原纤维蛋白-2干扰诱导小鼠视网膜病变及其可能的机制

张瑞雪; 蒋文君; 郭大东; 石永伟; 毕宏生; 温莹

doi:10.3760/cma.j.cn115989-20231014-00130

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中华实验眼科杂志

• 实验研究 •

ENGLISH ABSTRACT

原纤维蛋白-2干扰诱导小鼠视网膜病变及其可能的机制

作者及单位信息

出版日期： 2024 -09 -10 ·

DOI： 10.3760/cma.j.cn115989-20231014-00130

Fibrillin-2 interfering induced retinopathy and its possible mechanism

Authors Info & Affiliations

Zhang Ruixue

Shandong University of Traditional Chinese Medicine, Jinan 250014, China

Jiang Wenjun

The Eye Hospital Affiliated to Shandong University of Traditional Chinese Medicine, Key Laboratory of Eye Disease Prevention and Treatment Technology of Integrated Traditional Chinese and Western Medicine of Shandong Province, Shandong Academy of Eye Disease Prevention and Treatment, Jinan 250002, China

Guo Dadong

Shi Yongwei

Bi Hongsheng

Wen Ying

Published： 2024 -09 -10 ·

DOI： 10.3760/cma.j.cn115989-20231014-00130

236

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摘要

目的探索潜伏转化生长因子-β结合蛋白(LTBP)、转化生长因子-β(TGF-β)、周期蛋白依赖性激酶2(CDK2)、细胞周期素D2(CCND2)在原纤维蛋白-2( FBN2)缺陷诱导视网膜病变模型小鼠视网膜中的表达。

方法将27只8周龄C57BL/6J小鼠按照随机数字表法随机分为正常对照组、空载体组和FBN2干扰组，每组9只。正常对照组不做处理，空载体组和FBN2干扰组右眼玻璃体腔内分别注射3 μl空载体和3 μl携带sh-FBN2干扰质粒的腺相关病毒(AAV)。于注射后4周采用光学相干断层扫描(OCT)和全视野视网膜电图(ERG)检测视网膜结构形态和功能变化。采用免疫荧光染色法检测视网膜FBN2蛋白表达分布情况；采用实时荧光定量PCR、Western blot法检测小鼠视网膜中FBN2、LTBP-1、TGF-β2及CDK2、CCND2 mRNA和蛋白表达水平。

结果注射后4周，OCT检测结果显示，与正常对照组和空载体组相比，FBN2干扰组视网膜色素上皮层不规则，出现高密度反射区。全视野ERG结果显示，与空载体组和正常对照组比较，FBN2干扰组Rod-a、Rod-b、Max-a、Max-b波形振幅均下降，差异均有统计学意义(均 P<0.05)。免疫荧光染色结果显示FBN2表达在视网膜全层，FBN2干扰组中FBN2荧光强度均弱于正常对照组和空载体组。正常对照组、空载体组和FBN2干扰组小鼠FBN2荧光强度分别为16.21±2.21、15.57±3.63和5.32±1.06，总体比较差异有统计学意义( F＝66.03， P<0.05)，其中FBN2干扰组FBN2蛋白荧光强度较空载体组和正常对照组明显降低，差异均有统计学意义(均 P<0.05)。与空载体组和正常对照组相比，FBN2干扰组LTBP-1、TGF-β2 mRNA和蛋白相对表达量明显升高，FBN2、CDK2、CCND2 mRNA和蛋白相对表达量明显下降，差异均有统计学意义(均 P<0.05)。

结论LTBP-1、TGF-β2升高以及G1/S期相关蛋白CDK2、CCND2降低参与 FBN2缺陷型视网膜病变的发展。

关键词：原纤维蛋白-2;腺相关病毒;视网膜病变

ABSTRACT

ObjectiveTo investigate the expression of latent transforming growth factor-β-binding protein (LTBP), transforming growth factor-β (TGF-β), cyclin-dependent kinase 2 (CDK2) and cyclin D2 (CCND2) in fibrillin-2 ( FBN2) interfering induced mouse retinopathy.

MethodsTwenty-seven 8-week-old C57BL/6J mice were randomly divided into normal control group, empty vector group and FBN2 interference group according to the random number table method, with 9 mice in each group.The normal control group was not treated.The empty vector group and FBN2 interference group were intravitreally injected with 3 μl empty vector and 3 μl adeno-associated virus (AAV) carrying the sh-FBN2 interference plasmid in the right eye, respectively.The structural and functional changes of the retina were detected at 4 weeks after injection by optical coherence tomography (OCT) and full-field electroretinography (ERG).The expression and distribution of FBN2 protein in the retina were detected by immunofluorescence staining.The mRNA and protein expression levels of FBN2, LTBP-1, TGF-β2, CDK2 and CCND2 in mouse retina were detected by real-time fluorescence quantitative PCR and Western blot.All experiments complied with the ARVO statement.The research scheme was approved by the Experimental Animal Ethics Committee of Shandong University of Traditional Chinese Medicine (No.2019036).

