Notch1蛋白在人心脏瓣膜间质细胞凋亡与钙化关系中的作用

The role of Notch1 protein in the relationship between apoptosis and calcification of human heart valve interstitial cells

目的用人心脏瓣膜间质细胞(hVICs)在体外培养并用钙化诱导液建立瓣膜钙化模型,观察hVICs钙化过程中Notch1蛋白表达及细胞凋亡情况,探讨Notch1蛋白在hVICs凋亡与钙化关系中的作用。方法将体外培养的hVICs随机分为两组,钙化组用含钙化诱导液的培养基培养,以建立钙化模型,并按1~7天分别诱导细胞,观察钙化的动态过程;空白组用正常培养基(I-GRO PLUS)培养。所有hVICs均培养7天。检测两组细胞钙化结节数目、BMP-4蛋白、Notch1蛋白的表达及hVICs凋亡率。然后将钙化组中诱导钙化细胞再随机分脂多糖组(LPS)、Notch1抑制剂组(γ分泌酶抑制剂,DAPT)、对照组(只加钙化诱导液)体外培养3天,检测BMP-4、Notch1、半胱氨酸天冬氨酸蛋白酶3(Caspase3)的表达。结果与空白组细胞比较,钙化组细胞钙化及Notch1蛋白的表达均高于空白组,且随着诱导天数的增加而增加;细胞凋亡率在钙化诱导3天时最高,随后逐渐下降。LPS组与对照组比较,细胞凋亡和钙化均随着Notch1蛋白的表达增加而增加;DAPT组与对照组比较,细胞凋亡和钙化均减少。结论hVICs钙化与细胞凋亡有着密切的关系,且Notch1蛋白在hVICs凋亡与钙化关系中起着重要的作用。

Aim To establish valve calcification model , Human heart valve interstitial cells （hVICs） were cultured in vitro and induced by calcification liquid, observing the expression of Notchl protein , apoptosis in the process of hVICs calcification and revealing the role of Notchl protein in the relationship between hVICs apoptosis and calcification. Methods hVICs were randomly divided into two groups, the experimental group were treated with caleification induced liquid （beta glycerol phosphoric acid, ascorbic acid, dexamethasone, recombinant type human bone morphogenetic protein 2） to establish the models of calcification, hVICs were stimulated by calcification liquid for lday, 3days, 5days, 7days ; Control group was cultured with normal culture medium, all hVICs were cultured for 7 days, then detecting the number of calcification nodule ,the expression of BMP-4, Notchl and the Apoptosis rates of hVICs. Calcification induced cells in experimental group were randomly divided into three groups and cultured in vitro for 3 days, the lipopolysaccharide group （LPS）, the Notchl inhibitor group （γ secretase inhibitor, DAPT）, and the control group （ treated with calcification induced liquid）. The expression of BMP-4, Notchl, Cysteinyl aspartate specific proteinase 3 （Caspase3）were determined. Results The expression of Notchl protein and cell calcification in experimental group were higher than control group anti increased along with the induced days. The rate of cell apoptosis was the highest at the third day, then there was a gradual downward trend. Cell apoptosis and calcification in LPS group were aggravated with the increased expression of Notch1 compared with control group. In DAPT group compared with the control, cell apoptosis and calcification were reduced. Conclusion The relationship between apoptosis and calcification are close, Notchl protein plays an important role in the relationship between apoptosis and calcification of hVICs.