RGD多肽水凝胶对Tenon囊成纤维细胞活化和YAP信号的影响  

Effects of arginine-glycine-aspartic acid peptide hydrogels on the activation of Tenon capsule fibroblasts and Yes-associated protein signaling

作　　者：吕瑶 梁亮[1] 覃勉 蒋汝平 吴霞 Lü Yao;LIANG Liang;QIN Mian;JIANG Ruping;WU Xia(Department of Ophthalmology,Yichang Central People’s Hospital,the First College of Clinical Medical Science,China Three Gorges University,Yichang 443003,Hubei Province,China)

机构地区：[1]宜昌市中心人民医院眼科,三峡大学第一临床医学院,湖北省宜昌市443003

出　　处：《眼科新进展》2024年第9期686-691,共6页Recent Advances in Ophthalmology

基　　金：国家自然科学基金面上项目(编号:81770920);眼科学国家重点实验室开放课题(编号:303060202400383)。

摘　　要：目的 探究不同浓度的精氨酸-甘氨酸-天冬氨酸(RGD)多肽水凝胶,对Tenon囊成纤维细胞(HTF)活化和Yes相关蛋白(YAP)表达的影响。方法 制备0.5%、1.0%、2.0%三种浓度RGD多肽水凝胶,透射电子显微镜观察其内部微观结构,流变学检测弹性模量(E)。构建SD雄性大鼠结膜损伤模型后,分为空白组(不作处理)、低浓度RGD组(结膜下注射0.5%RGD多肽水凝胶)、中浓度RGD组(结膜下注射1.0%RGD多肽水凝胶)、高浓度RGD组(结膜下注射2.0%RGD多肽水凝胶),观察1周后取材,Masson染色观察大鼠结膜处胶原纤维增生情况,免疫组织化学检测大鼠结膜YAP、平滑肌肌动蛋白-α(α-SMA)表达情况。利用转化生长因子-β2(TGF-β2)刺激HTF活化构建瘢痕化细胞模型,按照实验要求分为对照组、 0.5%RGD水凝胶组、1.0%RGD水凝胶组、2.0%RGD水凝胶组、正常组,培养24 h后,Western blot检测HTF中YAP、结缔组织生长因子(CTGF)、α-SMA、纤连蛋白(FN)和I型胶原蛋白(Col I)的蛋白相对表达量;激光共聚焦显微镜观察细胞骨架蛋白(F-actin)和YAP蛋白表达定位。结果 电镜下观察可见,RGD多肽水凝胶内部纳米纤维交联密度随多肽浓度增加而升高,流变学测得0.5%、1.0%及2.0%RGD多肽水凝胶弹性模量(E)依次约为0.067 kPa、0.150 kPa、2.170 kPa。Masson染色结果显示,除中浓度RGD组大鼠结膜胶原纤维增生面积比明显低于空白组外(P<0.05),低浓度组、高浓度组与空白组差异均无统计学意义(均为P>0.05)。免疫组织化学结果显示,与空白组相比,低浓度RGD组、中浓度RGD组、高浓度RGD组大鼠YAP及α-SMA蛋白相对表达量均下降,中浓度RGD组下降最明显,差异均有统计学意义(均为P<0.05)。细胞免疫荧光染色结果显示,与对照组相比,0.5%RGD水凝胶组、1.0%RGD水凝胶组、2.0%RGD水凝胶组HTF的F-actin绿色荧光减弱,YAP红色荧光从细胞核转位于细胞质表达。Western blot检测结果显示,与对照组相比,0.5%RObjective To investigate the effects of arginine-glycine-aspartic acid(RGD)peptide hydrogels at different concentrations on the activation of Tenon capsule fibroblasts(HTF)and the expression of Yes-associated protein(YAP).Methods Three concentrations(0.5%,1.0%,2.0%)of RGD peptide hydrogels were prepared.Their internal microstructures were observed under a transmission electron microscope,and their elastic moduli(E)were measured using the rheological test.SD male rat models with conjunctival injury were established and divided into a blank group(no treatment),a low-concentration RGD group(subconjunctival injection of 0.5%RGD peptide hydrogel),a medium-concentration RGD group(subconjunctival injection of 1.0%RGD peptide hydrogel),and a high-concentration RGD group(subconjunctival injection of 2.0%RGD peptide hydrogel).After one week,tissues were collected,and collagen fiber proliferation in the rat conjunctiva was observed after Masson staining.Immunohistochemistry was performed to detect the expression of YAP andα-smooth muscle actin(α-SMA)in the rat conjunctiva.Scar formation cell models were constructed by stimulating HTF activation with transforming growth factor-β2(TGF-β2).According to experimental requirements,the cells were divided into a control group,a 0.5%RGD hydrogel group,a 1.0%RGD hydrogel group,a 2.0%RGD hydrogel group,and a normal group,and co-cultured for 24 hours.The relative expression levels of YAP,connective tissue growth factor(CTGF),α-SMA,fibronectin(FN),and type I collagen(Col I)proteins in HTF were detected by Western blot.The localization of cytoskeletal protein(F-actin)and YAP protein expression was observed under a confocal laser scanning microscope.Results Electron microscopy showed that the crosslinking density of the nanofibers inside the RGD peptide hydrogel increased with the rise of the peptide concentration.Rheological measurements indicated that the E values of the 0.5%,1.0%,and 2.0%RGD peptide hydrogels were approximately 0.067 kPa,0.150 kPa,and 2.170 kPa,respectively.Masson

关 键 词：TENON囊成纤维细胞 RGD多肽水凝胶 细胞外基质 Yes相关蛋白 瘢痕化 

分 类 号：R775.9[医药卫生—眼科]