Abstract: This study aimed to investigate the effect of autophagy on the stemness of canine bone marrow mesenchymal stem cells (cBMSCs). The isolated cBMSCs were cultured with ordinary medium, rapamycin-containing medium, 3-MA-containing medium. Cells were collected after 12, 24 and 48 hours' treatment, then autophagy microtubule-associated protein 1 light chain 3 Ⅱ (LC 3Ⅱ) were detected by indirect immunofluorescence; autophagy and stemness related genes, e.g. LC 3, Beclin 1, autophagy-related 7 (Atg 7), SRY-related high-monility-group (HMG)-box protein-2 (Sox 2), special AT-rich sequence-binding protein 2 (Satb 2), were detected by fluorescence quantitative PCR. The cBMSCs differentiation abilities were detected by the dyeing experiment after adipogenic (Oil red O staining) and osteoplastic (alizarin red staining) differentiation. Results were as follows:The LC 3Ⅱ protein expression and gene expression level at the rapamycin group was increased, while which was decreased in the 3-MA group. The results showed that there was a significant increase at the stemness and autophagy related mRNA expression levels of the rapamycin group, while which was decreased in the 3-MA group. The cBMSCs in the rapamycin group showed morphological alterations obviously and the larger mineralized area, while the cBMSCs in the 3-MA group showed the smaller mineralized area. There were fewer lipid droplets at the rapamycin group, while the 3-MA group was more. These results indicated that autophagy could enhance the stemness and osteogenic differentiation of cBMSCs, it was providing theoretical and technical evidence for cBMSCs in the treatment of diseases in dogs.

Key words: canine bone marrow mesenchymal stem cells, autophagy-related genes, stemness-related genes, osteogenesis induction, lipogenesis induction