【作者】 邹慧；

【导师】 赵劲松；

【作者基本信息】 吉林大学 ， 眼科学（专业学位）， 2022， 博士

【摘要】 增生性玻璃体视网膜病变(Proliferative Vitreoretinopathy,PVR)是一种致盲性眼底疾病,常见于孔源性视网膜脱离(Rhegmatogenous retinal detachment,RRD)、眼外伤和内眼手术后。PVR被认为是视网膜过度损伤修复的过程,其特点是在玻璃体腔和视网膜周围形成了可收缩的纤维增殖膜,目前,手术仍是PVR的主要治疗方法,尽管手术技术在不断提高,但PVR的手术效果仍不尽如人意,是视网膜脱离及眼外伤手术失败的主要原因。因此,从PVR的发病机理入手,探索预防及治疗PVR的新方法以提高术后视功能就显得尤为重要。PVR的发病机制错综复杂,生长因子,细胞因子,细胞外基质和多种细胞之间的相互作用均参与其中。目前研究多表明视网膜色素上皮(Retinal pigment epithelium,RPE)细胞是PVR发生发展的关键细胞之一。在正常生理状态下,分化成熟的RPE细胞一直处于静止不分裂状态,对维持视网膜和脉络膜的正常功能具有重要作用。然而当视网膜出现裂孔或眼球受到外伤引起视网膜脱离时,多种因素导致RPE细胞的连接复合体被破坏,与Bruch膜分离,迁移至视网膜神经上皮缺损处,发生增生并转化为肌成纤维细胞,形成纤维增殖膜,最终加剧视网膜脱离导致视力进一步下降。在这一过程中RPE细胞发生上皮-间质转化(Epithelial-mesenchymal transition,EMT),由上皮细胞转化为间质细胞,使其增殖、迁移、抗凋亡能力增强,并且分泌细胞外基质增多,在PVR形成过程中发挥着关键的作用。CD44为广泛表达于多种细胞的跨膜糖蛋白,参与生长发育、炎症、免疫反应、损伤修复、肿瘤转移等多种病理生理过程的调节。CD44不仅可通过与细胞外配体结合形成跨膜复合体,而且可通过与细胞内骨架蛋白结合来调节多条细胞信号通路的传导,从而对多种细胞功能进行调节,如细胞生长,细胞分化和细胞迁移。正常的RPE细胞处于静止不分裂状态,不表达CD44,而在病理状态如RRD中,RPE细胞发生增殖和迁移,出现CD44的表达,且RPE细胞CD44的表达变化与RRD的病理发展关系密切。有研究表明CD44可促进肺脏、肝脏、肾脏等其它脏器发生纤维化,但CD44在PVR发生发展过程中的作用还有待进一步研究。4-甲基伞形酮(4-methylumbelliferone,4MU)是一种香豆素衍生物,具有利胆和抗痉挛的作用。4MU不仅可以通过抑制透明质酸的合成来发挥抗肿瘤、抗炎及抗纤维化的作用,而且还可以使CD44的表达下调,抑制细胞内信号通路的传导,从而使细胞的增殖和迁移能力降低。因此,我们推测4MU可能也对RPE细胞的增殖和迁移具有抑制作用,从而阻止PVR中细胞纤维膜的形成。因此本研究先收集PVR患者的增殖膜,检测增殖膜中CD44的表达情况,然后利用TGF-β2建立ARPE-19细胞发生EMT的体外模型,并对CD44和4MU在RPE细胞EMT中的作用及分子机制进行研究,进一步阐明PVR的发生和发展机制,为临床上对该疾病的预防和治疗提供理论依据。目的:检测CD44在PVR增殖膜中的表达情况;研究CD44在ARPE-19细胞发生上皮间质转化中的作用及机制;并进一步探索4MU对ARPE-19细胞EMT的作用及机制。方法:1.临床手术中收集PVR患者的增殖膜样本,并利用免疫荧光技术检测CD44和细胞角蛋白CK-18的表达情况。2.利用TGF-β2建立ARPE-19细胞EMT体外模型,观察RPE细胞的形态变化情况,用CCK-8法检测TGF-β2处理24小时后ARPE-19细胞的增殖,通过细胞划痕实验和Transwell细胞迁移实验检测RPE细胞迁移能力,用Western Blot检测ARPE-19细胞间质型蛋白α-SMA、Fibronectin(FN)、N-cadherin和Vimentin的表达水平。另外,在TGF-β2处理的不同时间点,检测CD44蛋白及间质型蛋白的表达情况。3.向ARPE-19细胞转染CD44si RNA,在转染并经TGF-β2作用后检测各组RPE细胞的增殖活力及迁移能力;转染后并经TGF-β2作用48h时用Western Blot检测α-SMA、FN、N-cadherin、Vimentin、Smad2/Smad3和磷酸化Smad2/磷酸化Smad3的表达水平。4.利用CD44抗体抑制CD44的作用,先向ARPE-19细胞给予浓度为20μg/m L的Ig G抗体/CD44抗体作用后再给予TGF-β2处理,用Western Blot检测α-SMA、FN、N-cadherin、Vimentin、Smad2/Smad3和磷酸化Smad2/磷酸化Smad3的表达水平。5.向ARPE-19细胞加入不同浓度的4MU进行培养,检测RPE细胞的增殖及迁移情况,并通过Western Blot检测RPE细胞CD44及间质型蛋白α-SMA、FN、N-cadherin、Vimentin的表达情况,筛选合适的4MU浓度。6.我们选择0.8m M为4MU的作用浓度,将实验分为三组,分别为对照组,TGF-β2组和TGF-β2+4MU组,对照组为正常培养的ARPE-19细胞,TGF-β2组是给予浓度为10ng/m L的TGF-β2作用的ARPE-19细胞,TGF-β2+4MU组是同时给予浓度为10ng/m L的TGF-β2和浓度为0.8m M的4MU作用的ARPE-19细胞。应用CCK-8试剂盒检测各组RPE细胞的增殖能力,利用细胞划痕实验和Transwell细胞迁移实验来检测4MU对TGF-β2诱导的RPE细胞迁移的影响,用Western Blot检测各组RPE细胞CD44、α-SMA、FN、N-cadherin、Vimentin、Smad2/Smad3和磷酸化Smad2/磷酸化Smad3的表达情况。结果:1.收集了6例PVR患者的增殖膜样本,PVR增殖膜的免疫荧光检测结果显示:6例PVR患者的增殖膜上均能检测到CD44表达,且CD44和细胞角蛋白CK-18存在共定位。2.TGF-β2作用后的ARPE-19细胞由鹅卵石样上皮细胞形态转变为长梭形的纤维细胞样形态。CCK-8的结果显示与对照组相比,TGF-β2对RPE细胞的增殖有促进作用(P<0.0001)。划痕实验和Transwell迁移实验结果显示TGF-β2促进RPE细胞的迁移(P<0.05)。TGF-β2组RPE细胞的α-SMA、FN、N-cadherin和Vimentin蛋白表达水平较对照组明显增加,差异具有统计学意义(P<0.05)。TGF-β2使CD44蛋白表达上调,且在TGF-β2诱导24h时CD44表达最多,48h时CD44有下调的趋势,但与24h之间的差异无统计学意义(P=0.3919)。间质型蛋白α-SMA、FN、N-cadherin和Vimentin的表达水平随着TGF-β2作用时间延长而增加,都在TGF-β2诱导48h时表达最多。