基于Cx43蛋白介导的缝隙连接细胞间通讯研究先天肾虚影响骨发育的机制

According to the theory of Traditional Chinese Medicine, kidney, as the root of congenital constitution, governs growth. And therefore it governs bone development also. Founded on earlier investigations, we study the specific mechanism in baby rats’ bone development using a model in which horror-injured pregnant rats cause offspring kidney deficiency. Modern research suggested that Cx43-mediated gap junction constitutes the structural basis for intercellular message transfer in bone tissue. Its communication function controls the intercellular message and energy transfer, and gets involved in cell growth modulation. Therefore, it plays a fundamentally important role in bone development. We argue that congenital kidney deficiency affecting bone development is a result of Cx43 and Cx43-mediated GJIC loss. To approve this argument, we utilized advanced cellular- and molecular- biological techniques to do vivo/vitro experiment. Targeting osteoblasts, precartilaginous stem cells and bone marrow stromal cells, we specifically examined Cx43 expression, GJIC function and its influence on cell proliferation, cell differentiation and cell apoptosis. We also examined function-closely linked ERK signal passages and BMPs, Runx2, Sox9 and other growth genes to identify the key step in bone development mechanism. Gene transfection and RNA interference were used to change Cx43 expression levels and affect GJIC capability in vitro experiment. Finally, Zuogui Pill mediated serum was used in cell culture and test aftermath further proved the modulation function of Bushen Tianjing therapy and mechanism beneath.

肾为先天之本，主发育，肾又主骨，因此肾主骨发育。在前期研究基础上，我们采用恐伤孕鼠模型造成子代先天肾虚，研究其影响仔鼠骨发育的具体机制。现代研究发现，Cx43介导的缝隙连接是骨组织细胞间信息转导的结构基础,，其通讯作用调控细胞间信息和能量传递，参与细胞生长调控，在骨发育中具有至关重要作用。我们推测：先天肾虚影响骨发育的重要原因是Cx43及其介导的GJIC功能的缺少。为验证假说，我们运用先进的细胞、分子生物学技术，进行体内外实验，以成骨细胞、骨骺干细胞、骨髓间充质干细胞培养为依托，重点检测Cx43表达及GJIC功能，及其对细胞增殖、分化、凋亡的影响，并对与其功能密切相关之ERK通路及BMPs等发育基因检测，寻找其影响骨发育的关键环节。体外实验分别采用基因转染、RNA干扰技术，改变Cx43表达水平，影响GJIC能力，用左归丸含药血清，培养后检测，进一步论证补肾填精法对其的调控作用及机制研究。

Cx43是成骨细胞、骨髓间充质干细胞中最主要的缝隙连接蛋白，Cx43表达及其介导的GJIC功能在成骨分化及促骨形成中起重要作用。本研究以Cx43及GJIC 功能为切入点，采用恐吓复合应激刺激孕鼠，复制孕鼠肾虚模型，将孕鼠分为正常、模型、补肾3组，补肾组灌服左归丸浓缩剂。研究结果表明：模型组仔鼠存在先天肾虚，整体发育迟缓，成骨活性降低，影响长骨早期生长发育。中药左归丸补肾填精治疗，可有效改善先天肾虚仔鼠长骨早期发育不良。模型组仔鼠颅骨培养成骨细胞分化中后期Cx43、BGPmRNA表达减少，补肾治疗可提高Cx43、BGP表达，保证细胞正常功能，促进骨发育。体外血清药理学实验证明，左归丸含药血清能促进MC3T3-E1细胞成骨分化过程中Cx43表达，增强GJIC功能；进一步采用慢病毒介导的RNA干扰技术沉默Cx43基因，导致左归丸含药血清促MC3T3-E1细胞Runx2、Osterix mRNA表达作用下降，影响细胞成骨分化。另外左归丸含药血清亦能增强BMSCs成骨向诱导分化过程中Cx43表达及GJIC功能，并与β-catenin、BGP发挥协同作用。据此，我们推测Cx43及GJIC 功能异常与先天肾虚仔鼠骨早期发育不良有关，左归丸对其调节可能是肾主骨生长发育机制中的重要环节。