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目的 :探讨瘦素对主动脉瓣膜间质细胞(valvular interstitial cells,VICs)表型转化的影响,揭示瘦素在钙化性主动脉瓣疾病(calcific aortic value disease,CAVD)发生发展中所扮演的角色。方法:胶原酶消化法分离并培养猪主动脉瓣膜间质细胞,采用含不同浓度的瘦素分别刺激细胞24 h,检测细胞裂解液碱性磷酸酶(ALP)活性,并确定最佳刺激浓度。用最佳刺激浓度的瘦素刺激细胞24 h,提取细胞RNA以及蛋白,通过RT-PCR以及Western blot分别测定肌动蛋白(α-SMA)、骨桥蛋白(OPN)的表达情况。结果:瘦素刺激导致ALP表达上调且最大刺激浓度为100 ng/m L。相较于对照组,瘦素刺激组OPN mRNA及蛋白表达增高,而α-SMA表达无明显变化。结论:瘦素能够诱导VICs向成骨细胞分化,从而促进VICs钙化。
OBJECTIVE: To investigate the effect of leptin on the phenotype of aortic valvular interstitial cells (VICs) and to reveal the role of leptin in the development of calcific aortic value disease (CAVD) . Methods: Porcine aortic valve interstitial cells were isolated and cultured by collagenase digestion. The cells were stimulated with different concentrations of leptin for 24 h. The alkaline phosphatase (ALP) activity of cell lysate was determined and the optimal stimulation concentration was determined. The cells were stimulated with leptin at the optimal stimulus concentration for 24 h to extract RNA and protein. The expression of actin (α-SMA) and osteopontin (OPN) were detected by RT-PCR and Western blot respectively. RESULTS: Leptin stimulation resulted in up-regulation of ALP expression and maximum stimulation concentration of 100 ng / m L. Compared with the control group, the expression of OPN mRNA and protein in leptin-stimulated group increased, while the expression of α-SMA did not change significantly. Conclusion: Leptin can induce the differentiation of VICs into osteoblasts and thus promote the calcification of VICs.