【作者】 王鑫；

【导师】 彭广华；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2023， 硕士

【摘要】 背景和目的视网膜色素变性(Retinitis Pigmentosa,RP)是一种遗传性致盲性视网膜疾病,全球患病率约1/4000,是导致人类严重视力损害或失明的常见原因。其特点是随着视网膜感光细胞凋亡而逐渐丧失视功能。早期症状表现为夜间视力下降、视野缩小,最终导致失明,目前尚无有效的治疗方法。携带5号染色体Pde6b基因突变的rd1小鼠模拟人类RP进程,是一种经典的视网膜感光细胞变性的动物模型,适用于RP发病机制和治疗研究。miRNA是长约22 nt的内源性非编码小RNA分子,不参与编码蛋白质,在基因的转录后调控方面发挥重要作用。miRNA通过靶向特定的mRNA参与多种疾病的发生、进展和预后。视网膜感光细胞主要负责将外源性光信号转导为生物电信号,在感知光信号和视觉成像方面发挥着重要作用。miRNA-124-3p与视网膜感光细胞的发育成熟和功能维持密切相关,参与抑制视网膜炎症和光诱导的视网膜感光细胞变性的过程。本课题以rd1小鼠为研究对象,分析rd1小鼠视网膜变性过程中视网膜感光细胞的凋亡和炎症以及miRNA-124-3p表达水平的变化,研究miRNA-124-3p在视网膜感光细胞变性过程中的靶向干预效应,旨在探索和寻找RP治疗的新靶点。方法1.rd1小鼠视网膜变性过程中视网膜功能和组织形态变化:视网膜电图(Electroretinogram,ERG)检测rd1小鼠视网膜功能变化;视网膜相干断层扫描(Optical Coherence Tomography,OCT)和视网膜组织切片苏木素伊红(Hematoxylin and Eosin,H&E)染色观察rd1小鼠视网膜组织形态的变化。2.rd1小鼠视网膜感光细胞凋亡和炎症水平检测:TUNEL染色检测rd1小鼠视网膜感光细胞凋亡;Western Blot和免疫荧光染色检测rd1小鼠视网膜视杆细胞特异性标志物视紫红质(Rhodopsin,Rho)的表达变化;离子钙接头蛋白抗原-1(Ionized Calcium Binding Adaptor Molecule-1,IBA-1)免疫荧光染色观察rd1小鼠视网膜小胶质细胞形态和位置变化;Western Blot和qRT-PCR检测rd1小鼠视网膜中肿瘤坏死因子α(Tumor Necrosis Factor-α,TNF-α)、白介素1β(Interleukin-1β,IL-1β)的表达变化。3.miRNA-124-3p在rd1小鼠视网膜中的表达检测:出生第14天rd1小鼠和C57BL/6J小鼠视网膜总RNA进行miRNA高通量测序,分析差异表达的miRNA;qRT-PCR检测出生第14天rd1小鼠视网膜中miRNA-124-3p的表达水平。4.miRNA-124-3p在rd1小鼠视网膜变性过程中的作用:构建携带miRNA-124-3p前体序列的自互补腺相关病毒(Self-complementary Adeno-associated Virus,scAAV)载体和对照组scAAV载体,出生第10天的rd1小鼠视网膜下腔注射scAAV,8天后检测干预效果。绿色荧光蛋白(Superfolder green Fluorescent Protein,sf GFP)和DAPI染色确认转染位置;qRT-PCR检测转染效率;ERG检测视网膜功能;OCT和H&E染色观察视网膜组织形态;TUNEL染色检测视网膜感光细胞凋亡变化;免疫荧光染色和Western Blot检测Rho表达差异;IBA-1免疫荧光染色观察小胶质细胞活化数量变化;qRT-PCR检测促炎细胞因子表达变化。结果1.rd1小鼠视网膜变性过程中视网膜功能和组织形态的变化:与相应时间点C57BL/6J小鼠相比,rd1小鼠视网膜暗适应0.01 ERG的a波、暗适应3.0 ERG的a波和b波振幅显著降低;出生第14、18和21天的rd1小鼠视网膜总厚度和视网膜外核层(Outer Nuclear Layer,ONL)厚度明显变薄并且逐渐加重,ONL细胞形态紊乱,至出生第18天仅剩一层,出生第21天ONL细胞排列更为稀疏。2.rd1小鼠视网膜感光细胞凋亡和视网膜中炎症水平升高:出生第8天的rd1小鼠视网膜内核层(Inner Nuclear Layer,INL)开始出现TUNEL阳性细胞,出生第14天ONL中凋亡的感光细胞数达高峰;视杆细胞标志物Rho的免疫荧光染色和Western Blot结果发现,出生第14天、18天和21天的rd1小鼠视网膜中Rho的表达逐渐降低,出生第21天仅能观察到少量散在分布的Rho表达;IBA-1免疫荧光染色显示出生第14天、18天和21天的rd1小鼠视网膜中小胶质细胞活化,活化的小胶质细胞向ONL迁移,小胶质细胞活化数量于出生第14天达高峰;出生第14天、18天和21天的rd1小鼠视网膜中促炎细胞因子TNF-α、IL-1β的表达水平上调。3.miRNA-124-3p在rd1小鼠视网膜中的表达下调:miRNA124-3p在出生第14天的rd1小鼠和C57BL/6J小鼠视网膜中差异表达,qRT-PCR结果显示,出生第14天rd1小鼠视网膜中miRNA-124-3p的表达水平较同龄C57BL/6J小鼠显著降低,与miRNA测序结果一致。4.miRNA-124-3p延缓rd1小鼠视网膜变性:视网膜下腔注射scAAV过表达miRNA-124-3p,DAPI染色和绿色荧光蛋白显示病毒转染视网膜感光细胞层,qRT-PCR验证miRNA-124-3p在rd1小鼠视网膜中表达上调。过表达miRNA-124-3p显著提高rd1小鼠视网膜暗适应0.01 ERG的a波、暗适应3.0 ERG的a波和b波振幅;过表达miRNA-124-3p使rd1小鼠视网膜总厚度和ONL厚度增加;过表达miRNA-124-3p可减少rd1小鼠视网膜感光细胞凋亡数量,Rho蛋白表达升高;过表达miRNA-124-3p抑制rd1小鼠视网膜小胶质细胞的活化,抑制促炎细胞因子TNF-α的表达。结论1.rd1小鼠视网膜变性过程中,随着视网膜变性的进展,视网膜内炎症水平升高,miRNA-124-3p表达下调。2.rd1小鼠视网膜内过表达miRNA-124-3p,能够抑制视网膜炎症反应,改善视网膜功能和组织形态,延缓rd1小鼠视网膜变性过程。更多还原

【Abstract】 Background and PurposeRetinitis pigmentosa(RP)is an inherited blinding retinal disease with a global prevalence of 1/4000 and is a common cause of severe visual impairment or blindness in humans.It is characterized by progressive loss of vision with apoptosis of retinal photoreceptor cells.From early symptoms of night vision loss and visual field narrowing,it gradually progresses to eventually blindness,for which there is a lack of effective treatment.rd1 mice with a mutation in the Pde6b gene on chromosome 5mimic the human RP process,which is a classical animal model of retinal photoreceptor degeneration and suitable