| 段晓星,常永丽,张丽英,等.微小 RNA?9?5p调控脂多糖诱导小鼠树突状细胞免疫功能的机制研究[J].安徽医药,2021,25(1):17-22. |
| 微小 RNA?9?5p调控脂多糖诱导小鼠树突状细胞免疫功能的机制研究 |
| Study on the mechanism of mir?9?5p regulating lipopolysaccharide?induced the immune function of mouse dendritic cells |
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| DOI：10.3969/j.issn.1009?6469.2021.01.005. |
| 中文关键词: 微 RNAs 衔接蛋白质类，信号转导 脂多糖 转录激活因子 6 免疫系统 抗原， CD 白细胞介素类 miR?9?5p 免疫功能 |
| 英文关键词: MicroRNAs Adaptor proteins,signal transducing Lipopolysacharide Activating transcription factor 6 Im? mune system Antigens,CD Interleukins miR?9?5p Immune function |
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| 中文摘要: |
| 目的研究微小 RNA?9?5p(miR?9?5p)对脂多糖炎性损伤小鼠树突状细胞(DCs)免疫功能的影响，并探讨其机制。方法该研究于 2019年 1—9月完成。小鼠树突状细胞 DC2.4购于美国模式培养物中心。运用脂多糖(LPS)处理 DC2.4细胞，建立炎性树突状细胞损伤模型；将 miRNA模拟物阴性对照(miR?NC)、 miR?9?5p模拟物(mimics)、 miRNA抑制物阴性对照(anti? miR?NC)、 miR?9?5p抑制剂(anti?miR?9?5p)、小干扰 RNA无序对照(si?NC)组、信号转导和转录激活因子 6(STAT6)的小干扰 RNA(si?STAT6)、 miR?9?5p mimics+空载体质粒(pcDNA)、 miR?9?5p mimics+STAT6过表达质粒(pcDNA?STAT6)均用脂质体法转染至 LPS诱导的 DC2.4细胞；噻唑蓝(MTT)法检测细胞增殖；实时荧光定量逆转录聚合酶链反应(qRT?PCR)法检测细胞中 miR?9?5p、STAT6、白细胞分化抗原 80(CD80)、白细胞分化抗原 86(CD86)、白细胞介素 ?12(IL?12)的 mRNA表达；蛋白质印迹法(Western blot)检测细胞中 STAT6的蛋白表达；双荧光素酶报告基因实验检测细胞的荧光素酶活性。结果与正常 DC2.4相比，脂多糖炎性损伤的 DC2.4细胞中 miR?9?5p表达(0.16±0.01)比(1.00±0.08)降低， STAT6表达(3.02±0.26)比(1.00±0.07)升高(P＜0.05)；过表达 miR?9?5p可降低 LPS诱导的 DC2.4细胞中 CD80(0.31±0.03)比(1.00±0.08)、 CD86(0.29±0.02)比(1.01±0.06)、 IL?12(0.46±0.04)比(1.00±0.05)的表达；敲减 STAT6可降低 LPS诱导的 DC2.4细胞中 CD80(0.42±0.04)比(1.00±0.08)、 CD86(0.34±0.03)比(0.99±0.07)、 IL?12(0.29±0.02)比(1.01±0.08)的表达； miR?9?5p明显的抑制野生型 STAT6细胞的荧光活性，并负向调控 STAT6的表达水平；重要的是，过表达 STAT6可逆转过表达 miR?9?5p对 LPS诱导的 DC2.4细胞中 CD80［(1.68±0.13)比(1.00±0.06)］、 CD86［(1.79±0.15)比(0.99±0.08)］、 IL?12［(1.82±0.18)比(0.98±0.09)］的抑制作用。结论 miR?9?5p抑制 LPS诱导的 DC2.4细胞的免疫功能，其机制可能与靶向 STAT6有关，将可为炎性疾病的治疗提供新靶点。 |
| 英文摘要: |
| Objective To study the effect of microRNA?9?5p(miR?9?5p)on the immune function of dendritic cells(DCs)in mice with lipopolysaccharide inflammatory injury，and explore its mechanism.Methods The study was completed from January2019 to September 2019.Mice dendritic cells DC2.4 were purchased from the American Model Culture Center.DC2.4 cells weretreated with lipopolysaccharide(LPS)to establish a mice model of inflammatory dendritic cell(Nephritis DCs)injury.The miRNA mimic negative control(miR?NC)miR?9?5p mimic(mimics)miRNA inhibitor negative control(anti?miR?NC)，miR?9?5p inhibi? tor(anti?miR?9?5p)，smallinterfe，ring RNA disorder control(，si?NC) group，signal transducer and activator of transcription 6(STAT6)small interfering RNA(si?STAT6)miR?9?5p mimics + empty vector plasmid(pcDNA)，miR?9?5p mimics+STAT6 over? expressionplasmid(pcDNA?STAT6)weretrans，fected into LPS?induced DCs cells by liposome method；the proliferation was detect? ed by thiazole blue(MTT)method；real?time fluorescence quantitative reverse transcription polymerase detected the expression of miR?9?5p，STAT6，clusterdifferentiation 80(CD80)，clusterdifferentiation 86(CD86)，interleukin 12(IL?12)mRNA；the expres? sion of STAT6 protein was detected by western blotting(western blot)； the fluorescence activity of cells was detected by dual lucif?erase reporter gene assay.Results Compared with normal DC2.4 cells，the expression of miR?9?5p(0.16±0.01)vs.(1.00±0.08) was significantly decreased，STAT6(3.02±0.26)vs.(1.00±0.07)was significantly increased in LPS?induced DCs cells(P＜0.05). Overexpression miR?9?5p significantly decreased the expression of CD80(0.31±0.03)vs.(1.00±0.08)，CD86(0.29±0.02)vs.(1.01±0.06)and IL?12(0.46±0.04)vs.(1.00±0.05)in LPS?induced DC2.4 cells；STAT6 knockdown significantly reduced the ex? pression of CD80(0.42±0.04)vs.(1.00±0.08)，CD86(0.34±0.03)vs.(0.99±0.07)，and IL?12(0.29±0.02)vs.(1.01±0.08)in LPS?induced DC2.4 cells；miR?9?5p significantly inhibited the luciferase activity of wild?type STAT6 cells and negatively regulated the STAT6 expression；importantly，overexpression STAT6 could reverse the inhibition effect of overexpression miR?9?5p on CD80(1.68±0.13)vs.(1.00±0.06)，CD86(1.79±0.15)vs.(0.99±0.08)and IL?12(1.82±0.18)vs.(0.98±0.09)in LPS?induced DC2.4 cells.Conclusion MiR?9?5p inhibits the immune function of LPS?induced DC2.4 cells，and its mechanism may be related to target? ing STAT6，which will provide a new target for the treatment inflammatory diseases. |
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