TLR9参与调节高糖诱导的心肌纤维化
发布时间：2019-07-05 09:29
【摘要】：目的:本文旨在探索toll样受体9(TLR9)是否参与调节高糖诱导的心肌纤维化反应。方法:用高糖培养基培养心肌成纤维细胞,观察心肌成纤维细胞的增殖和炎症反应的变化,确定高糖刺激能否诱导心肌成纤维细胞发生增殖、炎症反应。然后用TLR9低表达慢病毒转染心肌成纤维细胞之后,同样用高糖培养基培养,观察心肌成纤维细胞的增殖和炎症反应的变化,从而证明高糖刺激可以通过调节TLR9的表达,诱导心肌成纤维细胞发生增殖和炎症反应,最终导致心肌纤维化。实验设计分为以下6组,正常对照组:用5.5mmol/L正常糖浓度培养基培养心肌成纤维细胞48小时;高糖组:用25mmol/L高糖浓度培养基刺激心肌成纤维细胞48小时;高糖+TLR9-RNAi-LV组:心肌成纤维细胞成功转染TLR9低表达慢病毒后,换用25mmol/L高糖浓度培养基继续培养48小时;正常糖+TLR9-RNAi-LV组:心肌成纤维细胞成功转染TLR9低表达慢病毒后,继续用5.5mmol/L正常糖浓度培养基继续培养48小时;正常糖+NC-LV组:心肌成纤维细胞成功转染空病毒后,用5.5mmol/L正常糖浓度培养基培养继续培养48小时;高糖+NC-LV毒组:心肌成纤维细胞成功转染空病毒后,换用25mmol/L高糖浓度培养基培养继续培养48小时。各组实验分别作用满48小时后,用RT-qPCR法检测各组心肌成纤维细胞TLR9的表达水平、各种炎症因子IL-6、IL-1β及TNF-a以及心肌纤维化因子TGF-1β、Col-1a及Col-3a的表达水平;用CCK8试剂盒检测各组心肌成纤维细胞的增殖情况;用酶联免疫吸附实验检测各组上清中炎症因子IL-6、IL-1β及TNF-a的表达水平。比较各组各种指标表达水平的变化,说明高糖刺激诱导的心肌纤维化与TLR9之间的关系。结果:RT-qPCR结果表明:与正常对照组相比,高糖组TLR9的mRNA表达水平明显增高(P0.01),说明高糖刺激可以诱导心肌成纤维细胞TLR9 mRNA的表达水平上调;高糖组炎症因子IL-6、IL-1β及TNF-a的mRNA表达水平较正常对照组明显升高(P0.01),而TLR9低表达慢病毒成功转染后的心肌成纤维细胞高糖刺激并未引起相关炎症因子mRNA表达的增加,说明TLR9参与调节高糖刺激诱导的心肌成纤维细胞的炎症反应;高糖组致纤维化相关因子TGF-1β、Col-1a及Col-3a的mRNA表达水平较正常对照组明显增高(P0.01),而TLR9低表达慢病毒成功转染后的心肌成纤维细胞高糖刺激并未引起致纤维化相关因子mRNA表达的增加,说明TLR9参与调节高糖刺激诱导的心肌成纤维细胞的致纤维化反应。CCK8结果显示:高糖组与正常对照组相比,心肌成纤维细胞出现了明显的增殖反应(P0.01),而高糖刺激作用下的TLR9低表达慢病毒成功转染后的心肌成纤维细胞增殖反应不明显,说明TLR9参与调节高糖刺激诱导的心肌成纤维细胞的增殖反应。酶联免疫吸附实验结果表明:高糖组炎症因子IL-6、IL-1β及TNF-a的表达水平均较正常对照组明显升高(P0.01),而TLR9低表达慢病毒成功转染后的心肌成纤维细胞高糖刺激并未引起相关炎症因子表达水平的增加,说明TLR9参与调节高糖刺激诱导的心肌成纤维细胞的炎症反应。结论:高糖刺激可以上调心肌成纤维细胞TLR9的表达,并且TLR9参与调节高糖诱导的心肌纤维化反应。
[Abstract]:Objective: To explore whether the Toll-like receptor 9 (TLR9) is involved in the regulation of high glucose-induced myocardial fibrosis. Methods: The changes of the proliferation and inflammatory response of the cardiac fibroblasts were observed with high glucose medium, and it was determined whether the high glucose stimulation could induce the proliferation and the inflammatory response of the cardiac fibroblasts. After the myocardial fibroblasts were transfected with the low-expression lentivirus of TLR9, the proliferation and the inflammatory response of the cardiac fibroblasts were also observed with a high-glucose medium, thereby demonstrating that the high glucose stimulation could be achieved by adjusting the expression of TLR9, Inducing the proliferation and the inflammatory reaction of the myocardial fibroblasts, and finally leading to myocardial fibrosis. The experimental design was divided into six groups: normal control group: cultured with 5.5 mmol/ L normal sugar concentration medium for 48 hours; high-sugar group: stimulation of myocardial fibroblasts with 25 mmol/ L high glucose concentration medium for 48 hours; high glucose + TLR9-RNAi-LV group: After successfully transfecting TLR9 low-expression lentivirus with a 25 mmol/ L high-sugar concentration medium, the normal sugar + TLR9-RNAi-LV group: after the myocardial fibroblasts were successfully transfected with TLR9 low-expression lentivirus, the normal sugar concentration medium of 5.5 mmol/ L was continued to be cultured for 48 hours; Normal sugar + NC-LV group: after the myocardial fibroblasts were successfully transfected with the empty virus, the culture was continued for 48 hours with 5.5 mmol/ L normal sugar concentration medium; the high glucose + NC-LV group: after the myocardial fibroblasts were successfully transfected with the empty virus, the culture was continued for 48 hours with a 25 mmol/ L high glucose concentration medium. The expression levels of TLR9, IL-6, IL-1, TNF-a and TGF-1, Chol-1a and Chol-3a of each group were detected by RT-qPCR, respectively. The levels of IL-6, IL-1 and TNF-a in