TLR9參與調(diào)節(jié)高糖誘導(dǎo)的心肌纖維化
發(fā)布時(shí)間：2019-07-05 09:29
【摘要】：目的:本文旨在探索toll樣受體9(TLR9)是否參與調(diào)節(jié)高糖誘導(dǎo)的心肌纖維化反應(yīng)。方法:用高糖培養(yǎng)基培養(yǎng)心肌成纖維細(xì)胞,觀察心肌成纖維細(xì)胞的增殖和炎癥反應(yīng)的變化,確定高糖刺激能否誘導(dǎo)心肌成纖維細(xì)胞發(fā)生增殖、炎癥反應(yīng)。然后用TLR9低表達(dá)慢病毒轉(zhuǎn)染心肌成纖維細(xì)胞之后,同樣用高糖培養(yǎng)基培養(yǎng),觀察心肌成纖維細(xì)胞的增殖和炎癥反應(yīng)的變化,從而證明高糖刺激可以通過(guò)調(diào)節(jié)TLR9的表達(dá),誘導(dǎo)心肌成纖維細(xì)胞發(fā)生增殖和炎癥反應(yīng),最終導(dǎo)致心肌纖維化。實(shí)驗(yàn)設(shè)計(jì)分為以下6組,正常對(duì)照組:用5.5mmol/L正常糖濃度培養(yǎng)基培養(yǎng)心肌成纖維細(xì)胞48小時(shí);高糖組:用25mmol/L高糖濃度培養(yǎng)基刺激心肌成纖維細(xì)胞48小時(shí);高糖+TLR9-RNAi-LV組:心肌成纖維細(xì)胞成功轉(zhuǎn)染TLR9低表達(dá)慢病毒后,換用25mmol/L高糖濃度培養(yǎng)基繼續(xù)培養(yǎng)48小時(shí);正常糖+TLR9-RNAi-LV組:心肌成纖維細(xì)胞成功轉(zhuǎn)染TLR9低表達(dá)慢病毒后,繼續(xù)用5.5mmol/L正常糖濃度培養(yǎng)基繼續(xù)培養(yǎng)48小時(shí);正常糖+NC-LV組:心肌成纖維細(xì)胞成功轉(zhuǎn)染空病毒后,用5.5mmol/L正常糖濃度培養(yǎng)基培養(yǎng)繼續(xù)培養(yǎng)48小時(shí);高糖+NC-LV毒組:心肌成纖維細(xì)胞成功轉(zhuǎn)染空病毒后,換用25mmol/L高糖濃度培養(yǎng)基培養(yǎng)繼續(xù)培養(yǎng)48小時(shí)。各組實(shí)驗(yàn)分別作用滿48小時(shí)后,用RT-qPCR法檢測(cè)各組心肌成纖維細(xì)胞TLR9的表達(dá)水平、各種炎癥因子IL-6、IL-1β及TNF-a以及心肌纖維化因子TGF-1β、Col-1a及Col-3a的表達(dá)水平;用CCK8試劑盒檢測(cè)各組心肌成纖維細(xì)胞的增殖情況;用酶聯(lián)免疫吸附實(shí)驗(yàn)檢測(cè)各組上清中炎癥因子IL-6、IL-1β及TNF-a的表達(dá)水平。比較各組各種指標(biāo)表達(dá)水平的變化,說(shuō)明高糖刺激誘導(dǎo)的心肌纖維化與TLR9之間的關(guān)系。結(jié)果:RT-qPCR結(jié)果表明:與正常對(duì)照組相比,高糖組TLR9的mRNA表達(dá)水平明顯增高(P0.01),說(shuō)明高糖刺激可以誘導(dǎo)心肌成纖維細(xì)胞TLR9 mRNA的表達(dá)水平上調(diào);高糖組炎癥因子IL-6、IL-1β及TNF-a的mRNA表達(dá)水平較正常對(duì)照組明顯升高(P0.01),而TLR9低表達(dá)慢病毒成功轉(zhuǎn)染后的心肌成纖維細(xì)胞高糖刺激并未引起相關(guān)炎癥因子mRNA表達(dá)的增加,說(shuō)明TLR9參與調(diào)節(jié)高糖刺激誘導(dǎo)的心肌成纖維細(xì)胞的炎癥反應(yīng);高糖組致纖維化相關(guān)因子TGF-1β、Col-1a及Col-3a的mRNA表達(dá)水平較正常對(duì)照組明顯增高(P0.01),而TLR9低表達(dá)慢病毒成功轉(zhuǎn)染后的心肌成纖維細(xì)胞高糖刺激并未引起致纖維化相關(guān)因子mRNA表達(dá)的增加,說(shuō)明TLR9參與調(diào)節(jié)高糖刺激誘導(dǎo)的心肌成纖維細(xì)胞的致纖維化反應(yīng)。CCK8結(jié)果顯示:高糖組與正常對(duì)照組相比,心肌成纖維細(xì)胞出現(xiàn)了明顯的增殖反應(yīng)(P0.01),而高糖刺激作用下的TLR9低表達(dá)慢病毒成功轉(zhuǎn)染后的心肌成纖維細(xì)胞增殖反應(yīng)不明顯,說(shuō)明TLR9參與調(diào)節(jié)高糖刺激誘導(dǎo)的心肌成纖維細(xì)胞的增殖反應(yīng)。酶聯(lián)免疫吸附實(shí)驗(yàn)結(jié)果表明:高糖組炎癥因子IL-6、IL-1β及TNF-a的表達(dá)水平均較正常對(duì)照組明顯升高(P0.01),而TLR9低表達(dá)慢病毒成功轉(zhuǎn)染后的心肌成纖維細(xì)胞高糖刺激并未引起相關(guān)炎癥因子表達(dá)水平的增加,說(shuō)明TLR9參與調(diào)節(jié)高糖刺激誘導(dǎo)的心肌成纖維細(xì)胞的炎癥反應(yīng)。結(jié)論:高糖刺激可以上調(diào)心肌成纖維細(xì)胞TLR9的表達(dá),并且TLR9參與調(diào)節(jié)高糖誘導(dǎo)的心肌纖維化反應(yīng)。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.23
本文編號(hào)：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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.23
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 翁艷潔;王永軍;石英;周文娟;王鴻雁;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
，本文編號(hào)：2510433
本文鏈接：http://sikaile.net/yixuelunwen/xxg/2510433.html
最近更新
教材專著
