ChREBP基因siRNA表达质粒构建及对原代 培养猪脂肪细胞生脂的影响

【目的】构建ChREBP基因siRNA表达质粒，干扰ChREBP在原代培养猪脂肪细胞的表达，研究其在葡萄糖诱导脂肪细胞生脂中的作用。【方法】合成4对靶向ChREBP基因的siRNA寡核苷酸，分别连接于pcDNA™6.2-GW/EmGFP载体构建siRNA表达质粒，测序验证后，采用脂质体介导法转染从3日龄仔猪皮下脂肪组织分离培养的脂肪细胞，荧光定量RT-PCR检测ChREBP基因沉默效率；以葡萄糖浓度为0—20 mmol·L-1的培养液培养转染细胞48 h，测定生脂及生脂基因表达变化。【结果】筛选出了1个转染效果好、ChREBP基因沉默效率达85%的siRNA表达质粒，转染原代培养猪脂肪细胞后，细胞生脂水平及生脂基因ACC1和FAS mRNA表达比阴性对照表达质粒转染细胞和未转染细胞显著降低（P＜0.05），且生脂水平不受葡萄糖水平的影响（P＞0.05）。【结论】构建的siRNA表达质粒能有效干扰猪脂肪细胞ChREBP表达，葡萄糖通过转录因子ChREBP调控猪脂肪细胞生脂及生脂基因表达。

Construction of siRNA Expression Plasmids Targeting ChREBP Gene and Its Effect on Lipogenesis in Primary Cultured Porcine Adipocytes

【Objective】To study the role of ChREBP in the regulation of lipogenesis by glucose in porcine adipocytes, siRNA expression plasmids targeting ChREBP gene were constructed and transfectd into primary cultured porcine adipocytes to silence its expression.【Method】The 4 pairs of oligonucleotides targeting ChREBP gene were synthesized and respectively inserted into linearized pcDNA™6.2-GW/EmGFP vector to construct siRNA expression plasmids. The plasmids were identified by sequencing, and then transfected into the adipocytes from subcutaneous adipose tissue obtained from 3-day-old piglets. Silencing efficiency was determined by fluorescence real-time PCR. The transfected adipocytes were exposed to 0-20 mmol·L-1 glucose for 48 h and lipogenesis and expression of lipogenic genes were measured.【Result】One siRNA expression plasmid with high transfection efficiency and 85% silencing efficiency for ChREBP gene was successfully constructed. Lipogenesis and expression of ACC1 and FAS mRNA were significantly decreased in the transfected adipocytes compared with in negative-siRNA plasmid transfected and non-transfected adipocytes (P＜0.05), and the glucose levels had no effect on this decrease of lipogenesis (P＞0.05).【Conclusion】The constructed siRNA expression plasmid could effectively interfere ChREBP expression in porcine adipocytes, and ChREBP mediates the regulation of lipogenesis by glucose in the cells.