for the study of RP pathogenesis and treatment.Mi RNA is an endogenous non-coding small RNA about 22 nt which is not participated in encoding proteins and plays an important role in the post-transcriptional regulation of genes.Mi RNAs are involved in the development,progression and prognosis of many diseases by targeting specific mRNAs.Retinal photoreceptor cells are responsible for transducing exogenous light signals into bioelectrical signals and play an integral role in the perception of light and visual signals.Mi RNA-124-3p is not only closely associated with the development and functional maintenance of retinal photoreceptors and also involved in inhibiting the retinal inflammation and the process of photoinduced retinal photoreceptor degeneration.In this study,we analyzed the apoptosis and inflammation of retinal photoreceptor cells and the changes of miRNA-124-3p expression level during retinal degeneration in rd1 mice,and investigated the targeting intervention effect of miRNA-124-3p in retinal photoreceptor cells degeneration,aiming to explore a new target for RP therapy.Methods1.Functional and histological changes in the retina of rd1 mice during retinal degeneration:Electroretinogram(ERG)was used to detect functional changes of rd1 mouse retina.Optical coherence tomography(OCT)and Hematoxylin eosin(H&E)staining of retinal tissue sections were performed to observe retina histological changes of rd1 mice.2.Detection of retinal photoreceptor cell apoptosis and inflammation level of rd1 mice:TUNEL staining was used to observe retinal photoreceptor cell apoptosis of rd1 mice;Western Blot and immunofluorescence staining were operated to detect the expression changes of retinal rod cell specific marker(Rhodopsin,Rho)in rd1 mice.Ionized calcium binding adaptor molecule-1(IBA-1)immunofluorescence staining was performed to observe the morphological and positional changes of retinal microglia cells in rd1 mice.Western Blot and qRT-PCR were used to detect the expression changes of pro-inflammatory cytokines Tumor necrosis factor-α(TNF-α)and Interleukin-1β(IL-1β)in the retina of rd1 mice.3.Detection of miRNA-124-3p expression in rd1 mice retina:Use the next generation sequencing to detect the differential expression of miRNA in retina between rd1 mice and C57BL/6J mice at postnatal day 14.The expression level of miRNA-124-3p in the retina of rd1 mice at postnatal day 14 was detected by qRT-PCR.4.Inhibiting effect of miRNA-124-3p on retinal degeneration in rd1 mice:Self-complementary adeno-associated virus(scAAV)vector carrying the miRNA-124-3p precursor gene for treatment group and scAAV vector for the control group were constructed.scAAV were injected in the subretinal cavity of rd1 mice at postnatal day10,and subsequent experiments were performed 8 days later.Super folder green fluorescent protein(sf GFP)and DAPI staining was used to confirm the transfection site.qRT-PCR was used to detect transfection efficiency.ERG was applied to detect retinal function.OCT and H&E staining were performed to observe retinal tissue morphology.TUNEL staining was carried out to detect apoptotic changes in retinal photoreceptor cells.The different expression of Rhodopsin was detected by immunofluorescence staining and Western Blot.Changes in the number of retinal microglia cells activation were observed