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

摘要/Abstract

摘要：

旨在建立鸽脂肪前体细胞的体外分离、培养、鉴定和成脂诱导分化的方法。本研究采集3只健康的1日龄银王鸽皮下脂肪组织，利用I型胶原酶进行消化以分离脂肪前体细胞，在脂肪前体细胞常规分离法基础上进行分离方法改良，对获得的脂肪前体细胞进行原代和传代培养，并观察细胞形态，通过免疫荧光鉴定特异性标记DLK1，确认脂肪前体细胞。通过在培养基中添加胰岛素和油酸钠进行成脂诱导分化，使用BODIPY493/503染色观察细胞中的脂滴分布情况，通过甘油三酯测定试剂盒检测细胞中甘油三酯的含量，采用实时荧光定量PCR和Western blot技术检测脂肪前体细胞成脂分化过程中分化相关基因的表达。研究结果表明，鸽脂肪前体细胞呈梭形，改良分离法比常规分离法能够获得更多的脂肪前体细胞；与37 ℃相比，在41 ℃培养温度下，获得的脂肪前体细胞数目显著增加(P < 0.001)。免疫荧光结果表明，DLK1表达为阳性，说明获得的是脂肪前体细胞。BODIPY493/503染色结果显示，分化6 d的细胞中产生大量的脂滴。且随着分化时间的增加，细胞中甘油三酯的相对含量也显著增加(P < 0.01)。qPCR结果显示，在添加诱导剂2 d时，PPARγ、SCD、DGAT2、PLIN2、FASN、AFABP、LPL基因的表达量显著上调(P < 0.05)，之后随分化时间的增加表达逐渐上调；SREBF1基因在分化2 d时表达量显著上调(P < 0.05)，之后表达量不变；ACACA基因在分化2 d时，表达量显著增加(P < 0.05)，在分化4 d时表达量达到高峰。Western blot结果表明，在分化2 d时，PPARγ、LPL和PLIN2的表达量显著上调(P < 0.05)，之后随着分化时间的延长，表达量进一步升高。综上所述，本研究改良了脂肪前体细胞常规分离法，成功分离获得鸽脂肪前体细胞，且筛选出最适培养温度。获得的鸽脂肪前体细胞经胰岛素和油酸钠诱导后能高效地分化为成熟的脂肪细胞。本研究为鸽脂肪代谢分子调控机制的研究提供了良好的细胞模型，同时也为鸽生物培育肉的制备提供种子细胞和技术指导。

关键词: 鸽, 脂肪前体细胞, 成脂分化, 成熟脂肪细胞, 细胞培育肉

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

中图分类号:

S836.2

梁小娟, 李雨爽, 付周, 唐铎, 李莹莹, 王守伟. 鸽脂肪前体细胞的分离培养及成脂诱导分化研究[J]. 畜牧兽医学报, 2024, 55(8): 3482-3492.

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.

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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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