关键词: 北京黑猪, 脂肪前体细胞, 成脂诱导分化, 成熟脂肪细胞, 三维培养, 细胞培育肉

Abstract:

The study aimed to establish the system for the isolation, culture and adipogenic differentiation of Beijing Black pig preadipocytes, to provide experimental materials for studying the molecular mechanism of fat deposition in pigs, and technical guidance for the preparation of cultured meat. The neck subcutaneous fat of a healthy 7-day-old male Beijing Black pig was collected, and preadipocytes were isolated using mechanical method combined with type Ⅰ collagenase digestion. The obtained preadipocytes were subjected to morphological observation, growth curve drawing, immunofluorescence identification, adipogenic differentiation, Oil Red O staining, BODIPY staining, triacylglycerol content determination, and detection of the expression of adipogenic differentiation-related genes using real-time quantitative PCR and Western blot techniques. The results showed that the isolated Beijing Black pig preadipocytes were spindle-shaped after adherence and the cell growth curve showed S type. The immunofluorescence results showed that PREF1 was positive, indicating that the isolated cells were indeed preadipocytes. Under the same combination of induction reagents, 10% FBS exhibited a better adipogenic differentiation effect than 2% FBS. Under the same serum concentration, the combined use of the cocktail with sodium oleate and rosiglitazone displayed a better differentiation effect. A large number of lipid droplets appeared in cells differentiated for 8 days, and the lipid droplets became red after Oil Red O staining and green after BODIPY staining. Moreover, the triacylglycerol content in cells differentiated for 8 days was significantly increased (P < 0.001). The qPCR results showed that the expression of PPARγ, CEBPα, ACCα, LPL, SCD, and FABP4 were all significantly increased after 2 days of differentiation (P < 0.05), reached a peak at 4-6 days, and decreased at 8 days. The Western blot results showed that the expression of PPARγ, CEBPα, FABP4, and APN gradually increased during cell differentiation. Expression of PREF1 gradually decreased during cell differentiation. Under three-dimensional culture condition, the combination use of 10% FBS, the cocktail, sodium oleate and rosiglitazone could also induce preadipocytes into mature adipocytes. The lipid droplets in cells differentiated for 8 days became green and red after BODIPY and Oil Red O staining, respectively, and the expression levels of PPARγ, CEBPα, ACCα, LPL, SCD, and FABP4 in cells differentiated for 8 days increased significantly compared with the control group (P < 0.001). In summary, this study successfully established an isolation and culture system for Beijing Black pig preadipocytes and screened out adipogenic differentiation methods suitable for two-dimensional and three-dimensional culture, laying a foundation for further studying the molecular mechanism of fat deposition in pigs and improving meat quality, and also providing technical guidance for the preparation of cultured meat.

Key words: Beijing Black pig, preadipocytes, adipogenic differentiation, mature adipocytes, three-dimensional culture, cultured meat