肾-骨应答调控骨骼VDR/RXR对糖尿病肾病动物模型FGF23分泌的影响及中药的干预作用

血管钙化是导致糖尿病肾脏病（DKD）继发心血管疾病的重要因素。前期研究发现其发生与肾脏表达的Kl基因不足和骨骼产生的FGF23增加相关。本研究基于肾主骨理论及DKD脾肾亏虚、浊毒内蕴病机，以Kl为调控基因，以骨骼为靶点，采用db/db小鼠模型，给以健脾补肾、通腑泄浊中药；运用qPCR、Western-blot、免疫组化及激光共聚焦等技术，解析肾脏产生的Kl、1,25(OH)2VitD3与骨骼表达的VDR/RXR、FGF23之间的相互作用；体外采用大鼠成骨细胞UMR-106与1,25(OH)2VitD3及中药含药血清共培养；构建VDRsiRNA及VDR质粒转染细胞，运用免疫共沉淀、免疫荧光及凝胶迁移等技术，验证VDR/RXR活化对FGF23基因启动子的影响；阐明在DKD中肾-骨应答调控骨骼分泌FGF23的分子机制及健脾补肾、通腑泄浊中药的干预作用，拓展肾主骨理论的深入研究及临床应用。

Vascular calcification is an important cause of cardiovascular disease secondary to diabetic kidney disease (DKD). Our previous studies have found that it is related to the insufficiency of Klotho (Kl) gene expressed in the kidneys and increase of fibroblast growth factor 23 (FGF23) produced by the bones. This study is based on the theory of “kidneys govern bones” and the pathogenesis “spleen and kidney deficiency, turbidity accumulation” of DKD. In the study, Kl is regarded as a regulatory gene and the bones as the target. Animal models are made by db/db mice, and treated with traditional Chinese medicine for tonifying spleen and kidney, purging bowels and expelling turbidity. Real-time PCR, Western blot, immunohistochemistry, and confocal laser scanning microscope will be used to analyze the interaction between Kl, 1,25(OH)2VitD3 produced by the kidneys and VDR/RXR, FGF23 secreted by the bones. In vitro, rat osteoblast UMR-106 cells will be co-cultured with 1,25(OH)2VitD3 and Chinese medicine-containing serum. Then, the cells will be transfected with VDRsiRNA and VDR plasmids, and analysed using techniques such as co-immunoprecipitation, immunofluorescence, and electrophoretic mobility shift assay to verify the effects of VDR/RXR activation on the promoter of the FGF23 gene. The purpose of this study is to clarify the molecular mechanism of kidney-bone axis to the osteocytic secretion of FGF23 in DKD and the effects of Chinese medicine of tonifying spleen and kidney, purging bowels and expelling turbidity to provide a basis for the in-depth study and application of the “kidneys govern bones” theory.