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目的:通过观察猪主动脉瓣成肌纤维细胞在受氧化型低密度脂蛋白(ox-LDL)作用时,阿托伐他汀(Ator)对细胞凋亡的影响以及是否表达骨相关蛋白,来探讨阿托伐他汀对心脏瓣膜钙化的防治机制。方法:以原代培养的猪主动脉瓣成肌纤维细胞作为研究对象,用ox-LDL处理细胞。将瓣膜间质细胞随机分为4组:对照组(Ctrl),ox-LDL组(25 mg·L-1ox-LDL),oxLDL+Ator组(25 mg·L-1ox-LDL+50 mg·L-1Ator),Ator组(50 mg·L-1Ator)。采用流式细胞学技术测定各组细胞在24,48,72 h的凋亡变化,利用Western印迹法和Real-time PCR检测72 h各组细胞的α-平滑肌蛋白(α-smooth muscle actin,α-SMA)活性,以及骨形成蛋白2(bone morphogenetic protein 2,BMP2),碱性磷酸酶(alkaline phosphatase,ALP)的基因表达。结果:ox-LDL分别处理成肌纤维细胞24,48,72 h后,细胞显示出时间依赖性的凋亡加速(P<0.05),加入Ator后,细胞的凋亡速度明显减低(P<0.05)。ox-LDL组α-SMA的表达明显增加(P<0.05),成为活性细胞,加用Ator并未对α-SMA蛋白产生影响(P>0.05)。骨相关蛋白BMP2、ALP和mRNA在对照组和Ator组仅有低水平表达;而在ox-LDL组表达显著增加(P<0.01),加入Ator后,BMP2、ALP的表达明显下降(P<0.05)。结论:ox-LDL促使心脏瓣膜成肌纤维细胞分化、凋亡加速以及向成骨细胞样表型转化;Ator可以抑制ox-LDL所诱导的细胞凋亡,并对细胞的激活,以及向成骨样细胞表型分化可以起到一定的抑制作用,对心脏瓣膜钙化有防治作用。
OBJECTIVE: To investigate the effects of ator on apoptosis and the expression of bone-related proteins in aorta myofibroblasts of pigs exposed to oxidized low density lipoprotein (ox-LDL) Mechanism of atorvastatin on heart valve calcification. Methods: Primary cultured porcine aortic valve myofibroblasts were used as the research object, and the cells were treated with ox-LDL. Valvular interstitial cells were randomly divided into 4 groups: control group (Ctrl), ox-LDL group (25 mg · L-1 ox-LDL), oxLDL + Ator group (25 mg · L -1 ox-LDL + 50 mg · L -1Ator), Ator group (50 mg · L-1Ator). Flow cytometry was used to determine the changes of apoptosis in each group at 24, 48 and 72 h. Western blotting and Real-time PCR were used to detect the expression of α-smooth muscle actin (α -SMA) activity, as well as the gene expression of bone morphogenetic protein 2 (BMP2) and alkaline phosphatase (ALP). Results: After treated with ox-LDL for 24,48,72 h, the cells showed a time-dependent increase in apoptosis (P <0.05). After addition of Ator, the apoptotic rate was significantly decreased (P <0.05) . The expression of α-SMA in ox-LDL group was significantly increased (P <0.05), and became active cells. Addition of Ator did not affect α-SMA protein (P> 0.05). BMP2, ALP and mRNA of osteopontin were only low expression in control group and Ator group, but were significantly increased in ox-LDL group (P <0.01), and the expression of BMP2 and ALP was significantly decreased after adding Ator (P <0.05 ). CONCLUSION: ox-LDL can promote the differentiation of cardiac valve myofibroblasts, accelerate the apoptosis and transform into osteoblast-like phenotype. Ator can inhibit the apoptosis induced by ox-LDL and activate the cells, Cell phenotypic differentiation can play a role in the inhibition of heart valve calcification have a preventive effect.