猪肌肉干细胞在三维水凝胶中的分化研究  

Differentiation of Porcine Muscle Stem Cells in Three-Dimensional Hydrogels

作　　者：陈彧 朱浩哲 陈益春 刘政 丁希 郭赟 丁世杰 周光宏[1] CHEN Yu;ZHU HaoZhe;CHEN YiChun;LIU Zheng;DING Xi;GUO Yun;DING ShiJie;ZHOU GuangHong(College of Food Science and Technology,Nanjing Agricultural University/National Meat Quality and Safety Control Engineering Technology Research Center/Key Laboratory of Meat Processing and Quality Control,Ministry of Education,Nanjing 210095)

机构地区：[1]南京农业大学食品科技学院/国家肉品质量安全控制工程技术研究中心/教育部肉品加工与质量控制重点实验室,南京210095

出　　处：《中国农业科学》2022年第22期4500-4512,共13页Scientia Agricultura Sinica

基　　金：江苏省青年科学家自然科学基金(BK20200537);江苏省重点研发计划(现代农业)(BE2020302);国家重点研发计划(2021YFC2101400);国家自然科学基金青年基金(32101991);江苏省肉类生产与加工质量安全控制协同创新中心。

摘　　要：【目的】探究猪肌肉干细胞在三维水凝胶中的分化效果,为体外诱导肌肉干细胞分化成为肌肉组织提供方法指导。【方法】将一定数目的猪肌肉干细胞分别在二维和三维条件下诱导分化(二维条件指在培养皿中培养细胞,三维条件指在水凝胶中培养细胞),分别收取增殖阶段、预分化阶段、分化初期、分化成熟、分化末期的二维培养细胞的RNA和蛋白样品,以及分化7和14 d的三维培养细胞的RNA和蛋白样品。利用RT-qPCR技术检测细胞在两种条件下分化至不同阶段时成肌相关基因MYOG、CAV-3、MyHC-slow、MyHC-2a的表达水平;利用Western Blot技术检测细胞在两种条件下分化至不同阶段时MYOG、MyHC蛋白的表达水平;利用免疫荧光染色技术观察猪肌肉干细胞在二维和三维条件下融合形成的肌管;使用氨基酸自动分析仪检测分化14 d培养肌肉组织的氨基酸含量及组成。【结果】二维培养的猪肌肉干细胞在分化第3天时开始发生肌融合,在分化第7天形成成熟肌管,随后进入分化末期,肌管开始脱落。三维培养的猪肌肉干细胞在分化第7天时还未完全伸展,细胞的MYOG和CAV-3表达水平低;分化第14天时水凝胶内已形成多核肌管,MYOG和CAV-3表达达到二维分化水平。三维分化有利于终末分化基因MyHC-slow和MyHC-2a的表达,分化14 d时MyHC-slow的表达量是二维分化7 d的12倍,MyHC-2a的表达量是二维分化7 d的4倍,但是MyHC蛋白的表达量仅为二维分化7 d时的1/6。氨基酸分析结果表明体外培养肌肉组织中17种水解氨基酸含量均低于猪肉,且必需氨基酸在总氨基酸的占比也低于猪肉,但是呈味氨基酸的占比相较猪肉更高。【结论】猪肌肉干细胞可以在三维胶原水凝胶中分化形成肌管,且三维条件有利于成肌分化相关基因表达,但要实现MyHC蛋白的高表达还需进一步研究,按此方法体外培养的肌肉组织有较高的呈味氨基酸含�【Objective】The objective of this study was to explore the differentiation effect of porcine muscle stem cells in three-dimensional hydrogels,and to provide a guidance for inducing muscle stem cells to differentiate into muscle tissue in vitro.【Method】Some porcine muscle stem cells were respectively induced to differentiate under the conditions of 2D and 3D(2D condition means culturing cells in culture dishes;3D condition means culturing cells in hydrogels).The RNA and protein samples of porcine muscle stem cells cultured in 2D were collected at proliferation,pre-differentiation,early differentiation,mature differentiation,and late differentiation,respectively,and those in 3D were also collected at day 7 and day 14 of differentiation,respectively.Then,RT-qPCR was used to compare the expression levels of the myogenic-related genes,including the genes of MYOG,CAV-3,MyHC-slow,and MyHC-2a,under 2D and 3D differentiation conditions.Correspondingly,the Western Blot was used to detect the expression levels of MyHC protein and MYOG protein in the two conditions.Moreover,the immunofluorescence staining was used to observe the myotubes formed in cell culture dishes and hydrogels.Further,the amino acid content and composition of the cultured muscle tissue were analyzed by an amino acid automatic analyzer at day 14 of differentiation.【Result】The porcine muscle stem cells started to fuse to form myotubes at day 3 of differentiation in 2D.The myotubes formed in 2D matured at day 7 and divorced from culture dish afterwards.The porcine muscle stem cells were still globe and had low expression of MYOG and CAV-3 at day 7 of differentiation in 3D.Multinucleated myotubes formed at day 14 and the expression of MYOG and CAV-3 reached levels of 2D differentiation.The cells in hydrogels had higher expression of terminal differentiation genes MyHC-slow and MyHC-2a than the cells in culture dishes.The expression of MyHC-slow was 12 times that at day 7 in 2D and the expression of MyHC-2a was 4 times that at day7 in 2D,but the ex

关 键 词：猪肌肉干细胞 分化 水凝胶 肌管 细胞培养肉 

分 类 号：TS219[轻工技术与工程—粮食、油脂及植物蛋白工程]