鸽脂肪前体细胞的分离培养及成脂诱导分化研究

doi:10.11843/j.issn.0366-6964.2024.08.021

Abstract

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

CLC Number:

S836.2

Xiaojuan LIANG, Yushuang LI, Zhou FU, Duo TANG, Yingying LI, Shouwei WANG. Isolation, Culture and Adipogenic Differentiation of Pigeon Preadipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3482-3492.

Figures/Tables 7

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References 45

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基因Gene	上游引物序列(5′→3′) Forward primer sequence	下游引物序列(5′→3′) Reverse primer sequence
PPARγ	CAAAGCTCACGAGGACCCTT	GCGTTATGCGACATCCCAAC
SREBF1	CCGTCAAGACAGATGCTGGT	TAGCGTTTCTCGATGGCGTT
ACACA	TGGGGTCTGAGGAGATGGAA	CTTCCCCCGGCTCTGATAAC
SCD	GGCTGGCGCTTCAACTTAAC	AAGCAGGAACTCAGCCACTC
LPL	AGAAGGGTCTTTGCCTGAGC	GGCATCTGAGCACGAGTCTT
AFABP	CCAGGAAAATGGCTGGTGTG	TCTTTCCCATCCCACTTCTGC
DGAT2	TGAGTCCCTGAATTGCACCC	TCCCCGAAGGAGTAGACAGG
PLIN2	CTGCCTCACACTGGTCTCAA	GGGTAAAAGGGACCTACCAGC
FASN	GAAGCTCCAAGCCAGTTTGC	CAGAGGCTTCACCACACCAT
18S	GTAACCCGTTGAACCCCATT	CCATCCAATCGGTAGTAGCG

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Isolation, Culture and Adipogenic Differentiation of Pigeon Preadipocytes

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