Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化

硫氧还蛋白互作蛋白（thioredoxin interacting protein, Txnip）是一种氧化还原调节蛋白质，与硫氧还蛋白结合并抑制其活性，调节细胞氧化还原状态，影响细胞多种生理过程，然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸，分别连接于重组慢病毒载体pGLV3/H1/GFP+Puro构建siRNA表达质粒。测序验证后，与包装质粒共转染293T细胞，获得滴度1×108 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞，转染率均达80%以上，其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后，每隔1 d检测细胞成脂分化及相关基因表达。结果发现，其分化比阴性对照质粒转染或未转染细胞显著增强（P<0.05），PPARγ和FAS mRNA表达水平显著提高（P<0.05）。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达，Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示，Txnip可能是猪脂肪细胞分化的抑制因子。

siRNA of Txnip Gene Constructed in Lentiviral Plasmids Promotes Porcine Preadipocytes Differentiation

Thioredoxin interacting protein (Txnip), a redox regulatory protein, binds to thioredoxin (Trx) and inhibits its activity. Txnip regulates the redox state in various physiological processes of cells, but its role in differentiation of porcine preadipocyte is not clear. In this study, 3 pairs of shRNA oligonucleotides targeting porcine Txnip gene were designed, synthesized and inserted to recombinant lentivirus vector pGLV3/H1/ GFP+ Puro to construct siRNA expression plasmids. After sequencing verification, the recombinant shRNA expression lentiviruses were co-transfected with packaging plasmids into 293T cells, and the packaged lentiviral interference plasmids with a titer of 1×108 pfu/mL were obtained. When the primary cultured porcine preadipocytes were transfected with these packaged plasmids, the transfection efficiencies were over 80%, with 75% silencing efficiency for Txnip gene in the Txnip-shRNA-2 transfected cells (P<0.01). After porcine preadipocytes transfected with Txnip-shRNA-2 were induced by adipogenic differentiation medium, the adipogenic differentiation of the cells was detected every other day. The results showed that the differentiation of the cells transfected with Txnip-shRNA-2 was significantly promoted (P<0.05) and expression level of PPARγ and FAS mRNA increased significantly (P<0.05) as compared with that of the cells transfected with/without negative control virus. The constructed siRNA lentiviral expression plasmid could effectively interfere with the Txnip expression of porcine preadipocytes, and Txnip expression-silencing stimulated differentiation of the cells through up-regulating PPARγ expression. It was speculated that Txnip could be an inhibitor of porcine adipocyte differentiation.