3.用CD44si RNA敲低了ARPE-19细胞CD44的表达,各组之间进行比较分析,结果显示与TGF-β2组和NCsi RNA+TGF-β2组相比,CD44si RNA+TGF-β2组RPE细胞的增殖能力降低(P<0.05);24h和48h时CD44si RNA+TGF-β2组细胞划痕愈合的面积较前两者的减小(P<0.05);24h时CD44si RNA+TGF-β2组迁移至下室的RPE细胞也明显减少(P<0.05);48h时CD44si RNA+TGF-β2组间质型蛋白α-SMA、FN、N-cadherin和Vimentin的表达水平下降(P<0.05),且磷酸化Smad2/磷酸化Smad3蛋白与总的Smad2/Smad3蛋白的比值也降低。4.将CD44m Ab+TGF-β2组分别与TGF-β2组、Ig Gm Ab+TGF-β2组进行组间比较,结果显示CD44m Ab+TGF-β2组α-SMA、FN、N-cadherin和Vimentin的表达水平及磷酸化Smad2/磷酸化Smad3蛋白与总的Smad2/Smad3蛋白的比值较后两组的都降低,差异有统计学意义(P<0.05)。5.在4MU作用24小时,与对照组相比,浓度为0.2m M的4MU对ARPE-19细胞的增殖无明显抑制作用(P=0.1237),另外4种浓度的4MU对RPE细胞的增殖均有抑制作用(P<0.05),而在作用48h时,不同浓度4MU组ARPE-19细胞的增殖能力都较对照组降低(P<0.05)。细胞划痕实验结果显示,与对照组进行比较,4MU抑制ARPE-19细胞的迁移,且呈现时间和浓度依赖性(P<0.05)。4MU使ARPE-19细胞CD44和间质型蛋白α-SMA、FN、N-cadherin和Vimentin的表达降低,且浓度为0.8m M的4MU的抑制作用最明显。6.CCK-8的结果显示TGF-β2+4MU组RPE细胞的增殖活性较TGF-β2组的降低,差异具有统计学意义(P<0.05)。细胞划痕实验结果显示在24h和48h时,TGF-β2+4MU组的RPE细胞划痕愈合面积较TGF-β2组的减小(P<0.05)。Transwell细胞迁移实验结果显示与TGF-β2组相比,TGF-β2+4MU组RPE细胞的迁移能力降低(P<0.05)。蛋白印迹结果显示TGF-β2+4MU组ARPE-19细胞的间质型蛋白α-SMA、FN、N-cadherin和Vimentin的表达水平及磷酸化Smad2/磷酸化Smad3蛋白与总的Smad2/Smad3蛋白的比值低于TGF-β2组的(P<0.05)。结论:1.PVR增殖膜中的RPE细胞有CD44的表达。2.TGF-β2促进体外ARPE-19细胞发生EMT,且ARPE-19细胞在发生EMT过程中伴随了CD44的上调。3.CD44si RNA和CD44抗体可能通过抑制Smad2和Smad3的磷酸化来抑制RPE细胞发生EMT。4.4MU可能通过SMAD通路抑制TGF-β2诱导的RPE细胞EMT。更多还原

【Abstract】 Proliferative vitreoretinopathy(PVR)is a blinding disease occurring secondary to rhegmatogenous retinal detachment(RRD),ocular trauma,or ocular surgical procedures.PVR has been considered as an exaggerated wound-healing process,and is characterized by the formation of contractile fibrocellular membranes within the vitreous cavity and on the preretinal and subretinal surfaces.At present,surgery remains the mainstay of treatment in PVR.Although there have been tremendous advancements in surgical techniques and management,PVR cannot be effectively treated and is still the primary cause of surgical failure when treating retinal detachment or ocular trauma.Therefore,in order to improve postoperative visual function,it is particularly important to explore new prophylactic and therapeutic approaches based on a deeper understanding of the pathogenesis of PVR.The mechanisms of PVR are orchestrated by multiple elements such as growth factors,cytokines,extracellular matrix proteins and various cells.So far,a growing body of evidence has been indicated that the retinal pigment epithelial(RPE)cell plays a crucial role in PVR.The terminally differentiated RPE cell is mitotically quiescent under physiological conditions.The RPE,which is located between the neural retina and the choroid,plays various roles indispensable for function of the neural retina and the choroid.However,when the eye suffers from a retinal break or trauma,multiple factors lead to the disruption of junctional complexes in RPE cells.Subsequently,RPE cells become detached from Bruch’s membrane,migrate through breaks in the neural retina,proliferate,and transform into