Roles Of Wnt/β-catenin Signaling In Adipogenic Differentiation Of Poultry Mesenchymal Stem Cells

Livestock and poultry genetic resources are valuable assets of China and a very important means of production in the animal husbandry industry.The protection and preservation of genetic resources are of great significance for promoting the sustainable development of animal husbandry and ensuring species diversity and genetic polymorphism in nature.The method of storing stem cells to preserve genetic resources has also opened up new ideas for the protection and utilization of livestock genetic resources.In this experiment,broiler adipose-derived stem cells and goose dermal mesenchymal stem cells were used as experimental materials to study the isolation,culture,biological characteristics and multi-directional differentiation potential of mesenchymal stem cells.The adipocyte differentiation process of livestock and poultry is differentiated from mesenchymal stem cells into preadipocytes and then differentiated into adipocytes.In this research,we analyzed the role of Wnt/β-catenin signaling pathway in the differentiation of adipose-derived stem cells to adipocytes from broilers,and obtained the following results:1.The broiler adipose-derived stem cells(ADSCs)were isolated by collagenase I and the morphology was long spindle-shaped.It is a typical mesenchymal cell morphology and cultured in vitro for 28 generations.The growth curves of P3,P15 and P25 ADSCs were tested for their ability to proliferate.They experienced latency,log phase,stationary phase and decay phase,showing a typical“S” shape growth.The proliferative capacity is gradually weakening.Similarly,the results of cell clone formation ability experiments show that ADSCs have self-renewal ability,but with the increase of generations,self-renewal ability will be weakened.The surface markers of the cells were detected by RT-PCR and immunofluorescence,and the positive rate of the surface markers was analyzed by flow cytometry,and the expression rate was above 98%.Under different induction conditions,ADSCs have multi-directional differentiation ability and differentiate into osteoblasts,adipocytes and chondrocytes under in vitro conditions.2.Goose dermal mesenchymal stem cells(DMSCs)were obtained by Dispase II and trypsin.The morphology was long spindle-shaped and cultured in vitro to 21 generations.The growth curves and cell clone formation ability of P3,P12 and P21 DMSCs were mapped.The experimental results showed that DMSCs had strong proliferative ability and self-renewal ability in low generation,and their ability would be weakened with the increase of generation.The surface markers of the cells were detected by RT-PCR and immunofluorescence,and the positive rate of surface markers was more than 99% by flow cytometry.Under different induction conditions,DMSCs differentiate into osteoblasts,adipocytes and chondrocytes in vitro.3.Using the Oil red O staining and extraction method to compare the adipogenic differentiation ability of ADSCs and DMSCs,the results showed that the adipogenic differentiation ability of ADSCs was greater than that of DMSCs;chicken embryo is a classic developmental biology model,it is easier to study in vitro.In addition,its genome sequencing has been completed,so the subsequent use of ADSCs as experimental materials to explore the role of Wnt/β-catenin signaling pathway in thedifferentiation of stem cells into adipocytes.4.Under the induction condition,the Wnt/β-catenin signaling pathway was activated by activator.The number of lipid droplets stained by oil red O was decreased,and the expression of adipogenic transcription factors C/EBPα and PPARγ were inhibited.Inhibition of Wnt/β-catenin signaling pathway,increased number of lipid droplets,increased expression of C/EBPα and PPARγ,promoted the potential of broiler ADSCs to differentiate into adipocytes,indicating that Wnt/β-catenin signaling pathway plays an inhibitory role in stem cells to fat.5.In summary,this experiment has successfully isolated and cultured broiler ADSCs and goose DMSCs.Both mesenchymal stem cells have self-renewal ability and multi-directional differentiation ability;by activating and inhibiting Wnt/β-catenin signaling pathway,This pathway was demonstrated to inhibit the differentiation of poultry adipose-derived stem cells into adipocytes.