Isolation,Culture and Adipogenic Differentiation of Pigeon Preadipocytes
The aim of this study was to establish methods for the isolation,culture,identification and adipogenic differentiation of pigeon preadipocytes in vitro.Subcutaneous adipose tissue was collected from 3 healthy 1-day-old silver king pigeons.Pigeon preadipocytes were isolated by typeⅠ collagenase digestion.The isolation method was improved based on the conventional isolation method of preadipocytes.Primary and passage cultures were performed,and cell morphology was observed.The preadipocytes were identified by immunofluorescence staining using specific marker DLK1.Adipogenic differentiation was induced by adding insulin and sodium oleate to the culture medium.The distribution of lipid droplets in the cells was indicated by staining with BODIPY493/503.The triglyceride content in the cells were measured by the triglyceride assay kit.Quantitative real-time PCR(qPCR)and Western blot were used to detect the expression of adipogenic-related genes during preadipocytes differentiation.The results showed that the pigeon preadipocytes displayed spindle-shape.The modified isolation method yielded more preadipocytes compared to the conventional isolation method.Compared with 37 ℃,the number of preadipo-cytes was significantly increased at 41 ℃(P＜0.001).Immunofluorescence staining confirmed positive DLK1 expression,indicating the obtained cells were indeed preadipocytes.The results of BODIPY493/503 staining revealed abundant lipid droplets in cells after 6 days of differentiation.The relative triglyceride content in the cells was significantly increased with differentiation time(P＜0.01).The qPCR data indicated that the expression of PPARγ,SCD,DGAT2,PLIN2,FASN,AFABP,and LPL genes was significantly upregulated after 2 days of adipogenic induc-tion(P＜0.05),and continued to increase with longer differentiation time.The expression of SREBF1 gene was significantly up-regulated after 2 days of differentiation(P＜0.05),and re-mained unchanged thereafter.The expression of ACACA gene was significantly increased after 2 days of differentiation(P＜0.05),and reached its peak after 4 days of differentiation.The West-ern blot results showed that the relative expression of PPARγ,LPL and PLIN2 were significantly up-regulated after 2 days of differentiation(P＜0.05),and then further increased with the pro-longation of differentiation time.In conclusion,this study successfully modified the conventional isolation method to isolate pigeon preadipocytes and screened the optimal culture temperature.The obtained pigeon preadipocytes were efficiently differentiated into mature adipocytes after in-duction with insulin and sodium oleate.This study provides a good cell model for investigating the molecular regulation mechanism of fat metabolism of pigeon,and also provided seed cells and technical guidance for the preparation of pigeon cell cultured meat.
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