myofibroblasts.These cells participate in the growth of fibrotic membranes,which contract and lead to further retinal detachment and blurring vision.In the process,RPE cells undergo epithelial-mesenchymal transition(EMT),which plays a pivotal role in the development of PVR.During EMT,RPE cells transdifferentiate into mesenchymal cells that are characterized by enhanced proliferative and migratory capacity,and resistance to apoptosis.They produce extracellular matrix proteins and lead to PVR.CD44 is a ubiquitous transmembrane glycoprotein which is expressed on a variety of cell types and is implicated in many physiological and pathological processes including development,inflammation,immune response,wound healing and cancer progression.CD44 participates in signal-transduction processes by establishing specific transmembrane complexes with extracellular ligands and by organizing signaling cascades through association with the actin cytoskeleton,thereby regulating many cellular processes,such as cell growth,differentiation and migration.CD44 has not been identified on normal RPE cells which remain mitotically quiescent.Under pathologic conditions,such as RRD,CD44 is expressed on proliferating and migrating RPE cells.The upregulation of CD44 in RPE cells has been correlated with the pathological progression of RRD.CD44 has been proposed to promote tissue fibrosis in lung,liver,kidney and so on.But the role of CD44 in PVR needs to be further studied.4-methylumbelliferone(4MU)is a derivative of coumarin that has choleretic and antispasmodic effects.4MU has anti-tumor,anti-inflammatory and anti-fibrotic effects by inhibiting the synthesis of hyaluronic acid.And 4MU abrogates cell proliferation and migration via downregulation of CD44 expression and its mechanisms of effects involve the inhibition of intracellular signal-transduction pathways.Therefore,we speculate that 4MU may prevent the formation of fibrocellular membranes in PVR by inhibiting the proliferation and migration of RPE cells.Therefore,in this study,we collected PVR membranes and detected the expression of CD44 in the membranes.Then the in vitro model of ARPE-19 cell EMT was established by using TGF-β2,and the effects and mechanisms of CD44 and 4MU in RPE cell EMT were investigated.This study further clarified the pathogenesis of PVR,and provided a theoretical basis for the prevention and treatment of PVR.ObjectiveThis study aimed to detect the expression of CD44 in the PVR membrane,and to explore the effects and mechanisms of CD44 in the epithelial-mesenchymal transition of ARPE-19 cells.We further investigated the effects and mechanisms of 4MU in ARPE-19 cell EMT.Methods1.PVR membranes were obtained from patients during routine surgical procedures and were evaluated by indirect immunofluorescence for CD44 and cytokeratin-18(CK-18).2.In vitro model of ARPE-19 cell EMT was created by incubating with TGF-β2.The morphological changes of RPE