml in the calcification inhibition group and （80.41 ± 2.96） pg/ml in calcified control group （P =0.02）.Conclusions Upregulated Notch 1 expression promotes BMP-2/4 secretion in LPS stimulated hVICs,and contributes to osteogenic changes in hVICs.Inhibiting Notch1 can decrease the BMP-2/4 secretion and calcification in hVICs,which may serve as a novel therapeutic option for treating calcific valve disease.