Yap基因对牙周膜干细胞的干性维持及增殖分化的影响

Periodontal ligament stem cells(PDLSCs)，key cells of maintaining periodontal tissue homeostasis and regeneration，play an important role in adult regeneration of periodontal tissue. But the underlying mechanisms of how PDLSCs maintain stem cell characteristics and regulate stem cell proliferation, differentiation and orderly regulation are still unclear. YAP protein (Yes-associated protein) is a switch protein that plays a central role in the Hippo signaling pathway. YAP protein when binding to certain transcription factors, such as TEAD, can upregulate the expression of downstream protein, thus promoting cell proliferation and inhibit cell apoptosis. Yap, as a gene transcription coactivator, plays an important role in maintaining stem cell self-renewal and differentiation and most likely regulates the PDLSCs also. On the basis of successfully culturing PDLSCs, our group will establish stable cell lines overexpressing or knocking down YAP protein using lentiviral plasmid. We will characterize the effect of YAP protein on cell differentiation, aging-related protein expression and Hippo signaling pathway regulation. We will assess the mechanisms of regulate stem cell proliferation and differentiation, to better understanding the mechanism by which how these stem cells promote tissues regeneration .

牙周膜干细胞是维持牙周组织动态平衡和缺损修复的关键细胞，对牙周组织再生发挥重要作用。但牙周膜干细胞的干性维持机制，干细胞增殖、分化与有序的调控机制尚不明确。YAP蛋白(Yes associated protein)是Hippo信号通路中一个起中心作用的开关蛋白，在细胞正常生长发育过程中，YAP结合转录因子TEAD等促进下游目的基因的表达从而促进细胞生长、抑制细胞凋亡。Yap作为一种基因转录共激活因子在维持干细胞自我更新及分化方面起着重要作用。我们推测Yap对牙周膜干细胞具有重要调控作用。本课题组在成功培养牙周膜干细胞的基础上，构建人的Yap慢病毒过表达/干涉载体并获得稳定高表达/干涉Yap的人牙周膜干细胞株，研究对牙周膜干细胞增殖、分化，衰老调控机制表达及信号传导通路的影响。研究牙周膜干细胞增殖、分化调控机制，为通过调控干细胞促进组织再生提供重要的理论依据。

牙周膜干细胞（Periodontal ligament stem cells，PDLSCs)是维持牙周组织动态平衡和缺损修复的关键细胞，对牙周组织再生发挥重要作用。Hippo-YAP信号通路作为高度保守的细胞抑制生长性信号通路，在多种肿瘤及间充质干细胞中报道与细胞增殖与分化密切相关，但其对PDLSCs增殖、分化的调控机制尚不明确。本课题首先采用western blot技术验证PDLSCs中Hippo-YAP信号通路的表达状况，YAP与p-YAPS127表达表达强度不同，免疫荧光技术检测YAP可表达于胞核与胞质中。构建过表达及干扰YAP的慢病毒载体并转染PDLSCs获得可稳定高表达及干扰YAP表达的PDLSCs。干扰YAP后CCK-8及EdU结果表明细胞增殖活性降低；细胞早期及晚期凋亡率均增加；G0/G1期细胞比例增多,S期以及G2/M期比例减少；β-gal衰老细胞数量增多；干性标记物Nanog、C-myc表达降低。干扰YAP后磷酸化Erk通路受到抑制；细胞凋亡相关蛋白Bcl-2家族中表达升高；周期蛋白依赖性激酶CDK4/6-Cyclin D等表达增高，周期蛋白抑制因子p18表达增高；成骨诱导后ALP,COL-1,Runx2,OCN的mRNA表达量增高,细胞成骨分化能力增强。过表达YAP后CCK-8及EdU结果表明细胞增殖活性增高；细胞早期及晚期凋亡率均降低；G1期细胞比例减少,G2/M期细胞比例明显增多；β-gal衰老细胞数量降低。过表达YAP后起到Erk通路激活；细胞凋亡相关蛋白Bcl-2家族中表达降低；周期蛋白依赖性激酶CDK4/6-Cyclin D等表达降低，周期蛋白抑制因子p18、p21成表达降低趋势；干性标记物Oct4、Sox2、Nanog等维持不变；成骨诱导后ALP,COL-1,Runx2,OCN的mRNA表达量减少,细胞成骨分化能力减弱。mRNA基因芯片分析发现，YAP与MAPK pathway、Apoptosis、Pathways in cancer 以及Wnt/β-Catenin信号通路关系最为密切，并进行验证分析验证。综上所述：YAP在PDLSCs中发挥促增殖、抑凋亡两方面的作用，YAP可以延缓PDLSCs的细胞衰老，抑制细胞分化并与Wnt/β-Catenin、Erk、Bcl-2等信号通路发挥交叉调控作用。