颗粒蛋白前体抑制钙盐诱导主动脉瓣膜间质细胞成骨分化

该文主要研究颗粒蛋白前体(progranulin,PGRN)对猪主动脉瓣膜间质细胞(valve interstitial cells,VICs)成骨分化的影响及机制,为钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)的早期干预及治疗提供理论依据。采用免疫组化检测正常组和CAVD组中Runx2、OPN的表达,Western blot检测PGRN、纤维化指标α-SMA、钙化指标(Runx2、OPN)的表达以及AKT磷酸化水平。采用胶原酶连续消化法分离VICs,并用免疫荧光染色行表型鉴定。体外实验加入人PGRN重组蛋白,采用ALP染色、茜素红S染色、qPCR和Western blot检测细胞早期及晚期成骨分化能力以及AKT的磷酸化水平;并加入AKT的激活剂SC-79进行反向验证。结果表明,与正常组织相比,CAVD瓣膜组织中PGRN明显降低,α-SMA、Runx2、OPN和p-AKT在CAVD组中表达均明显高于正常组。成功分离出原代VICs,α-SMA和vimentin阳性,vWF阴性。PGRN可使VICs的ALP活性降低、钙盐沉积明显减少;PGRN可下调纤维化/钙化指标,且AKT的磷酸化水平降低;SC-79可减弱PGRN对纤维化/钙化指标的下调作用。提示PGRN能够抑制静止的VIC向肌纤维母细胞样的活化VIC乃至成骨样VIC进行转化,AKT信号通路可能在该过程中发挥重要作用。

PGRN Inhibits Osteogenic Differentiation of Aortic Valve Interstitial Cells Induced by Osteogenic Induction Medium

The aim of this study was to investigate the effect and mechanism of PGRN(progranulin)on osteogenic differentiation of porcine aortic VICs(valve interstitial cells),which could provide theoretical basis for early intervention and treatment of CAVD(calcific aortic valve disease).The expression levels of Runx2 and OPN in the Normal group and CAVD group were tested by immunohistochemistry.The expression levels of PGRN,fibrosis markers(α-SMA)/calcification markers(Runx2,OPN)and p-AKT were detected by Western blot.VICs were isolated by continuous collagenase digestion,their morphological characteristics were observed and the phenotypes were identified by immunofluorescence staining.VICs were treated by increasing concentration of PGRN.ALP(alkaline phosphatase)staining,Alizarin red S staining,qPCR,Western blot were used to evaluate the cell early and late osteogenic differentiation abilities.The protein level of p-AKT was determined by Western blot.SC-79,an activator of the AKT,was used for reverse verification.The results showed that fibrosis/calcification markers in CAVD group were significantly higher than that in Normal group.However,the expression of PGRN remarkably decreased.VICs were successfully isolated,the staining ofα-SMA and vimentin were positive,the staining of vWF was negative.The ALP activity and deposition of calcium salts of VICs were significantly decreased by PGRN.The mRNA and protein levels of fibrosis/calcification markers were reduced.Meanwhile,the phosphorylation level of AKT was down-regulated.The down-regulation of PGRN on fibrosis/calcification markers was attenuated by SC-79.We concluded that PGRN could inhibit the conversion of quiescent VICs to activated myofibroblast-like VICs and even osteogenesis-like VICs.The AKT signaling pathways may play an important role in these processes.