小干扰RNA-TLR9对高糖下大鼠视网膜神经节细胞凋亡的抑制作用及其机制  

Inhibitory effect of small interfering RNA-TLR9 on high glucose-induced retinal ganglion cell apoptosis

作　　者：杨慧慧[1] 阚全娥[1] 于璐[1] 李旭晴[1] 黄爱国 袁慧娟[1] Yang Huihui;Kan Quane;Yu Lu;Li Xuqing;Huang Aiguo;Yuan Huijuan(Department of Endocrinology,Henan Provincial People's Hospital,Zhengzhou 450003,China;Henan Provincial People's Hospital,Henan Eye Hospital,Henan Eye Institute,Henan Key Laboratory of Ophthalmology and Visual Sciences,People's Hospital of Zhengzhou University,People's Hospital of Henan University,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院内分泌科,郑州450003 [2]河南省人民医院河南省立眼科医院河南省眼科研究所河南省眼科与视觉科学重点实验室郑州大学人民医院河南大学人民医院,郑州450003

出　　处：《中华实验眼科杂志》2020年第1期38-44,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：河南省科技厅创新人才项目(164100510022);河南省医学科技攻关计划项目(SBGJ2018078)。

摘　　要：目的探讨Toll样受体9(TLR9)对高糖下大鼠视网膜神经节细胞(RGC)-5凋亡的影响及小干扰RNA-TLR9(si-TLR9)对凋亡的抑制作用。方法将体外培养的RGC-5细胞分为正常对照组、高糖组、高糖+阴性对照组和高糖+si-TLR9组,分别行正常培养、高糖培养、高糖下空质粒转染和高糖下siRNA-TLR9质粒转染细胞。采用实时PCR法检测各组细胞中TLR9 mRNA表达;采用MTT法检测各组细胞存活率;采用流式细胞仪检测各组细胞凋亡率;采用caspase-3活性检测试剂盒检测各组细胞中含半胱氨酸天冬氨酸蛋白水解酶3(caspase-3)活性;采用Western blot法检测TLR9及B淋巴细胞瘤-2(bcl-2)、bcl-2相关X蛋白(bax)、p38丝裂原活化蛋白激酶(p38MAPK)和磷酸化(p)-p38MAPK蛋白的表达。结果与高糖+阴性对照组比较,高糖+si-TLR9组细胞中TLR9 mRNA和蛋白的相对表达量明显下降,差异均有统计学意义(均P<0.05)。高糖组和高糖+阴性对照组细胞存活率分别为(78.36±5.13)%和(75.12±4.25)%,较正常对照组的(95.48±7.25)%明显降低,差异均有统计学意义(均P<0.05);高糖+si-TLR9组细胞存活率为(86.58±5.32)%,明显高于高糖+阴性对照组的(75.12±4.25)%,差异均有统计学意义(均P<0.05)。高糖组和高糖+阴性对照组细胞凋亡率分别为(13.23±1.22)%和(12.52±1.38)%,较正常对照组的(2.26±0.15)%明显升高,差异均有统计学意义(均P<0.05);高糖+si-TLR9组细胞凋亡率为(7.15±0.24)%,明显低于高糖+阴性对照组的(12.52±1.38)%,差异有统计学意义(P<0.05)。与高糖+阴性对照组比较,高糖+si-TLR9组细胞中bax蛋白表达量明显下降,bcl-2蛋白相对表达量明显增加,差异均有统计学意义(均P<0.05)。高糖+si-TLR9组细胞中caspase-3活性明显低于高糖+阴性对照组,差异有统计学意义(P<0.05)。与高糖+阴性对照组比较,高糖+si-TLR9组细胞中p-p38MAPK蛋白相对表达量明显下降,差异有统计学意义(P<0.05)。结论下调TLR9表达可抑制高糖诱导�Objective To investigate the effect of Toll-like receptor 9(TLR9)on high glucose-induced retinal ganglion cells-5(RGC-5)apoptosis and the inhibitory effects of small interfering RNA-TLR9(si-TLR9)on apoptosis.Methods RGC-5 cells were divided into normal control group,high glucose group,high glucose+negative control group and high glucose+si-TLR9 group which cells were respectively dealt with normal culture medium,high glucose medium,transfection of non-specific siRNA under high glucose and transfection of siRNA-TLR9 under high glucose.The expression of TLR9 mRNA was detected by real time PCR;the survival rate of the cells was evaluated by MTT assay;the apoptotic rate of the cells was detected by flow cytometry;the caspase-3 activity in the cells was detected by related kit,and the expressions of TLR9,B cell lymphoma(bcl-2),bcl-2 associated X protein(bax),p38 mitogen-activated protein kinases(p38MAPK)and phosphorylated(p)-p38MAPK proteins were detected by Western blot.Results The expressions of TLR9 mRNA and protein in the high glucose+si-TLR9 group were significantly decreased in comparison with the high glucose+negative control group(both at P<0.05).The cell survival rate of the high glucose group and high glucose+negative control group was(78.36±5.13)%and(75.12±4.25)%,respectively,which was significantly lower than(95.48±7.25)%in the normal control group,and that in the high glucose+si-TLR9 group was(86.58±5.32)%,which was significantly reduced in comparison with the high glucose+negative control group(all at P<0.05).The apoptotic rate of the cells in the high glucose group and high glucose+negative control group was(13.23±1.22)%and(12.52±1.38)%,respectively,which was significantly higher than(2.26±0.15)%of the normal control group,and apoptotic rate in the high glucose+si-TLR9 group was(7.15±0.24)%,which was significantly lower than that in high glucose+negative control group(all at P<0.05).The expression of bax in the cells of the high glucose+si-TLR9 group was significantly decreased,and the expressio

关 键 词：TOLL样受体9 小干扰RNA 视网膜神经节细胞 高糖 凋亡 细胞培养 

分 类 号：R774.1[医药卫生—眼科]