基于“肾主骨”探讨肾细胞外泌体靶向调控成骨细胞生长的机理

The theory of "kidney dominating bone" suggested the existence of communication between the kidney and bone, but the mechanism is not clear. The exosome has the function of regulating the distal target cells with cell information. Previous studies have shown that the exosomes from renal tubular epithelial cells can be absorbed by osteoblasts and promote their proliferation, and so it is suggested that the exosomes from renal tubular epithelial cells can target-regulate the growth of osteoblasts. This project intends to add the exosomes of renal tubular epithelial cell to the transwell systern of the osteoblasts with osteoclasts in vitro, to observe the effect of the exosomes on the osteoblast and osteoclast. In vivo the exosomes intervene bone defect model mice, to observe bone growth by X light and micro-CT, to position the exosomes signal in cells of bone defect tissue by biological imaging and laser capture microscopy, to determine the molecules related with OPG/RANKL/RANK and Sema4D/plexin of B1 biological axis between osteoblasts and osteoclasts by RT-PCR and Western Bloting. So to reveal the molecular mechanism of renal cells derived exosomes target-regulating bone growth and repair, to explain the scientific connotation of the theory of "kidney dominating bone", and to provide the basis for the "kidney dominating bone" to guide the exosomes targeted research. This is of great significance.

“肾主骨”理论暗示肾与骨之间存在信息传递，然机制未明。外泌体具有携带细胞信息靶向调控远端靶细胞的功能。前期研究显示肾小管上皮细胞外泌体可被成骨细胞吸收并促进其增殖，据此提出：肾小管上皮细胞外泌体靶向调控成骨细胞生长。本项目体外拟将肾小管上皮细胞外泌体加入到成骨与破骨细胞transwell共培养体系，观察外泌体对成骨或破骨细胞的影响。体内采用外泌体干预骨缺模型小鼠，X光与micro-CT观察骨生长状况，活体成像术和激光捕获显微细胞术定位骨缺部位外泌体信号，RT-PCR和Western Blot测定成骨与破骨细胞之间 OPG/RANKL/RANK和Sema4D/plexin B1生物轴的相关分子，以此揭示肾细胞外泌体靶向调控骨生长修复的分子机制，阐释“肾主骨”理论的科学内涵，为“肾主骨”指导外泌体的靶向研究提供依据，具有重大意义。

“肾主骨”理论暗示肾与骨之间存在信息传递，而外泌体具有携带细胞信息靶向调控远端靶细胞的功能，因此，肾细胞外泌体能否行使部分肾脏功能，将信息传递至骨组织，有待研究然。本项目通过体内外试验，采用超高速离心法、纳米粒径分析、电镜、WB、激光共聚焦显微镜、高分辨率成像、无标记细胞分析及骨折小鼠模型等技术平台，探索肾小管皮细胞外泌体（HK-2-Exo）在体外靶向成骨细胞，研究其促进成骨细胞生长及机理，并观察对其在体内的分布和对骨折模型小鼠的影响。结果表明：HK-2-Exo为直径30～150nm的囊泡，电镜下呈现圆形或椭圆形杯状结构，其特征蛋白ALIX、TSG101、HSP70有表达，Calnexin无表达，表明不是细胞器囊泡。3D显微图显示HK-2-Exo被人成骨细胞hFOB1.19摄取并分布在核周质中；HK-2-Exo在0.003-2.0μg/ml时促进细胞生长，大于2.0μg/ml后抑制细胞生长，具有双向调节作用。HK-2-Exo通过调控骨生长相关基因蛋白Runx2、OPGL、RANKL、plexinB1、SEMA4D、BGLAP、RANKL、BMP2的表达，促进成骨细胞生长，抑制破骨细胞分化；小鼠尾静脉注射给药HK-2-Exo，剂量为0.02ug/ml/只，隔天1次，共4次，能加速骨折模型小鼠腿部骨折的愈合；且HK-2-Exo静脉给药后迅速分布于肝、肾、骨、脑组织，符合中医“肾主骨主髓”理论，表明肾细胞外泌体有一定的选择性和靶向性，并通过调控成骨细胞和破骨细胞的生长分化来促进骨组织损伤的修复。