the supernatant of each group were detected by enzyme-linked immunosorbent assay. The relationship between myocardial fibrosis and TLR9 induced by high glucose was explained. Results: The results of RT-qPCR showed that the expression of TLR9 mRNA in the high-sugar group was significantly higher than that in the normal control group (P0.01). The expression level of TLR9 mRNA in the myocardium was up-regulated by high glucose stimulation, and the anti-inflammatory factor IL-6 in the high-sugar group was higher than that of the normal control group. The mRNA expression level of IL-1 and TNF-a was significantly higher than that in the normal control group (P0.01), while the high glucose stimulation of the myocardial fibroblasts after the low expression of TLR9 low-expression lentivirus did not cause an increase in the expression of the relevant inflammatory factor mRNA. The expression of TGF-1, Chol-1a and Chol-3a in the high-glucose group was significantly higher than that in the normal control group (P0.01). The high glucose stimulation of the myocardial fibroblasts after the successful transfection of the TLR9 low-expression lentivirus did not cause an increase in the expression of the fibrosis-related factor mRNA, indicating that the TLR9 was involved in the regulation of the fibrotic response of the cardiac fibroblasts induced by high glucose stimulation. The results of CCK8 showed that the hyperglycemic group had a significant proliferation response (P0.01) compared with the normal control group, while the low expression of TLR9 low expression of TLR9 in high glucose stimulation was not obvious after the successful transfection of the slow virus. TLR9 is indicated to be involved in the regulation of the proliferation of cardiac fibroblasts induced by high glucose stimulation. The results of the enzyme-linked immunosorbent assay showed that the levels of IL-6, IL-1 and TNF-a in the high-sugar group were significantly higher than those in the normal control group (P0.01). TLR9 is indicated to be involved in the regulation of the inflammatory response of myocardial fibroblasts induced by high glucose stimulation. Conclusion: High glucose stimulation can upregulate the expression of TLR9 in cardiac fibroblasts, and TLR9 is involved in the regulation of high glucose-induced myocardial fibrosis.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.23
本文编号：2510433
[Abstract]:Objective: To explore whether the Toll-like receptor 9 (TLR9) is involved in the regulation of high glucose-induced myocardial fibrosis. Methods: The changes of the proliferation and inflammatory response of the cardiac fibroblasts were observed with high glucose medium, and it was determined whether the high glucose stimulation could induce the proliferation and the inflammatory response of the cardiac fibroblasts. After the myocardial fibroblasts were transfected with the low-expression lentivirus of TLR9, the proliferation and the inflammatory response of the cardiac fibroblasts were also observed with a high-glucose medium, thereby demonstrating that the high glucose stimulation could be achieved by adjusting the expression of TLR9, Inducing the proliferation and the inflammatory reaction of the myocardial fibroblasts, and finally leading to myocardial fibrosis. The experimental design was divided into six groups: normal control group: cultured with 5.5 mmol/ L normal sugar concentration medium for 48 hours; high-sugar group: stimulation of myocardial fibroblasts with 25 mmol/ L high glucose concentration medium for 48 hours; high glucose + TLR9-RNAi-LV