藏绵羊KLF7基因表达特征分析及其过表达对前脂肪细胞增殖分化的影响

【目的】分析藏绵羊Krüppel样因子7(Krüppel-like factor 7,KLF7)基因表达特征,研究过表达该基因对前脂肪细胞增殖及分化的影响。【方法】从藏绵羊脂肪组织中分离前脂肪细胞进行培养及成脂诱导,应用实时荧光定量PCR技术检测KLF7基因在藏绵羊7个组织(大脑、皮下脂肪、肾脏、背最长肌、瘤胃、睾丸和回肠)和前脂肪细胞不同分化阶段(第0、2、4和8天)的mRNA相对表达水平;应用RT-PCR方法从藏绵羊脂肪组织中扩增KLF7基因CDS区序列,并将其连接到pcDNA3.1(+)真核表达载体获得pcDNA3.1-KLF7过表达质粒,转染前脂肪细胞;应用实时荧光定量PCR方法检测脂肪细胞增殖及分化标志基因mRNA表达水平;采用EdU和CCK-8方法分别检测过表达KLF7基因对EdU阳性细胞数和细胞活力的影响;采用油红O染色检测过表达KLF7基因后脂肪细胞脂滴生成量。【结果】KLF7基因在藏绵羊7个组织中均有表达,其中在大脑中的表达量最高,其次为皮下脂肪和肾脏,均显著高于其他组织(P<0.05);诱导分化第2、4和8天脂肪细胞mRNA表达量均显著高于分化前(P＜0.05),且分化第2天表达量最高;pcDNA3.1-KLF7过表达质粒转染前脂肪细胞2 d后显著或极显著抑制增殖标志基因CDK4、CyclinB1和CyclinD1的表达水平(P＜0.05;P＜0.01),极显著降低细胞活力及EdU阳性细胞数量(P＜0.01);pcDNA3.1-KLF7过表达质粒转染前脂肪细胞,诱导分化8 d后,脂肪细胞分化标志基因PPARγ、Glut4和ELOVL6的mRNA相对表达水平显著或极显著下调(P＜0.05;P＜0.01),且脂质沉积极显著减少(P＜0.01),表明过表达KLF7基因可抑制藏绵羊前脂肪细胞增殖及分化。【结论】KLF7基因在藏绵羊多个组织中广泛表达,且大脑、皮下脂肪、肾脏中表达量较高;诱导分化后脂肪细胞表达量显著高于分化前,且分化第2天表达量最高;过表达KLF7基因可抑制藏绵羊前脂肪细胞的增殖及分化。试验结果为阐明藏绵羊脂肪沉积的分子调控机制提供了基础数据。

Expression Characterization Analysis of KLF7 Gene and Effects of Its Overexpression on Proliferation and Differentiation of Preadipocytes in Tibetan Sheep

【Objective】 The objective of this study was to analyze the expression profile of Krüppel-like factor 7 (KLF7) gene,and explore the effect of its overexpression on proliferation and differentiation of preadipocytes in Tibetan sheep.【Method】 Preadipocytes that isolated from adipose tissue in Tibetan sheep were cultured and induced differentiation.The mRNA relative expression of KLF7 gene in 7 tissues (brain,subcutaneous fat,kidney,longissimus dorsi muscle,rumen,testis and ileum) and preadipocytes at different differentiation stages (0,2nd,4th and 8th days) in Tibetan sheep were analyzed by Real-time quantitative PCR.The CDS sequence of KLF7 gene was amplified from adipose tissue by RT-PCR,and ligated into the pcDNA3.1(+) eukaryotic expression vector to conduct pcDNA3.1-KLF7 overexpression plasmid.The pcDNA3.1-KLF7 overexpression plasmid was transfected into preadipocytes,and the mRNA expression of adipocyte proliferation and differentiation marker genes were detected by Real-time quantitative PCR.The EdU-positive cell,cell viability and production of lipid droplets in KLF7 gene overexpressing preadipocytes were detected by EdU,CCK-8 and Oil red O staining methods,respectively.【Result】 KLF7 gene was expressed in 7 tissues of Tibetan sheep,and the highest expression was found in brain,followed by subcutaneous fat and kidney,which were significantly higher than other tissues (P<0.05).The mRNA expression of adipocytes which was induced at the 2nd,4th and 8th days were significantly higher than that of undifferentiated cell (P<0.05),and the highest expression was identified in adipocytes at the 2nd day after differentiation.In KLF7 gene overexpressing preadipocytes at the 2nd day,the expression of CDK4,CyclinB1 and CyclinD1 genes were significantly or extremely significantly inhibited (P<0.05 or P<0.01),and the cell viability and the number of EdU positive cell were also extremely significantly decreased (P<0.01).In KLF7 gene overexpressing preadipocytes at the 8th day,the expression of PPARγ,Glut4 and ELOVL6 genes were significantly or extremely significantly down-regulated (P<0.05 or P<0.01),and the lipid deposition was extremely significantly reduced (P<0.01). The results indicated that KLF7 gene overexpression inhibited the proliferation and differentiation of preadipocytes in Tibetan sheep.【Conclusion】 The widely expressed of KLF7 gene was found in tissues of Tibetan sheep,and there was a higher expression in brain,subcutaneous fat and kidney.The expression of differentiated adipocyte was significantly higher than that of undifferentiated,and the highest expression was observed at the 2nd day after differentiation.KLF7 gene overexpression could inhibit the proliferation and differentiation of preadipocytes in Tibetan sheep,which provided basic data for elucidating the molecular regulation mechanism of fat deposition in Tibetan sheep.