【作者】 李越；

【导师】 惠延年；

【作者基本信息】 第四军医大学 ， 眼科学， 2008， 博士

【摘要】 研究背景:年龄相关性黄斑变性(AMD)是全球范围内老年人群中的主要致盲性眼病,其发病机制尚不清楚。视网膜色素上皮细胞的老化及伴随发生的功能障碍被认为是AMD发生中关键的早期病变。20S蛋白酶体在维持细胞内环境平衡和调节细胞基本生理功能中具有重要作用,与细胞的老化和衰老有密切关系。已证明在多种细胞和组织的老化进程中,20S蛋白酶体活性、含量和表达水平发生改变,这些改变具有高度的细胞和组织特异性;同时,20S蛋白酶体活性受到特异性的抑制或加强时,可诱导细胞老化进程的加速或减缓。20S蛋白酶体在人RPE细胞的老化和衰老中很可能也具有相似的效应,但目前尚未见相关报道。目的:通过检测人RPE细胞老化中的20S蛋白酶体活性、含量和表达水平的变化,了解20S蛋白酶体的氧化蛋白质降解功能与人RPE细胞老化间的相关性;进一步通过特异性抑制20S蛋白酶体活性,分析其对RPE细胞老化进程的影响及可能的机制。方法:(1)参照本实验室已有方法,建立原代培养的人RPE细胞体外复制衰老模型,常规传代并记录传代次数,选择第2~4代、12~14代和22~24代细胞分别作为RPE细胞体外老化的少年期、中年期和老年期细胞,对各期细胞的老化和衰老特征进行如下鉴定:倒置相差显微镜观察形态特征、电子显微镜观察亚细胞特征、XTT法检测增生活性、β-半乳糖苷酶染色检测衰老细胞比例和流式细胞仪检测自发荧光水平。(2)在经鉴定的人RPE细胞复制衰老模型中,研究20S蛋白酶体的年龄相关性改变,包括:荧光标记蛋白质底物法测定20S蛋白酶体的降解活性,DNPH比色法检测细胞内氧化蛋白质水平,western blot检测20S蛋白酶体的含量,细胞免疫荧光法分析RPE细胞内的氧化蛋白质和20S蛋白酶体的分布和相对定量,以及real-time PCR分析20S蛋白酶体活性亚基的表达水平。(3)用不同浓度的蛋白酶体特异性抑制剂MG132处理少年期RPE细胞,检测短暂抑制、持续抑制或抑制后恢复三种情况下的RPE细胞的老化和衰老特征,并进一步测定MG132干预后RPE细胞的蛋白酶体活性和氧化蛋白质水平,分析20S蛋白酶体活性抑制对人RPE细胞老化进程的影响效应及其可能的作用机理。结果:(1)以少年期细胞作为对照,随着RPE细胞体外传代次数的增加,中年期和老年期RPE细胞部分出现体积变大、形态和排列失规则;电子显微镜观察可见老年期的细胞胞浆中有空泡形成、尘样致密物聚集和细胞器结构模糊等改变;同时细胞增生活性明显降低,β-半乳糖苷酶染色阳性细胞比例显著增加,细胞内自发荧光水平升高。提示人RPE细胞经过重复的体外传代后逐渐老化,具备衰老表型。(2)蛋白酶体活性检测发现,老年期RPE细胞中20S蛋白酶体的PGPH、CT-L和T-L三种特异性水解活性较少年期均显著降低,蛋白酶体对FITC-酪蛋白的降解能力也减弱至少年期细胞活性的50%左右。蛋白酶体功能的削弱伴随着细胞内氧化蛋白质水平显著升高,然而,细胞内20S蛋白酶体的含量无显著改变。进一步的细胞免疫荧光定位和定量分析提示,RPE细胞内的氧化蛋白质随着老化由细胞核周区域重分布至核内,老化相关的氧化蛋白质水平升高在细胞核周区域和核内最为显著;而20S蛋白酶体的分布始终以细胞核内为主。20S蛋白酶体比氧化蛋白质的比率反映了蛋白酶体的水解效率,少年期RPE细胞核中这一比率的值最大,到老年期降低至不到50%,而细胞浆中的这一比率较为低且稳定。实时定量PCR分析显示RPE细胞中,20S蛋白酶体β5亚基的基因表达随老化呈双相改变,老年期表达水平显著降低,分别是少年期和中年期的83%和68%。(3)亚毒性剂量的MG132处理后,年轻RPE细胞出现不可逆的生长停滞、形态和排列失规则、胞浆空泡化和β-半乳糖苷酶染色增强的衰老表型,这一诱导衰老效应具有剂量和时间依赖性。进一步的蛋白酶体功能检测和细胞内氧化蛋白质定量分析提示,5μM浓度的MG132首先抑制RPE细胞中的PGPH活性和CT-L活性,并显著降低20S蛋白酶体对FITC标记酪蛋白的降解能力,但T-L活性对这一浓度的MG132较为不敏感。同时,这一浓度的MG132并不引起RPE细胞内氧化蛋白质水平的明显增加。10μM浓度的MG132可显著抑制蛋白酶体的3种水解活性,并使RPE细胞内的氧化蛋白质含量显著升高。结论:(1)体外培养的RPE细胞经过重复多次的传代后表现出典型的衰老表型,提示RPE细胞的复制衰老模型可用于老化和衰老相关研究;(2)人RPE细胞中的20S蛋白酶体功能随着老化而降低,伴随细胞内氧化蛋白质含量升高。这一蛋白酶体功能的老化相关性减弱可能是活性亚基的表达水平改变和/或发生氧化性修饰造成的。RPE细胞中的20S蛋白酶体集中分布在细胞核内,细胞核内的蛋白酶体降解效率随老化显著降低,提示蛋白酶体在细胞核中的作用与RPE细胞的老化进程关系最为密切。(3)蛋白酶体特异性抑制剂MG132可诱导年轻RPE细胞出现不可逆的衰老表型,这一诱导效应的原因至少包括氧化蛋白质聚集和蛋白酶体对多种细胞因子调节障碍,后者所占的比重很可能超过预期,仍待进一步探索。总之,我们的实验提示蛋白酶体在RPE细胞老化、功能失调和AMD的发病中具有重要的调节效应。这些研究结果目前尚未见于报道。更多还原

