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ENGLISH ABSTRACT
miR-31-5p过表达对兔自身免疫性干眼外周血Th17细胞的免疫调控作用
高敏
赵璐
陈思思
魏瑞华
粘红
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20210809-00452
Immunoregulatory effects of miR-31-5p overexpression on peripheral blood Th17 cell in a rabbit model of autoimmune dry eye
Gao Min
Zhao Lu
Chen Sisi
Wei Ruihua
Nian Hong
Authors Info & Affiliations
Gao Min
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Zhao Lu
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Chen Sisi
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Wei Ruihua
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Nian Hong
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
·
DOI： 10.3760/cma.j.cn115989-20210809-00452
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摘要

目的探讨慢病毒介导微小RNA(miR)-31-5p过表达对兔自身免疫性干眼模型外周血辅助性T细胞17(Th17)的免疫调控作用。

方法构建miR-31-5p重组慢病毒载体。包装miR-31-5p过表达及其对照慢病毒，进行浓缩和滴度测定。建立兔自身免疫性干眼模型，并分离模型兔外周血单个核细胞(PBMC)，分别感染miR-31-5p及阴性对照慢病毒颗粒作为miR-31-5p过表达组和对照组，采用实时荧光定量PCR法检测miR-31-5p的表达水平。将2个组PBMC与经γ射线照射后的泪腺上皮细胞共培养，采用实时荧光定量PCR法检测2个组PBMC中Th17细胞特异性转录因子维甲酸相关孤儿核受体C(RORC)、标志性细胞因子白细胞介素-17(IL-17)及Th17细胞极化相关细胞因子IL-1β、IL-6和IL-23的mRNA表达水平；采用Western blot法检测PBMC中IL-17的蛋白表达水平。

结果通过测序验证，成功构建miR-31-5p重组慢病毒载体。包装并浓缩miR-31-5p过表达和对照慢病毒颗粒，滴度测定结果分别为3.82×10 7 TU/ml和3.50×10 7 TU/ml，满足后续实验需求。实时荧光定量PCR结果显示，miR-31-5p过表达组miR-31-5p相对表达量较对照组显著升高，差异有统计学意义( t＝－9.696， P<0.001)。在兔泪腺上皮细胞与PBMC共培养体系中，miR-31-5p过表达组PBMC中的RORC、IL-17 mRNA相对表达量分别为0.33±0.03和0.28±0.09，明显低于对照组的1.00±0.00和1.00±0.00，差异均有统计学意义( t＝46.256、13.810，均 P<0.05)。Western blot检测结果显示，miR-31-5p过表达组PBMC中IL-17蛋白相对表达量较对照组明显降低，差异有统计学意义( t＝4.977， P＝0.008)。实时荧光定量PCR结果显示，miR-31-5p过表达组PBMC中IL-6、IL-23和IL-1β mRNA相对表达量较对照组显著下降，差异均有统计学意义( t＝220.076、6.641、13.271，均 P<0.05)。

结论miR-31-5p过表达可能通过下调免疫微环境中IL-6、IL-23、IL-1β等细胞因子的表达负向调控Th17细胞免疫反应。

干眼;干燥综合征;Th17细胞;miR-31-5p;动物模型;外周血单个核细胞
ABSTRACT

ObjectiveTo investigate the immunoregulatory effects of lentivirus-mediated microRNA (miR)-31-5p overexpression on peripheral blood T helper cell 17 (Th17) in a rabbit model of autoimmune dry eye.

MethodsThe miR-31-5p recombinant lentiviral vector was constructed.Lentivirus overexpressing miR-31-5p and its control virus were packaged.The concentration measurement and lentiviral titer determination were carried out.A rabbit model of autoimmune dry eye was established and the peripheral blood mononuclear cells (PBMC) of the rabbits were isolated.PBMC infected with miR-31-5p and negative control lentivirus particles were assigned as the miR-31-5p overexpression group and control group, respectively.The miR-31-5p expression level was detected using quantitative real-time PCR (qRT-PCR). Then PBMC in the two groups were co-cultured with γ-ray irradiated lacrimal gland epithelial cells.The expressions of Th17 cell related transcription factor retinoic acid-receptor-related orphan receptor C (RORC) and interleukin-17 (IL-17) mRNA, IL-1β, IL-6 and IL-23 were determined by qRT-PCR.The IL-17 protein expression level was detected by Western blot.The use and care of animals complied with Regulation for the Administration of Affair Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Tianjin Medical University Eye Hospital (No.TJYY20201221036).

