为研究转化生长因子β1 (TGF-β1)对脂多糖(LPS)刺激小鼠乳腺上皮细胞(MECs)分泌炎症因子的影响,本实验利用siRNA转染技术干扰MECs TGF-β1基因,采用荧光定量RT-PCR方法筛选最佳转染条件,并检测其干扰后LPS介导其下游信号smad3 mRNA的表达量,采用ELISA法检测其下游信号smad3蛋白含量和LPS介导细胞分泌前炎症细胞因子TNF-α和IL-6的含量.结果显示,选用50 nM浓度的TGF-β1-mus-1212片段转染组能够极显著抑制TGF-1的mRNA表达(p＜0.01);TGF-β1被干扰后,LPS刺激其下游smad3 mRNA的表达量与对照组相比呈显著性降低(p＜0.05),smad3蛋白含量与对照组相比均极显著性降低(p＜0.01);细胞分泌TNF-α的量与对照组相比显著降低(p＜0.05),分泌IL-6的量各组无变化(p＞0.05).本实验结果表明干扰TGF-β1能降低LPS诱导乳腺上皮细胞TNF-α的分泌量,因此TGF-β1可作为靶基因深入研究乳腺上皮细胞的免疫功能,本研究为乳腺炎症、肿瘤等乳腺相关疾病的治疗提供新的思路及实验数据,为新药的开发和应用提供理论依据.%To examine the effect of silencing transforming growth factor beta-1 (TGF-β1) on the inflammatory responses in mouse mammary epithelial cells (MECs) with lipopolysaccharide(LPS)-induced secreted inflammatory factor,in this study,siRNA transfection technique was used to interfere with MECs TGF-β1,TGF-β1-knockdown efficiency and the smad3 mRNA was determined by real-time quantitative RT-PCR.The levels of smad3 protein,IL-6 and TNF-α in the mouse MECs were measured by ELISA.The results showed that 1) 50 nM TGF-β1-mus-1212 could significantly inhibit TGF-1 mRNA expression (p＜0.01).2) When the mouse MECs were transfected with siRNA of TGF-β1 and stimulated with LPS,the expression of smad3 mRNA was reduced significantly (p＜0.05),and the expression of smad3 protein was prominently reduced (p＜0.05).3) The secretion of TNF-α was prominently reduced (p＜0.05),but there was no change (p＞0.05) on the IL-6 in each group.This study suggested that silencing of TGF-β1 could alter the secretion of TNF-in LPS-stimulated MECs,thus TGF-β1 could be used as a target gene to further explore the immune function of mouse MECs.Our study could provide insight and experimental data for treatment of mammary gland inflammation,tumor and other related mammary gland disease,and provide a experimental basis for the development and application of new drugs.
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