北京黑猪脂肪前体细胞的分离培养及成脂诱导分化研究  

Isolation,Culture and Adipogenic Differentiation of Beijing Black Pig Preadipocytes

作　　者：梁小娟 李雨爽 李莹莹[1] 王守伟[1] LIANG Xiaojuan;LI Yushuang;LI Yingying;WANG Shouwei(China Meat Food Research Center,Beijing Institute of Food Science,Beijing 100068,China)

机构地区：[1]中国肉类食品综合研究中心,北京食品科学研究院,北京100068

出　　处：《畜牧兽医学报》2024年第7期2877-2889,共13页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划项目(2021YFC2101404);中国工程院战略研究与咨询项目(2023-XZ-79)。

摘　　要：旨在建立北京黑猪脂肪前体细胞的分离培养及成脂诱导分化培养体系,为研究猪脂肪沉积的分子机制提供试验材料,为生物培育肉的制备提供技术指导。本研究采集1头7日龄健康的北京黑猪公猪的颈部皮下脂肪组织,使用机械法联合I型胶原酶消化法分离脂肪前体细胞。对获得的脂肪前体细胞进行形态观察、生长曲线绘制、免疫荧光鉴定、成脂诱导分化、油红O染色、BODIPY染色、甘油三酯含量测定及使用实时荧光定量PCR和Western blot技术检测成脂分化相关基因的表达。结果表明,分离的北京黑猪脂肪前体细胞贴壁后呈长梭形,细胞生长曲线呈S型。免疫荧光结果表明,PREF1表达为阳性,表明分离的细胞的确为脂肪前体细胞。在相同的诱导试剂组合条件下,使用10%FBS比2%FBS成脂诱导分化效果更好。在相同血清浓度的情况下,鸡尾酒法与油酸钠、罗格列酮共同联用的细胞分化效果较好。分化8 d的细胞中出现大量的脂滴,脂滴经油红O染色呈红色,经BODIPY染色呈绿色。且分化8 d的细胞中甘油三酯的含量显著升高(P<0.001)。qPCR结果显示,PPARγ、CEBPα、ACCα、LPL、SCD和FABP4在分化2 d时表达量均显著升高(P<0.05),4~6 d达到高峰,8 d时表达量降低。Western blot结果表明,PPARγ、CEBPα、FABP4、APN在细胞分化过程中表达逐渐上调。PREF1在细胞分化过程中表达逐渐下调。在三维培养情况下,使用10%FBS,鸡尾酒法与油酸钠、罗格列酮共同联用也能使脂肪前体细胞分化为成熟的脂肪细胞,分化8 d的细胞中脂滴经BODIPY和油红O染色后分别呈绿色和红色,且PPARγ、CEBPα、ACCα、LPL、SCD和FABP4在分化8 d的细胞中的表达量显著上调(P<0.001)。综上所述,本研究成功建立了北京黑猪脂肪前体细胞的分离与培养体系,并筛选出适合二维和三维培养的成脂诱导分化方法,为进一步研究猪脂肪沉积的分子机制和改善肉品质奠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 FABP 4 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 differ

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 

分 类 号：S828.1[农业科学—畜牧学]