by IBA-1 immunofluorescence staining.qRT-PCR was used to detected changes in expression of pro-inflammatory cytokines.Results1.Changes in retinal function and histology during retinal degeneration in rd1 mice:Compared with C57BL/6J mice at the corresponding time,the a-wave of dark-adapted 0.01 ERG,the a-wave and b-wave amplitudes of dark-adapted 3.0 ERG of rd1 mice retina were significantly lower.The total retinal thickness and Outer nuclear layer(ONL)of rd1 mice at postnatal day 14,postnatal day 18,and postnatal day 21 were significantly thinner and the cell morphology of ONL was disordered.Only one-layer cells remained at postnatal day 18 and the cell arrangement of ONL was more sparse at postnatal day 21.2.Apoptosis of retinal photoreceptor cells and increased inflammation in the retina of rd1 mice:TUNEL-positive cells began to appear in the Inner nuclear layer(INL)of the retina in rd1 mice at postnatal day 18,and the number of apoptotic retinal photoreceptor cells in the ONL reached a peak at postnatal day 14.Immunofluorescence staining and Western Blot results showed that the expression of Rho was reduced in the retinas of rd1 mice at postnatal day 14,postnatal day 18 and postnatal day 21,and only a small amount of scattered Rhodopsin expression could be observed at postnatal day 21.IBA-1 immunofluorescence staining showed that the retinal microglial cells in rd1 mice were activated at postnatal day 14,postnatal day 18and postnatal day 21 and the number of activated retinal microglia cells peaked at postnatal day 14.The expression of pro-inflammatory cytokines TNF-αand IL-1βin the retinas of rd1 mice at postnatal day 14,postnatal day 18 and postnatal day 21increased.3.The expression of miRNA-124-3p was down-regulated in the retinas of rd1mice:The next generation sequencing revealed that miRNA124-3p was differentially expressed in the retina of rd1 and C57BL/6J mice at postnatal day 14.qRT-PCR results showed that the expression of miRNA-124-3p was significantly lower in the retinas of rd1 mice at postnatal day 14 compared with that of C57BL/6J mice.It was consistent with miRNA sequencing results.4.miRNA-124-3p retarded the retinal degeneration in rd1 mice:Subretinal injection of scAAV was performed to overexpress miRNA-124-3p.sf GFP and DAPI staining showed that the scAAV transfected retinal photoreceptor cell layer and qRT-PCR verified that the expression of miRNA-124-3p was upregulated in the retina of rd1 mice.Overexpression of miRNA-124-3p significantly increased both the a-wave of dark-adapted 0.01 ERG and the a-wave and b-wave amplitudes of dark-adapted 3.0ERG of rd1 mice retina.Overexpression of miRNA-124-3p increased the total retinal thickness and thickness of ONL in rd1 mice.Overexpression of miRNA-124-3p decreased the number of retinal photoreceptor cell apoptosis in rd1 mice.Overexpression of miRNA-124-3p inhibited the activation of retinal microglia cells and suppressed the expression of the pro-inflammatory cytokine TNF-αin rd1 mice.Conclusions1.The inflammation level is increased and miRNA-124-3p expression is down-regulated with the progression of retinal degeneration in rd1 mice.2.Overexpression of miRNA-124-3p in rd1 retina suppresses the inflammatory response of retina,improves the retinal function and histomorphology,and delays rd1mice retinal degeneration.更多还原