【Abstract】 Background: Age-related macular degeneration (AMD) remains high incidence and accounts for a main cause of blindness in aging people, but its mechanism is still poorly understood. Aging and associated dysfunction of retinal pigment epithelial (RPE) cells are believed to be the pathological onset of AMD. The 20S proteasome has been tightly correlated with cell aging due to its fundamental role in maintaining cellular homeostasis, but its implication in human RPE cell aging was seldom concerned.Purpose: This study aimed to demonstrate the interconnections between proteasome and RPE aging by characterizing age-dependent changes of the 20S proteasome in primarily cultured human RPE cells, and further investigate the impact of specific proteasome inhibition on RPE aging process.Methods: (1) A replicative aging RPE cell model was established based on previous work of our lab. The primarily cultured human RPE cells were maintained and subcultured as usual. Passages 2~4, 12~14 and 22~24 were selected and defined as the young, middle and old passage group, respectively. For each passage group, RPE cells went through inverted phase contrast microscope and electro-microscope observation, XTT test for cell viability,β-galactosidase staining for senescence detection, and flow cytometry assay for cellular autofluorescence intensity. (2) The fluorogenic substrates (LLE-AMC, suc-LLVY-AMC, LSTR-AMC and FITC-casein) were used to determine the proteolytic activities of the 20S proteaosome in RPE lysates. Colorimetric carbonyl assay and western blot were employed for measurement of oxidized protein and 20S proteasome content, respectively. Immunofluorescence assay was applied for intracellular localization and quantification of both the oxidized proteins and 20S proteasome. Real-time PCR was used to detect the gene expression of the proteasomal proteolytic subunits. (3) RPE cells of early passage group were treated with MG132, a specific proteasome inhibitor, and the age-related characters were investigated through microscope observation, XTT test andβ-galactosidase staining. The proteasome function and intracellular oxidized protein content was determined with aforementioned methods.Results: (1) RPE cells in culture exhibited typical features of senescent at the late stage of cell growth, as they were enlarged, did not line up in parallel arrays, showed plasmic vaculation, had reduced cell viability, turned positive toβ-galactosidase senescence biomarker, and had elevated cellular autofluorescence. (2) Significant decline in all the three specific activities and degradation of FITC-casein of the 20S proteasome was found in aging RPE cells. The malfunctions were accompanied with remarkably increased content of oxidized proteins in the old RPE cells, and stable content of the 20S core. Immunofluorescence assay revealed that a most significant elevation of the oxidized proteins content occurred in the peri-nuclear regions and nucleus, while the 20S proteasome was concentrated in RPE nucleus regardless of passages. Proteasome-to-oxidized protein ratio indicated functional efficiency of the 20S proteasome. The highest value of this ratio was found in the nucleus of young RPE passages, which declined to less than 50% in the old passages. Real-time PCR assay revealed an up-regulated expression of the proteasomalβ5 subunit followed by a down-regulated level during RPE aging process. (3) Treatment with sub-toxic dose of MG132 elicited irreversible senescence-like features in young RPE cells, including growth arrest, typical senescence morphology, plasmic vaculation, and enhancedβ-galactosidase staining. These inducible features appeared to be dose- and time-dependent. While 5μM of MG132 inhibited a large part of PGPH activity, CT-L activity and FITC-casein degradation of the proteasome, the same dosage exhibited little effect on T-L activity and intracellular oxidized protein level. 10μM of MG132 diminished proteasomal activities and lead to significantly higher content of oxidized proteins in young RPE cells.Conclusions: An aging model of human RPE cells was validated for age- and senescence-related investigation in vitro. In such a model, we testified age-related malfunction of the 20S proteasome, concomitant with increased oxidized protein level. Partial inhibition of proteasomes in young RPE cells caused by treatment with specific inhibitors induced a senescence-like phenotype. Thus we demonstrated the fundamental importance of the proteasome in human RPE cells aging. Future researches on the mechanism of these events are still strongly recommended.To sum up, our research supported a fundmental role of the proteasome in RPE cell aging. To the best of our knowledge, similar findings have not been reported yet at home and abroad.更多还原