Abstract: BACKGROUND: Programmed cell death protein 1 belongs to the immunoglobulin gene superfamily and can regulate the differentiation of osteoblasts and affect bone homeostasis. However, there are few studies on the regulatory role and mechanism of programmed cell death protein 1 in diabetic osteoporosis.
OBJECTIVE: To investigate the regulatory role and mechanism of programmed cell death protein 1 on osteogenic differentiation of rat bone marrow mesenchymal stem cells under high-glucose environment. 
METHODS: (1) Animal experiment: A total of 12 Sprageu-Dawley rats were randomized into a control group (n=6) and a model group (n=6). The control group was fed routinely, whereas the model group was injected intraperitoneally with streptozotocin to establish a model of type 1 diabetes mellitus, and the high-fat feed was fed for 8 weeks to establish a model of type 1 diabetic osteoporosis. After 8 weeks of feeding, the femurs of rats in the two groups were taken and subjected to hematoxylin-eosin staining and micro-CT assay. The mRNA expression of programmed cell death protein 1 and programmed death ligand 1 was detected. (2) Cell experiment: Passage 3 rat bone marrow mesenchymal stem cells were randomly divided into four groups: normal control group, high-glucose model group cultured in low glucose medium, programmed cell death protein 1-silenced group transfected with programmed cell death protein 1 siRNA, and programmed cell death protein 1-silenced null group transfected with siRNA-NC. After 48 hours of transfection, the normal control group was cultured in a new low-glucose medium, and the other three groups were cultured in a high-glucose medium for another 48 hours of culture followed by osteogenic induction. After 21 days of osteogenic induction, alizarin red staining, and qRT-PCR (programmed cell death protein 1 and RUNX2 mRNA expression) and western blot (β-catenin, GSK-3β, p-GSK-3β and Axin2 protein expression) were performed.
RESULTS AND CONCLUSION: In the animal experiment, hematoxylin-eosin staining and micro-CT assay showed successful modeling of type 1 diabetic osteoporosis in the model group. qRT-PCR assay showed that the mRNA expression of programmed cell death protein 1 and programmed cell death ligand 1 was higher in the model group than the control group (P < 0.05). In the cell experiment, the results of alizarin red staining showed that the ability of mineralized nodule formation was lower in the high-glucose model group and the programmed cell death protein 1-silenced null group than in the control group and the programmed cell death protein 1-silenced group. Compared with the normal control group, the programmed cell death protein 1 mRNA expression and GSK3β and Axin2 protein expression were elevated in the high-glucose model group and the programmed cell death protein 1-silenced null group (P < 0.05), and the RUNX2 mRNA expression and p-GSK3β and β-catenin protein expression were decreased (P < 0.05). Compared with the high-glucose model group and the programmed cell death protein 1-silenced null group, programmed cell death protein 1 mRNA expression and GSK3β and Axin2 protein expression were decreased in the programmed cell death protein 1-silenced group (P < 0.05), and RUNX2 mRNA expression and p-GSK3β and β-catenin protein expression were elevated (P < 0.05). To conclude, programmed cell death protein 1 silencing can activate the Wnt/β-catenin and improve the osteogenic differentiation of rat bone marrow mesenchymal stem cells under high-glucose conditions.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: bone marrow mesenchymal stem cells, diabetes mellitus, osteoporosis, osteogenic differentiation, programmed cell death protein 1, Wnt/β-catenin pathway, engineered tissue construction