Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (8): 3482-3492.doi: 10.11843/j.issn.0366-6964.2024.08.021

• Animal Genetics and Breeding • Previous Articles     Next Articles

Isolation, Culture and Adipogenic Differentiation of Pigeon Preadipocytes

Xiaojuan LIANG(), Yushuang LI, Zhou FU, Duo TANG, Yingying LI, Shouwei WANG*()   

  1. China Meat Food Research Center, Beijing Institute of Food Science, Beijing 100068, China
  • Received:2024-01-29 Online:2024-08-23 Published:2024-08-28
  • Contact: Shouwei WANG E-mail:xyskylxj8907@163.com;cmrcwsw@126.com

Abstract:

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 type I 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 preadipocytes 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 induction (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 remained 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 Western 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 prolongation 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 induction 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.

Key words: pigeon, preadipocytes, adipogenic differentiation, mature adipocytes, cell cultured meat

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