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骨髓间充质干细胞来源的外泌体miR-183靶向调控视网膜脱氢酶11对视网膜色素上皮细胞的影响

Bone marrow mesenchymal stem cell-derived exosomes miR-183 target regulation of retinal dehydrogenase 11 to inhibit the development of retinitis pigmentosa
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摘要 目的观察骨髓间充质干细胞(BMSC)来源的外泌体上miR-183、视网膜脱氢酶11(RDH11)的表达,初步探讨两者靶向关系以及对视网膜色素上皮(RPE)细胞的影响。方法分离培养C57BL/6(C57)小鼠BMSC,鉴定BMSC来源的外泌体。将BMSC分为空白组、模拟空白对照组(mimic-NC组)、miR-183模拟组(miR-183-mimic组)。参照文献方法培养C57小鼠、视网膜变性10(rd10)小鼠RPE细胞。来自rd10小鼠的RPE细胞转染BMSC外泌体并共培养后分为对照组、外泌体组、mimic-NC-外泌体组(mimic-NC-exo组)、miR-183-mimic-外泌体组(miR-183-mimic-exo组)。采用实时荧光定量聚合酶链反应、蛋白免疫印迹法检测C57小鼠、rd10小鼠以及各组RPE细胞中miR-183、RDH11 mRNA和蛋白相对表达量。生物信息学网站及双荧光素酶报告分析miR-183与RDH11的靶向关系。细胞计数试剂盒8检测BMSC外泌体上miR-183对RPE细胞增生的影响;原位末端标记法检测RPE细胞凋亡情况。多组间比较采用单因素方差分析。结果与C57小鼠比较,rd10小鼠RPE中miR-183相对表达量下调,RDH11 mRNA相对表达量上调,差异均有统计学意义(t=5.230、8.548,P=0.006、0.001)。与空白组、mimic-NC组比较,miR-183-mimic组外泌体中miR-183 mRNA相对表达量显著上升(F=60.130,P<0.05)。共培养24 h时,外泌体进入RPE细胞。与mimic-NC-exo组比较,miR-183-mimic-exo组RPE细胞中miR-183 mRNA相对表达量显著升高(t=7.311,P=0.002),细胞增生能力增强(F=10.949,P=0.012)、凋亡数量减少(t=4.571,P=0.002),差异均有统计学意义。生物信息学网站及双荧光素酶报告证实miR-183与RDH11具有靶向关系。与mimic-NC组比较,miR-183-mimic组外泌体中RDH11 mRNA及蛋白相对表达量均降低,差异有统计学意义(t=5.361、6.591,P=0.006、0.003)。共培养后,与对照组比较,外泌体组RPE细胞中RDH11 mRNA及蛋白相对表达量差异无统计学意义(t=0.169、1.134,P=0.874、0.320);与mimic-NC-exo组比较,miR-183-mimic-exo组RPE细胞中RDH11 mRNA及蛋白相对表达量均降低,差异有统计学意义(t=5.554、5.546,P=0.005、0.005)。结论上调BMSC来源的外泌体miR-183通过靶向抑制RDH11的表达促进RPE细胞体外增生,减少细胞凋亡数量。 Objective To observe the expressions of miR-183 and retinal dehydrogenase 11(RDH11)in exosomes derived from bone marrow mesenchymal stem cells(BMSC),and to preliminarily explore their targeting relationship and their effects on retinal pigment epithelial(RPE)cells.Methods BMSC from C57BL/6(C57)mice were isolated and cultured,and BMSC-derived exosomes were identified.BMSC were divided into blank group,simulation blank control group(mimic-NC group),miR-183 simulation group(miR-183-mimic group).C57 mice and retinal degeneration 10(rd10)mouse RPE cells were cultured with reference to literature methods.RPE cells from rd10 mice were transfected with BMSC exosomes and co-cultured and divided into control group,exosome group,mimic-NC-exosome group(mimic-NC-exo group),miR-183-mimic-exosome group(miR-183-mimic-exo group).The relative expression levels of miR-183,RDH11 mRNA and protein in C57 mice,rd10 mice and RPE cells in each group were detected by real-time quantitative polymerase chain reaction and western blotting.The targeting relationship between miR-183 and RDH11 was analyzed by bioinformatics website and dual luciferase reporter.Cell counting kit 8 was used to detect the effect of miR-183 on BMSC exosomes on RPE cell proliferation;in situ labeling end labeling method was used to detect RPE cells apoptosis.One-way ANOVA was used to compare multiple groups.Results Compared with C57 mouse RPE cells,the relative expression of miR-183 in rd10 mouse RPE cells was down-regulated,and the relative expression of RDH11 mRNA was up-regulated,and the differences were statistically significant(t=5.230,8.548;P=0.006,0.001).Compared with the blank group and the mimic-NC group,the relative expression of miR-183 mRNA in the exosomes of the miR-183-mimics group was significantly increased(F=60.130,P<0.05).After 24 h of co-culture,exosomes entered RPE cells.Compared with the mimic-NC-exo group,the relative expression of miR-183 mRNA in RPE cells in the miR-183-mimic-exo group was significantly increased,the proliferation ability was enhanced(t=7.311,P=0.002),and the number of apoptotic cells was decreased(F=10.949,P=0.012),and the differences were statistically significant(t=4.571,P=0.002).Bioinformatics website and dual-luciferase report confirmed that miR-183 has a targeting relationship with RDH11.Compared with the mimic-NC group,the relative expression of RDH11 mRNA and protein in the exosomes of the miR-183-mimic group was decreased,and the difference was statistically significant(t=5.361,6.591;P=0.006,0.003).After co-culture,compared with the control group,there was no significant difference in the relative expression of RDH11 mRNA and protein in RPE cells in the exosome group(t=0.169,1.134;P=0.874,0.320);The relative expressions of RDH11 mRNA and protein in RPE cells in-183-mimic-exo group were decreased,and the difference was statistically significant(t=5.554,5.546;P=0.005,0.005).Conclusion Up-regulation of BMSC-derived exosomal miR-183 promote the proliferation of RPE cells in vitro by targeting the expression of RDH11 and reduce the number of apoptosis.
作者 李媛 秦廷玉 郭龙 李淑珍 侯习武 Li Yuan;Qin Tingyu;Guo Long;Li Shuzhen;Hou Xiwu(Department of Ophthalmology,Shangqiu First People's Hospital,Zhengzhou 476000,China;Department of Ophthalmology,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)
出处 《中华眼底病杂志》 CAS CSCD 北大核心 2022年第8期688-695,共8页 Chinese Journal of Ocular Fundus Diseases
基金 河南省高等学校重点科研项目(19A320059)。
关键词 视网膜色素上皮 骨髓细胞 间质干细胞 细胞凋亡 miR-183 视网膜脱氢酶11 Retinal pigment epithelium Bone marrow cells Mesenchymal stem cells Apoptosis miR-183 Retinal dehydrogenase 11
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