为分析鸡PPARγ基因的组织表达特性及其在脂肪细胞增殖和分化过程中的功能, 文章以东北农业大学高、低腹脂双向选择品系肉鸡为实验材料, 利用Western blotting方法, 检测PPARγ基因的组织表达特性及其在高、低脂系肉鸡腹部脂肪组织间的表达差异; 采用RNAi技术, 在鸡原代脂肪细胞中抑制PPARγ基因的表达后, 通过MTT和油红O提取比色的方法, 研究鸡PPARγ基因对脂肪细胞增殖和分化的调控作用; 利用Real-time PCR和Western blotting技术, 分析PPARγ基因表达下调后, 其他脂肪细胞分化转录因子以及与脂肪细胞分化相关的重要基因的表达变化情况。结果表明, PPARγ基因在7周龄高脂系肉鸡腹部脂肪组织、肌胃、脾脏、肾脏组织中表达量较高, 在心脏中表达量较低, 在肝脏、胸肌、腿肌、十二指肠中未检测到表达信号; 与高脂系相比, PPARγ基因在5和7周龄低脂系肉鸡腹部脂肪组织中的表达量较低(P<0.05); PPARγ基因的表达量下降后, 鸡脂肪细胞的增殖能力增强, 分化能力减弱; 同时, C/EBPα、SREBP1、A-FABP、Perilipin1、LPL、IGFBP-2基因的表达量均下降(P<0.05)。由此可见, PPARγ基因的表达可能与肉鸡腹部脂肪的沉积有一定的关系, 该基因可能是调控鸡脂肪细胞增殖与分化的关键因子。
To characterize the chicken PPARγ gene expression and its impact on chicken adipocyte proliferation and differentiation, western blotting approach was conducted to investigate the expression of PPARγ in various chicken tissues and the difference of expression level in abdominal adipose tissues between the NEAU broiler lines divergently selected for abdominal fat content. The expression of PPARγ gene was suppressed in chicken adipocytes using RNAi technology, and the roles of PPARγ gene in the adipocytes proliferation and differentiation were investigated by MTT assay and Oil Red O staining extraction assay, respectively. After PPARγ gene was downregulated, the expression level of other transcript factors and marker genes related to the adipocyte differentiation was detected by Real-time PCR and Western blotting analyses. The results showed that PPARγ highly expressed in abdominal adipose tissue, gizzard, spleen, kidney, lowly expressed in heart, and not expressed in liver, breast muscle, leg muscle, and duodenum. Meanwhile, PPARγ expressed much higher in fat birds than in lean ones in abdominal adipose tissue at 5 and 7 weeks of age (P<0.05). RNAi analysis showed that knockdown of PPARγ gene increased chicken adipocyte proliferation and decreased cell differentiation and significantly decreased the expression levels of C/EBPα, SREBP1, A-FABP, Perilipin1, LPL, and IGFBP-2 (P<0.05). In summary, PPARγ gene may be related to the broiler abdominal fat deposition, and be probably a key regulator of chicken adipocyte proliferation and differentiation.
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