ResultsFour weeks after injection, the results of OCT examination showed that compared with normal control and empty vector groups, the retinal pigment cortex of the FBN2 interference group was irregular with high density reflection areas.Full-field ERG results showed that compared with normal control and empty vector groups, the amplitude of Rod-a, Rod-b, Max-a and Max-b waveforms in FBN2 interference group decreased, and the differences were statistically significant (all at P<0.05).The results of immunofluorescence staining showed that FBN2 was expressed in the whole retina, and the fluorescence intensity of FBN2 was weaker in FBN2 interference group than that in normal control and empty vector groups.The fluorescence intensity of FBN2 in normal control group, empty vector group and FBN2 interference group was 16.21±2.21, 15.57±3.63 and 5.32±1.06, respectively, with a statistically significant overall difference ( F=66.03, P<0.05).The fluorescence intensity of FBN2 protein in FBN2 interference group was significantly lower than that in empty carrier group and normal control group (both at P<0.05).Compared with normal control and empty vector groups, the relative expression levels of LTBP-1 and TGF-β2 mRNA and protein were significantly increased in FBN2 interference group, while the relative expression levels of FBN2, CDK2 and CCND2 mRNA and protein were significantly decreased, and the differences were statistically significant (all at P<0.05).

ConclusionsThe increase of LTBP-1 and TGF-β2 and the decrease of G1/S phase related proteins CDK2 and CCND2 are involved in the development of FBN2-deficient retinopathy.

KEYWORDS： Fibrillin-2;Adeno-associated virus;Retinopathy

Corresponding authors: Bi Hongsheng, Email: mocdef.6ab21ibgnehsgnoh;

Corresponding author:Wen Ying, Email: mocdef.3ab61eyegniynew

FUNDING:

National Key R&D Plan Project（2021YFC2702103）

Key R&D Plan Project in Shandong Province（2021LCZX09）

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No content published by the journals of Chinese Medical Association may be reproduced or abridged without authorization. Please do not use or copy the layout and design of the journals without permission.

All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

引用本文

张瑞雪,蒋文君,郭大东,等. 原纤维蛋白-2干扰诱导小鼠视网膜病变及其可能的机制[J]. 中华实验眼科杂志,2024,42(09)：798-805.

DOI:10.3760/cma.j.cn115989-20231014-00130

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原纤维蛋白-2(fibrillin-2，FBN2)是微纤维的重要组成部分，参与全身结缔组织中弹性纤维的形成。研究表明， FBN2基因变异可引起黄斑变性疾病，包括年龄相关性黄斑变性(age-related macular degeneration，AMD)和早发性黄斑变性，导致视物变形、黄斑变性、色素上皮萎缩和萎缩性黄斑 ^{[
1
]}。Duvvari等 ^{[
2
]}在AMD患者中发现 FBN2基因罕见错义变异c.4141G>T (p.His1381Asn)，并确认该变异具有致病性。 FBN2基因可以调节转化生长因子-β(transforming growth factor-β，TGF-β)表达 ^{[
3
]}。据报道，在新生血管性AMD中， FBN2缺失会异常激活TGF-β，这表明TGF-β在视网膜纤维化过程中起关键作用，并直接诱导人原代视网膜色素上皮(retinal pigment epithelium，RPE)细胞增殖，并活化成纤维细胞 ^{[
4
]}。前期研究表明，玻璃体腔注射FBN-2抗体后视网膜中TGF-β表达水平升高 ^{[
5
]}。成熟TGF-β的同源二聚体可与潜伏相关蛋白非共价结合，形成潜伏TGF-β结合蛋白(latent TGF-β binding protein，LTBP-1)，使成熟的TGF-β失去活性。FBN已被证实可以通过与LTBP结合来调节TGF-β活性，进而调节大鼠肝细胞系细胞周期 ^{[
6
,

7
]}。TGF-β在控制细胞增殖和分化中起关键作用，可以调节G1/S期细胞周期 ^{[
8
]}。周期蛋白依赖性激酶2(cyclin-dependent kinases2，CDK2)、细胞周期素D2(cyclin D2，CCND2)是G1/S期细胞周期标志物 ^{[
9
]}。本课题组前期研究发现，小鼠玻璃体腔注射腺相关病毒(adeno-associated virus，AAV)介导 FBN2基因干扰后4周，视网膜FBN2表达水平降低、视网膜结构及功能发生改变 ^{[
10
]}。本研究在前期研究的基础上，进一步明确LTBP/TGF-β和G1/S期细胞周期相关蛋白CDK2、CCND2在 FBN2缺陷型视网膜病变发病过程中的作用机制，为 FBN2缺陷型视网膜病变的精准治疗提供依据。

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参考文献

参考文献

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