cells were observed.Cell proliferation was analyzed by Cell Counting Kit-8(CCK-8)assays at 24 h after the treatment of TGF-β2.The migratory capacity of RPE cells was evaluated by cell scratch assay and Transwell cell migration assay,and the expression of α-SMA,Fibronectin(FN),N-cadherin and Vimentin in ARPE-19 cells were detected by Western Blot.In addition,the expression of CD44 and mesenchymal proteins were detected at different time points of TGF-β2treatment.3.ARPE-19 cells were transfected with CD44 si RNA.After transfection with si RNA,ARPE-19 cells were incubated with TGF-β2,and cell proliferation and migration in each group were detected.Western blot was used to measure the expression of α-SMA,FN,N-cadherin,Vimentin,Smad2/Smad3 and phospho-Smad2/phosphoSmad3 at 48 h after TGF-β2 treatment.4.CD44 antibody was used to inhibit the effect of CD44.ARPE-19 cells were preincubated with 20μg/m L antibody to Ig G or CD44 and then treated with TGF-β2.Western Blot was used to detect the expression of α-SMA,FN,N-cadherin,Vimentin,Smad2/Smad3 and phospho-Smad2/phospho-Smad3.5.ARPE-19 cells were incubated with different concentrations of 4MU,and the proliferation and migration of RPE cells were analyzed.Western Blot was used to detect the expression of CD44,α-SMA,FN,N-cadherin,and Vimentin.To screen for the suitable concentration of 4MU.6.We chose 0.8m M as the appropriate concentration of 4MU,and divided the experiment into three groups,including control group,TGF-β2 group and TGF-β2+4MU group.The control group was normally cultured ARPE-19 cells.The TGF-β2group was ARPE-19 cells treated with 10ng/m L TGF-β2.ARPE-19 cells were treated with 10ng/m L TGF-β2 in the presence of 0.8m M 4MU in the TGF-β2+4MU group.The proliferation ability of RPE cells in each group was measured by CCK-8 kit.RPE cell migration was evaluated by cell scratch assay and Transwell cell migration assay.Western Blot was used to detect the expression of CD44,α-SMA,FN,N-cadherin,Vimentin,Smad2/Smad3 and phospho-Smad2/phospho-Smad3.Results1.The PVR membrane samples were obtained at vitrectomy from 6 patients with PVR.All 6 samples contained cells positive for CD44.And there are identifiable cells positive for both CD44 and CK-18.2.ARPE-19 cells treated with TGF-β2 changed from cobblestone-like epithelial appearance to long-spindle fibroblast-like shape.The results of CCK-8 assay showed that compared with the control group,TGF-β2 promoted RPE cell proliferation(P<0.0001).The results of scratch assay and Transwell migration assay showed that TGF-β2 induced RPE cell migration(P<0.05).Compared to the control group,TGF-β2 significantly stimulated the upregulation of α-SMA,FN,N-cadherin and Vimentin in RPE cells,and the difference was statistically significant(P<0.05).TGF-β2increased the expression of CD44,and the expression of CD44 was the highest at 24 h of TGF-β2 induction,and there was a downward trend of CD44 at 48 h,but there was no significant difference between 24 h and 48h(P=0.3919).