group: After successfully transfecting TLR9 low-expression lentivirus with a 25 mmol/ L high-sugar concentration medium, the normal sugar + TLR9-RNAi-LV group: after the myocardial fibroblasts were successfully transfected with TLR9 low-expression lentivirus, the normal sugar concentration medium of 5.5 mmol/ L was continued to be cultured for 48 hours; Normal sugar + NC-LV group: after the myocardial fibroblasts were successfully transfected with the empty virus, the culture was continued for 48 hours with 5.5 mmol/ L normal sugar concentration medium; the high glucose + NC-LV group: after the myocardial fibroblasts were successfully transfected with the empty virus, the culture was continued for 48 hours with a 25 mmol/ L high glucose concentration medium. The expression levels of TLR9, IL-6, IL-1, TNF-a and TGF-1, Chol-1a and Chol-3a of each group were detected by RT-qPCR, respectively. The levels of IL-6, IL-1 and TNF-a in the supernatant of each group were detected by enzyme-linked immunosorbent assay. The relationship between myocardial fibrosis and TLR9 induced by high glucose was explained. Results: The results of RT-qPCR showed that the expression of TLR9 mRNA in the high-sugar group was significantly higher than that in the normal control group (P0.01). The expression level of TLR9 mRNA in the myocardium was up-regulated by high glucose stimulation, and the anti-inflammatory factor IL-6 in the high-sugar group was higher than that of the normal control group. The mRNA expression level of IL-1 and TNF-a was significantly higher than that in the normal control group (P0.01), while the high glucose stimulation of the myocardial fibroblasts after the low expression of TLR9 low-expression lentivirus did not cause an increase in the expression of the relevant inflammatory factor mRNA. The expression of TGF-1, Chol-1a and Chol-3a in the high-glucose group was significantly higher than that in the normal control group (P0.01). The high glucose stimulation of the myocardial fibroblasts after the successful transfection of the TLR9 low-expression lentivirus did not cause an increase in the expression of the fibrosis-related factor mRNA, indicating that the TLR9 was involved in the regulation of the fibrotic response of the cardiac fibroblasts induced by high glucose stimulation. The results of CCK8 showed that the hyperglycemic group had a significant proliferation response (P0.01) compared with the normal control group, while the low expression of TLR9 low expression of TLR9 in high glucose stimulation was not obvious after the successful transfection of the slow virus. TLR9 is indicated to be involved in the regulation of the proliferation of cardiac fibroblasts induced by high glucose stimulation. The results of the enzyme-linked immunosorbent assay showed that the levels of IL-6, IL-1 and TNF-a in the high-sugar group were significantly higher than those in the normal control group (P0.01). TLR9 is indicated to be involved in the regulation of the inflammatory response of myocardial fibroblasts induced by high glucose stimulation. Conclusion: High glucose stimulation can upregulate the expression of TLR9 in cardiac fibroblasts, and TLR9 is involved in the regulation of high glucose-induced myocardial fibrosis.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.23
【参考文献】
相关期刊论文 前1条
1 翁艳洁;王永军;石英;周文娟;王鸿雁;王常玉;;TLR9 Expression and Its Role in Chemosensitivity to DDP in Human Cervical Cancer Cells in vitro[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2011年04期
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