ResultsThe construction of the miR-31-5p recombinant lentiviral vector was verified by DNA sequencing.The lentiviral titer of lentivirus overexpressing miR-31-5p and control lentivirus particles was 3.82×10 7 TU/ml and 3.50×10 7 TU/ml, respectively.The miR-31-5p relative expression level of PBMC was significantly increased in miR-31-5p overexpression group in comparison with control group, showing a statistically significant difference ( t=-9.696, P<0.001). When PBMC were co-cultured with lacrimal gland epithelial cells in vitro, the relative expression levels of RORC and IL-17 mRNA in miR-31-5p overexpression group were 0.33±0.03 and 0.28±0.09, which were significantly decreased in comparison with 1.00±0.00 and 1.00±0.00 in control group, with statistically significant differences between them ( t=46.256, 13.810; both at P<0.05). The relative expression level of IL-17 protein in miR-31-5p overexpression group was significantly reduced than control group ( t=4.977, P=0.008). The relative expression levels of IL-1β, IL-6 and IL-23 mRNA were significantly lower in miR-31-5p overexpression group than control group ( t=220.076, 6.641, 13.271; all at P<0.05).

ConclusionsThe overexpression of miR-31-5p can inhibit the Th17-immune response via down-regulating the expression of IL-6, IL-1β and IL-23.

Dry eye;Sjögren syndrome;Th17 cells;miR-31-5p;Disease models, animal;Peripheral blood mononuclear cell
Nian Hong, Email: mocdef.6ab21gnohnain
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引用本文

高敏,赵璐,陈思思,等. miR-31-5p过表达对兔自身免疫性干眼外周血Th17细胞的免疫调控作用[J]. 中华实验眼科杂志,2022,40(08)：716-723.

DOI:10.3760/cma.j.cn115989-20210809-00452

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干燥综合征(Sjögren syndrome，SS)相关干眼是常见的自身免疫性干眼，严重者可继发角膜溃疡，造成视力损伤 [ 1 ]。由于SS发病机制尚未完全明确，临床上仍缺乏有效的治疗方案 [ 2 , 3 ]。辅助性T细胞17(T helper cell 17，Th17)是自身免疫性干眼中关键的致炎细胞群 [ 4 ]，研究发现Th17细胞在SS患者外周血中比例上调，并且与SS疾病活动指数ESSDAI密切相关 [ 5 ]。多项研究证实微小RNA(microRNA，miRNA)参与调控Th17细胞免疫反应，进而影响自身免疫性疾病的发生和发展 [ 6 ]。miR-31-5p是一种进化高度保守的miRNA，在多种自身免疫性疾病中发挥免疫调控作用。研究表明miR-31可通过促进T细胞中白细胞介素2(interleukin-2，IL-2)的异常表达参与系统性红斑狼疮的发病 [ 7 ]，或通过抑制Gprc5a影响调节性T细胞的分化，促进实验性自身免疫性脑脊髓炎的炎症反应 [ 8 ]；但其在自身免疫性干眼中的调控作用及机制尚不明确。本课题组前期研究结果表明，miR-31-5p在兔自身免疫性干眼模型泪腺以及外周血单个核细胞(peripheral blood mononuclear cell，PBMC)中表达显著下调 [ 9 ]。本研究进一步探讨miR-31-5p对兔自身免疫性干眼模型外周血Th17细胞的免疫调控作用。
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备注信息
A
粘红，Email： mocdef.6ab21gnohnain
B

高敏：实验设计、实施研究、采集数据、分析数据、起草文章；赵璐、陈思思：实验设计、实施研究、文章修改；魏瑞华：参与选题、研究设计及文章定稿；粘红：参与选题、研究设计、文章修改及定稿

C
所有作者均声明不存在利益冲突
D
国家自然科学基金项目 (81970793、82070929)
天津市临床重点学科（专科）建设项目 (TJLCZDXKT003)
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