| 摘要: |
| 为探究Ilk对LPS诱导小鼠乳腺上皮细胞(MECs)分泌炎性因子IL-8、TNF-α的影响，采用siRNA技术筛选最佳转染条件，qPCR法和ELISA法检测siRNA转染后下游蛋白Akt1的表达来评价Ilk沉默，ELISA法检测Ilk沉默后对小鼠MECs分泌炎性因子IL-8、TNF-α的影响。结果显示：选用Ilk-mus-595片段的20 nM转染组可极显著抑制Ilk的mRNA表达(P＜0.01)，转染效率最好，达84.83%；Ilk转染后可显著减少LPS介导的Akt1 mRNA的转录和蛋白表达(P＜0.05)；可显著减少LPS介导的TNF-α分泌(P＜0.05)，但各组IL-8的量无变化(P＞0.05)。结果提示：利用siRNA成功转染的Ilk可以影响细胞内Akt1的表达，降低细胞分泌的TNF-α含量，Ilk可能与小鼠MECs的抗炎有关，可作为靶基因进行更加深入的研究，以期为乳腺炎症的治疗提供新的思路及数据参考。 |
| 关键词: 乳腺上皮细胞 Ilk基因 炎性因子 |
| DOI： |
| 投稿时间：2016-10-08修订日期：2016-10-14 |
| 基金项目:国家自然科学基金项目（31260618; 31470128） |
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| Effects of ILK on Secretion of IL-6 and TNF-α in LPS-stimulated Mammary Epithelial Cell |
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| (College of Animal Science,Guizhou University,Guiyang Guizhou) |
| Abstract: |
| To investigate the effect of Ilk on secretion of IL-8 and TNF-α in LPS-stimulated mammary epithelial cells (MECs)，Ilk gene was transfected with RNA interference, then the best transfection concentration was obtained by real-time quantitative RT-PCR. Meanwhile, the transfection efficiency was evaluated by the Akt1 relative transcript levels assayed by RT-PCR and the content of Akt1 protein measured by ELISA.The content of IL-8 and TNF-α in the mouse MECs were measured by ELISA.The results showed that the transfection efficiency of Ilk-mus-595 in 20 nM was highest (84.83%),which could significantly inhibited Ilk mRNA expression(P＜0.01). When the mouse MECs was transfected with siRNA of Ilk and stimulated with LPS, compared with the control group, the expression of Akt1 mRNA and protein was significantly reduced after the silence of Ilk gene (P＜0.05). The secretion of TNF-α was prominently reduced (P＜0.05), but there was no change (P＞0.05) on the IL-8 content in each group.These results indicated that the silence of Ilk could change the expression of Akt1 and reduce the secretion of TNF-α in LPS-stimulated MECs, which proved that its anti-infection effect maybe related to Ilk,so Ilk could be chosen as a target gene to further explore the immune function of mouse MECs. Which can provide new train of thought and experimental data for treatment of mammary gland inflammation and provide theoretical basis for the development and application of new drugs. |
| Key words: MECs Ilk cytokine |
