猪脂肪源间充质干细胞诱导成脂分化及Krüppel样因子2的表达  

Differentiation of porcine adipose mesenchymal stem cells into adipocytes and the expression of Krüppel like factor 2

作　　者：刘京霞[1] 李方正[1] 朴英姬[1] 姜忠玲[1] 宋学雄[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109

出　　处：《解剖学报》2015年第5期616-622,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(31372392)

摘　　要：目的旨在阐明猪脂肪源间充质干细胞(AMSCs)在诱导成脂分化过程中Krüppel样因子2(KLF2)的表达模式。方法采用I型胶原酶消化法处理猪脂肪组织,经原代和传代培养分离、纯化和扩增AMSCs,用流式细胞仪检测AMSCs纯度,在倒置显微镜下用形态学和油红O染色法检测AMSCs的成脂分化,用实时定量PCR检测KLF2的表达模式,并与过氧化物酶体增殖物激活受体2(PPARγ2)的表达进行了比较。结果分离、纯化的AMSCs,其间充质干细胞表面抗原CD29、CD44和CD105表达量分别为91.7%、95.1%和95.6%,而造血干细胞表面抗原CD34表达量仅为3.39%;在诱导分化2d后,个别细胞质中出现小脂滴,且含脂滴的细胞数量和脂滴量随诱导时间的延长而增加,在诱导分化第18天成脂分化率达48.2%;在诱导分化第2天、第8天和第16天,KLF2的表达量分别为1.84±0.206、1.07±0.072和0.83±0.095,而PPARγ2 mRNA的表达量分别为3.06±0.542、13.22±0.5736和15.11±1.073。结论获得纯度较高的AMSCs,具有良好的成脂分化潜能,KLF2的表达量在成脂分化早期的前脂肪细胞阶段增加,成脂分化开始后逐步减少,KLF2作为脂肪分化负调控转录因子而发挥作用。Objective To investigate the gene expression changes of Kruppe-1 like factor （KLF）2 during the process of induced adipogenic differentiation from porcine adipose mesenchymal stem cells （AMSCs） in vitro and to reveal the foundation for regulating the differentiation of adipocyte. Methods The porcine AMSCs were isolated by type I collagen enzyme method and subcuhured. F3 culture cell surface antigen was detected by flow cytometry. The differentiated adipocytes were identified by morphology and oil red 0 staining; The expression of KLF2 mRNA and adipogenic marker genes proliferator-activated receptor γ（PPARγ）2 were measured by Real-time PCR. Results The AMSCs expressed the MSCs cellular markers, CD29, CD44 and CD105 with a marking rate a of 91.7% ,95.1% and 95.6% respectively,but did not express CD34 with a marking rate of 3.39%. Some adipocytes appeared when the AMSCs induced and differentiated for 2 days. When the induced differentiation time extended, the number of adipocytes increased. The adipogenic conversion rate reached 48.2% in the induction of 18 days. In the induction of 2,8 and 16 days, the expressions of KLF2 mRNA were 1.84 ±0. 206,1.07 ±0. 072 and 0. 83 ± 0. 095, and the expressions of PPARγ2 mRNA were 3.06± 0. 542,13.22 ± 0. 5736 and 15. 11 ± 1. 073. Conclusion This study successfully obtained AMSCs which showed good adipogenic differentiation potential. The expressions of KLF2 was increased in early adipogenic differentiation phase, and then gradually reduced. The results suggest that the KLF2 is the negative regulation of transcription factors.

关 键 词：脂肪间充质干细胞 表面鉴定 诱导 分化 Krüppel样因子2 实时定量PCR 猪 

分 类 号：Q291[生物学—细胞生物学]