KLF2对山羊肌内前体脂肪细胞分化的影响

旨在克隆山羊KLF2基因的锌指结构域序列，明确其在组织及细胞表达模式，最终阐明其对山羊肌内前体脂肪细胞分化的影响及可能发挥作用的方式。本研究以5只1周岁左右的健康简州大耳羊为试验动物，利用RT-PCR、细胞培养、实时荧光定量PCR（real-time quantitative PCR，qPCR）、RNA干扰等方法克隆KLF2基因锌指结构，明确KLF2基因的时空表达特性并阐明其对肌内前体脂肪细胞分化的影响。结果显示，本研究获得的山羊KLF2锌指结构域为454 bp（KU041748.1），且该结构域与绵羊、牛、小鼠的同源性为100%。KLF2在山羊各组织中存在广泛表达，且在肺和腹间脂肪中存在较高水平表达（P<0.05）。KLF2在山羊肌内前体脂肪细胞分化过程中的表达呈先下降又上升然后又下降趋势，且在成脂诱导分化12 h呈最低表达水平，显著低于在未分化的前体脂肪细胞中的表达水平（P<0.05）。油红O染色结果显示，干扰KLF2基因可明显促进山羊肌内脂肪细胞的脂滴积聚；并且PPARγ和C/EBPβ表达量伴随着这个过程分别极显著上调48.5%（P<0.01）和显著上调43.6%（P<0.05）；同时该家族中的KLF1、KLF13、KLF14、KLF15和KLF16表达水平极显著升高（P<0.01），KLF4显著下降（P<0.05），KLF3、KLF6、KLF8、KLF9及KLF11极显著下降（P<0.01）。山羊KLF2的锌指结构域与其他物种同源性高度保守，且其在山羊肺和腹间脂肪中表达量较高（P<0.05）。同时KLF2是山羊肌内脂肪细胞分化的负调控因子，并且可能通过PPARγ和C/EBPβ基因来发挥作用，且与其他KLFs基因存在等级调控现象。

The Effect of KLF2 on the Differentiation of Goat Intramuscular Preadipocyte

The aim of this study was to clone the sequence of zinc finger domain of goat KLF2, to clarify the tissue and cell expression profiles of KLF2, and finally to elucidate the effect of interfering KLF2 gene on the goat intramuscular preadipocytes differentiation and the possible ways of action. In this study, 5 healthy, 1-year-old Jianzhou Big Ear goats were selected as experiment animals. By using RT-PCR, cell culture, real-time quantitative PCR (qPCR), RNA interference and other methods, the sequence of the zinc finger domain of goat KLF2 gene was cloned, the tissue and cell temporal expression of KLF2 gene was detected and futher the effect of KLF2 gene on goat intramuscular preadipocytes differentiation was elucidated. The results showed that the length of goat KLF2 zinc finger domain was 454 bp (KU041748.1), and the domain had 100% homology with sheep, cattle and mice. KLF2 was widely expressed in goat tissues with the higher expression level in the lung and abdominal fat (P<0.05). The expression of KLF2 decreased first, then increased, and decreased again during the intramuscular preadipocytes differentiation, the expression level reached to the lowest after differentiation 12 h, which was significantly lower than that in undifferentiated preadipocytes (P<0.05). Oil red O staining result showed that interfering KLF2 significantly promoted the accumulation of lipid droplets in adipocytes; and the expression of PPARγ and C/EBPβ were extremely significantly up-regulated by 48.5% (P<0.01) and significantly up-regulated by 43.6%(P<0.05); At the same time, the expression level of KLF1, KLF13, KLF14, KLF15 and KLF16 were extremely markedly increased (P<0.01), the expression level of KLF4 was significantly decreased (P<0.05), and the expression level of KLF3, KLF6, KLF8, KLF9 and KLF11 were extremely significantly decreased (P<0.01). The zinc finger domain of goat KLF2 was highly conserved with other species, and KLF2 was highly expressed in lung and abdominal fat of goat(P<0.05). Meanwhile, KLF2 was a negative regulator for goat intramuscular preadipocytes differentiation, and might play an important role through targeting PPARγ and C/EBPβ genes, what's more it could hierarchically regulated other KLFs members.