α-SMA,FN,N-cadherin and Vimentin were significantly up-regulated in a time-dependent manner and were the highest at 48 h after TGF-β2 treatment(P<0.05).3.CD44 si RNA was used to knock down CD44 in ARPE-19 cells.Compared with the TGF-β2 group and the NCsi RNA+TGF-β2 group,RPE cells proliferation was reduced in the CD44 si RNA+TGF-β2 group(P<0.05).The scratched healing area of the CD44 si RNA+TGF-β2 group was reduced at 24 h and 48h(P<0.05).The migrated RPE cells were also significantly decreased in the CD44 si RNA+TGF-β2 group(P<0.05).At48 h,the expression of α-SMA,FN,N-cadherin and Vimentin decreased in CD44 si RNA+TGF-β2 group(P<0.05),and the ratio of phospho-Smad2/phosphoSmad3 to total Smad2/Smad3 was also reduced.4.Compared with the TGF-β2 group and the Ig Gm Ab+TGF-β2 group,the X expression of α-SMA,FN,N-cadherin,Vimentin and the ratio of phosphoSmad2/phospho-Smad3 to total Smad2/Smad3 decreased in the CD44 m Ab+TGF-β2group,and the difference was statistically significant(P<0.05).5.After 24 h of 4MU treatment,compared with the control group,0.2 m M 4MU had no significant inhibitory effect on the proliferation of ARPE-19 cells(P=0.1237),and the other four concentrations of 4MU significantly inhibited RPE cells proliferation(P<0.05).The proliferation of ARPE-19 cells in different concentrations of 4MU groups was reduced compared with the control group at 48h(P<0.05).The results of the cell scratch assay showed that compared with the control group,4MU significantly hampered ARPE-19 cells migration in a time and dose-dependent manner(P<0.05).Compared with the control group,4MU reduced the expression of CD44,α-SMA,FN,N-cadherin and Vimentin in ARPE-19 cells,and the inhibitory effect of 4MU at a concentration of 0.8 m M was the most obvious.6.The results of CCK-8 showed that RPE cells proliferation in the TGF-β2+4MU group was reduced compared to the TGF-β2 group,and the difference was statistically significant(P<0.05).The results of cell scratch assay showed that the scratched healing area in the TGF-β2+4MU group was smaller than that in the TGF-β2 group at 24 h and48h(P<0.05).The results of Transwell cell migration assay showed that compared with TGF-β2 group,RPE cells migration was reduced in TGF-β2+4MU group(P<0.05).The results of Western blotting showed the expression of α-SMA,FN,N-cadherin and Vimentin and the ratio between phospho-Smad2/phospho-Smad3 and total Smad2/Smad3 in ARPE-19 cells in TGF-β2+4MU group were lower than that of the TGF-β2 group(P<0.05).Conclusions1.The RPE cells in the PVR membrane have the expression of CD44.2.TGF-β2 promoted EMT in ARPE-19 cells in vitro,and ARPE-19 cells were accompanied by up-regulation of CD44 during EMT.3.CD44 si RNA and CD44 antibody may inhibit RPE cell EMT by inhibiting the phosphorylation of Smad2 and Smad3.4.4MU may inhibit TGF-β2-induced EMT in RPE cells through the SMAD pathway.更多还原