连接蛋白43调节牙髓干细胞向成牙本质细胞分化的机制研究

Odontogenic differentiation of dental pulp cells play a key role in repair following the dental injury,but the mechanism of differentiation is still not completely clear.Connexin43(Cx43) is the major component of intercellular gap junction.Research showed that Cx43 could promote the repair after skin tissue injury,this prompt us that Cx43 may be benefit to repair follow dental injury either.So,it was found in our preliminary work that expression of Cx43 was significant in dental pulp cells which were in the process of repair follow dental injury,and dramatically higher than the normal ones.And the latest research was found that the growth of dental pulp and mineralization were inhibited when the expression of Cx43 was down-regulated.These findings indicated that Cx43 participated and regulated the odontogenic differentiation of dental pulp cells,which was closely related to repair follow the dental injury,but the pathway of Cx43 regulation was not clear.Therefore,we plan to study the effect of Cx43 in odontogenic differentiation of dental pulp stem cells(DPSCs)and the expression of related signal pathway in order to investigate the the regulation mechanism of Cx43 in differentiation from DPSCs into odontoblast,to provide a new heoretical basis for clarification of mechanism of repair following dental injury by overexpression and inhibition of Cx43 in human DPSCs.

牙髓细胞成牙本质分化是牙髓损伤后修复的关键，但分化机制尚未完全清楚。连接蛋白43（Cx43）是构成细胞间缝隙连接通道的主要组成部分。研究显示，Cx43可以促进皮肤组织的损伤后修复，提示我们，Cx43也可能对牙髓的损伤后修复起到一定作用。于是，我们在前期研究中发现，Cx43在损伤后修复过程中的牙髓细胞中表达显著，明显高于正常牙髓细胞。并且近来研究显示，下调Cx43的表达会抑制牙髓细胞的生长和矿化形成。结果表明，Cx43参与调控了牙髓细胞成牙本质分化，与牙髓的损伤后修复密切相关，但通过什么样的途径还未可知。为此，我们拟通过在人的牙髓干细胞（DPSC）中过表达和抑制Cx43，研究其对DPSC成牙本质分化的作用及相关信号通路的表达变化，探讨Cx43调节DPSC向成牙本质细胞分化的分子机理，为阐明牙髓损伤后修复的机制提供新的理论基础。

牙髓干细胞（DPSCs）的成牙本质分化是牙髓损伤后修复的关键。连接蛋白43（Cx43）是构成细胞间缝隙连接通道的主要组成部分。牙髓损伤后Cx43表达明显升高，阻断Cx43会抑制DPSCs的生长。结果表明，Cx43对牙髓的损伤后修复起到重要作用，但Cx43对DPSCs成牙本质分化的作用及机制尚不清楚，阐明Cx43的调控机制对理解和掌握牙髓的损伤后修复机制具有积极的促进作用。为此，我们通过在DPSCs内过表达和抑制Cx43，观察Cx43对DPSCs成牙本质分化的影响，探讨其调控机制。结果显示，胞外Ca2+通过L型Ca2+通道内流，上调胞内Ca2+浓度，促进成牙本质细胞分化标志性基因DSPP的表达，并通过BMP-2介导的Smad1/5/8和Erk1/2通路促进DPSCs向成牙本质细胞分化和矿化。过表达Cx43不会对DPSCs的成牙本质分化有影响，但是可以通过Erk1/2信号增强Ca2+诱导的DSPP表达。流式细胞仪检测显示，在Ca2+诱导下，Cx43可以增加磷酸化Erk1/2阳性细胞的百分比。结果表明，在DPSCs中，Cx43可能通过其构成的细胞间缝隙连接通道，促使信号在偶联的细胞间传递，从而使更多的DPSCs感受到胞外Ca2+的刺激。另外，抑制Cx43功能以及缝隙连接通道会抑制Ca2+诱导的DSPP表达，说明Cx43需要细胞间的直接接触来促进Ca2+诱导的细胞分化。同样，DSPCs高低密度培养实验也揭示了细胞间的直接接触对于Cx43促进Erk1/2活性和DSPP表达的必要性。总之，本项研究阐明了Ca2+通过L型Ca2+通道内流，上调胞内Ca2+浓度，激活BMP-2介导的Smad1/5/8和Erk1/2通路并促进DPSCs向成牙本质细胞的分化和矿化。在DPSCs中，Cx43组成细胞间的缝隙连接通道，可能通过其介导的细胞间通信，使整个细胞集落感受到胞外Ca2+刺激，增强Erk1/2信号活性， 促进Ca2+诱导的DSPP表达和DPSCs的成牙本质分化。