[1]令狐绍容,罗泓扬.视网膜色素上皮细胞通过分泌含有miR-4488的外泌体抑制糖尿病视网膜病变进展的机制研究[J].眼科新进展,2022,42(5):353-358.[doi:10.13389/j.cnki.rao.2022.0071]
　LINGHU Shaorong,LUO Hongyang.Inhibition mechanism of retinal pigment epithelial cells-induced miR-4488-containing exosomes on diabetic retinopathy[J].Recent Advances in Ophthalmology,2022,42(5):353-358.[doi:10.13389/j.cnki.rao.2022.0071]
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视网膜色素上皮细胞通过分泌含有miR-4488的外泌体抑制糖尿病视网膜病变进展的机制研究/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年5期
页码:
353-358
栏目:
实验研究
出版日期:
2022-05-05

文章信息/Info

Title:
Inhibition mechanism of retinal pigment epithelial cells-induced miR-4488-containing exosomes on diabetic retinopathy
作者:
令狐绍容; 罗泓扬
563000　贵州省遵义市，遵义医科大学附属医院眼科中心（令狐绍容）；563000　贵州省遵义市，遵义医科大学附属医院眼科临床技能实验室（罗泓扬）
Author(s):
LINGHU Shaorong1; LUO Hongyang2
1.Department of Ophthalmology，Affiliated Hospital of Zunyi Medical University，Zunyi 563000，Guizhou Province,China
2.Ophthalmic Clinical Skills Laboratory，Affiliated Hospital of Zunyi Medical University，Zunyi 563000，Guizhou Province,China
关键词:
miR-4488; 转化生长因子β; 糖尿病视网膜病变; 内皮细胞间充质转化; 外泌体; 视网膜色素上皮细胞
Keywords:
miR-4488;  transforming growth factor β;  diabetic retinopathy;  endothelial-to-mesenchymal transition;  exosome;  retinal pigment epithelial cells
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2022.0071
文献标志码:
A
摘要:
目的　探讨视网膜色素上皮细胞通过分泌含有miR-4488的外泌体抑制糖尿病视网膜病变进展的分子机制。方法　将人视网膜色素上皮细胞系ARPE-19细胞分为空白组、高糖组。将人视网膜微血管内皮细胞（HRMECs）分为NG组、HG组、NG+外泌体组和HG+外泌体组。空白组、NG组和NG+外泌体组细胞用含5.5 mmol·L-1正常浓度葡萄糖的培养基培养24 h；高糖组、HG组和HG+外泌体组细胞用含30 mmol·L-1高浓度葡萄糖的培养基培养24 h；NG+外泌体组和HG+外泌体组细胞在培养基中分别加入空白组或高糖组ARPE-19细胞分泌的外泌体40 μg·L-1。PKH67绿色荧光标记外泌体；利用CCK-8法、Transwell小室法检测HRMECs增殖、迁移和血管生成。取空白组和高糖组ARPE-19细胞外泌体进行miRNA微阵列分析。采用双荧光素酶报告分析检测miR-4488和转化生长因子β受体Ⅱ（TGFβR2）基因序列的靶向关系。采用Western blot或免疫荧光染色检测各组HRMECs中TGFβR2、Smad2、p-Smad2ser467或Desmin、α-平滑肌肌动蛋白（α-SMA）的表达。结果　空白组和高糖组ARPE-19细胞分泌的被PKH67标记的外泌体加入培养基后都可被HRMECs吸收。与NG组相比，HG组HRMECs细胞增殖率、迁移率、血管结点数和血管总长度、TGFβR2和p-Smad2ser467蛋白表达量、Desmin和α-SMA蛋白荧光强度均增加（均为P<0.05）；与HG组相比，HG+外泌体组上述指标均被逆转（均为P<0.05）；NG+外泌体组与NG组上述指标相比差异均无统计学意义（均为P>0.05）。与空白组相比，高糖组ARPE-19细胞分泌的外泌体中miR-4488相对表达量增加，且miR-4488模拟物和TGFβR2wt共转染可增加双荧光素酶活性（均为P<0.05）。结论　在高糖条件下，ARPE-19细胞释放的外泌体可通过miR-4488/TGFβR2/Smad信号通路抑制HRMECs间充质转化。
Abstract:
Objective　To investigate the inhibition mechanism of retinal pigment epithelial (RPE) cells-induced miR-4488-containing exosomes on diabetic retinopathy (DR). Methods　ARPE-19 cells were divided into the blank group and high glucose group. Human retinal microvascular endothelial cells (HRMECs) were divided into the NG group, HG group, NG+Exo group, and HG+Exo group. Cells in the blank group, NG group, and NG+Exo group were cultured in medium containing 5.5 mmol·L-1 glucose for 24 hours, cells in the high glucose group, HG group, and HG+Exo group were cultured in medium containing 30 mmol·L-1 glucose for 24 hours, and cells in the NG+Exo group and HG+Exo group were cultured in medium added with 40 μg·L-1 of exosomes secreted by ARPE-19 cells in the blank group and high glucose group, respectively. Exosomes were labeled with PKH67 green fluorescence, and the proliferation, migration and angiogenesis of HRMECs were detected by CCK-8 and Transwell assays. The ARPE-19 cells-induced exosomes in the blank group and high glucose group were analyzed by miRNA microarray. Dual-luciferase reporter gene was used to detect the targeting relationship between miR-4488 and transforming growth factor beta receptor II (TGFβR2) gene sequences. The expression levels of TGFβR2, Smad2, p-Smad2ser467 or Desmin, and α-smooth muscle actin (α-SMA) in HRMECs were detected by Western blot and immunofluorescence staining. Results　PKH67-labeled exosomes secreted by ARPE-19 cells in the blank group and high glucose group were absorbed by HRMECs after being added to the medium. Compared with the NG group, the proliferation and migration of HRMECs, number of vascular nodes, length of vessels, protein expression of TGFβR2 and p-Smad2ser467, and the fluorescence intensity of Desmin and α-SMA increased in the HG group (all P<0.05). However, the above indexes were reversed in the HG+Exo group compared with the HG group (all P<0.05). There was no significant difference in the above indexes between the NG+Exo group and the NG group (all P>0.05). Compared with the blank group, the relative expression of miR-4488 was increased in the high glucose group, and the cotransfection of miR-4488 mimics and TGFβR2wt increased the dual-luciferase activity (all P<0.05). Conclusion　Under the high glucose, ARPE-19 cells-induced exosomes can inhibit HRMECs mesenchymal transition via miR-4488/TGFβR2/Smad signaling pathway.

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更新日期/Last Update: 2022-05-08