正常幼儿及不同年龄段白内障患者晶状体中常见miRNA的表达

目的：检测并分析miRNA在正常幼儿及不同年龄段白内障患者晶状体中的表达差异,初步探讨其在维持晶状体正常功能和不同年龄段白内障形成中的可能作用。

方法：使用stem-loop RT-PCR法检测正常幼儿和幼儿(先天性白内障)、中青年及老年(年龄相关性白内障)白内障患者晶状体中miRNA的表达情况,比较各组间miRNA表达差异。

结果：正常幼儿晶状体中miR-184表达量最高。与正常幼儿相比,白内障幼儿晶状体中miR-184、miR-182表达升高,miR-124、miR-204表达降低。与幼儿患者相比,中青年患者晶状体中miR-204、miR-124和let-7d表达升高,miR-184、miR-183和let-7a表达降低,而老年患者晶状体中所有被检测miRNA表达均有改变,其中miR-182、miR-204、miR-124表达均升高,miR-184、miR-181b、miR-183、miR-125b、let-7a/b/d表达均降低。

结论：正常幼儿及不同年龄段白内障患者晶状体中miRNA表达差异显著,部分miRNA与晶状体的正常形态、功能及某些病理状态相关,这为进一步研究miRNA在维持幼儿晶状体正常功能及不同年龄段白内障形成过程中的可能作用机制提供了理论依据。     

水通道蛋白1在老年性白内障及透明晶状体前囊膜上皮细胞的表达

目的明确水通道蛋白1(aquaporin1,AQP1)在透明晶状体及老年性白内障晶状体上皮细胞及前囊膜的表达及分布,并初步探讨AQP1在老年性白内障发病中的重要作用。方法收集52例老年性白内障及10例正常透明晶状体前囊膜,通过HE染色和免疫组化方法检测AQP1的表达。结果老年性白内障及正常透明晶状体上皮细胞中均可见AQP1的表达,透明晶状体组中AQP1的阳性细胞表达率(100%)显著高于老年性白内障组(53.8%)(P<0.05)。而在晶状体纤维中未见表达。结论晶状体上皮细胞是晶状体代谢最活跃的部位,终身进行有丝分裂,并移行至赤道部分化成晶状体纤维。晶状体上皮细胞有AQP1的活性表达,这对于维持晶状体上皮细胞一定的增殖潜能,保证晶状体的生理动态平衡和晶状体的透明性起到了重要的作用。提示AQP1在老年性白内障晶状体前囊膜及上皮细胞中的异常表达与老年性白内障的发生密切相关,具体调控机制有待进一步研究。     

miR-204调控眼科疾病的研究进展

微小RNA(miRNA)是一类广泛存在于真核生物体内、长度为20～ 25个核苷酸、具有调控功能的非编码单链RNA,参与机体的各种生命进程,包括细胞的生长、分化、增生、凋亡和自噬.miRNA-204-5p(miR-204-5p)是由位于染色体9q21.12上的TRPM3大内含子6表达.研究发现,miR-204在角膜损伤愈合过程中起着十分重要的作用,亦能够保持静止状态下血-视网膜屏障的稳定,并且在人小梁网细胞中,miR-204与细胞的凋亡、生存能力以及炎症介质的表达有着重要联系.这些研究都表明miR-204在眼部呈多维表达,提示miR-204很可能是不同眼部疾病的关键miRNA.本文从miRNA的生物合成,miR-204与糖尿病性角膜病变、视网膜色素上皮细胞、人小梁网细胞、年龄相关性白内障、糖尿病视网膜病变、视网膜母细胞瘤的关系,以及miR-204与自噬的相关研究等几个方面,就miR-204调控眼科疾病的研究进展进行综述,为探寻眼部难治性疾病的防治方法寻找新的靶点.     

Altered microRNA expression profiles in retinas with diabetic retinopathy

Rats with streptozotocin (STZ)-induced diabetes were studied in order to identify abnormal microRNA (miRNA) expression profiles in diabetic retinopathy (DR) and to ascertain miRNAs associated with DR. Histopathologically, we observed characteristic features of DR in rats at 10 weeks after STZ injection. Investigation of miRNA expression profiles in the retinas of control and diabetic rats using miRNA microarrays revealed that many miRNAs were abnormally expressed in DR. On the basis of their fold changes and probability values, a total of 37 miRNAs were selected for further validation by real-time PCR analysis. The results showed that 11 miRNAs were significantly upregulated and 6 miRNAs were notably downregulated in DR. Furthermore, these changes in retinal miRNA expression levels paralleled the course of DR. Levels of miR-182, miR-96, miR-183, miR-211, miR-204, and miR-124 were significantly increased during the progress of DR, whereas miR-10b, miR-10a, miR-219-2-3p, miR-144, miR-338, and miR-199a-3p were significantly decreased. Our data indicate that the aberrant miRNA expression profiles in DR are associated with the development of DR. Modulation of retinal miRNA expression levels may provide a potential therapeutic strategy for DRs.     

Prediction and verification of miRNA expression in human and rat retinas

PURPOSE: MicroRNAs (miRNAs) play a global role in regulating gene expression and have important tissue-specific functions. Little is known about their role in the retina. The purpose of this study was to establish the retinal expression of those miRNAs predicted to target genes involved in vision. METHODS: miRNAs potentially targeting important "retinal" genes, as defined by expression pattern and implication in disease, were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). The presence of candidate miRNAs in human and rat retinal RNA was assessed by RT-PCR. cDNA levels for each miRNA were determined by quantitative PCR. The ability to discriminate between miRNAs varying by a single nucleotide was assessed. The activity of miR-124 and miR-29 against predicted target sites in Rdh10 and Impdh1 was tested by cotransfection of miRNA mimics and luciferase reporter plasmids. RESULTS: Sixty-seven miRNAs were predicted to target one or more of the 320 retinal genes listed herein. All 11 candidate miRNAs tested were expressed in the retina, including miR-7, miR-124, miR135a, and miR135b. Relative levels of individual miRNAs were similar between rats and humans. The Rdh10 3'UTR, which contains a predicted miR-124 target site, mediated the inhibition of luciferase activity by miR-124 mimics in cell culture. CONCLUSIONS: Many miRNAs likely to regulate genes important for retinal function are present in the retina. Conservation of miRNA retinal expression patterns from rats to humans supports evidence from other tissues that disruption of miRNAs is a likely cause of a range of visual abnormalities.     

IGFBP-5和Lamp-2在年龄相关性白内障和透明晶状体上皮细胞的蛋白表达

目的:研究胰岛素样生长因子结合蛋白5(insulin-likegro-wthfactorbindingprotein5,IGFBP-5)和溶酶体膜糖蛋白2(lysosomeassociatedmembraneprotein2,Lamp-2)在年龄相关性白内障和透明晶状体上皮细胞(LECs)的表达,为了解白内障发生机制提供实验依据。方法:术中获取白内障和透明晶状体前囊,免疫组化检测LECsIGFBP-5和Lamp-2蛋白的表达。结果:成熟期年龄相关性白内障与核性年龄相关性白内障LECs表达IGFBP-5蛋白阳性率分别低于透明晶状体对照组(40.0%,42.1%vs79.0%,P<0.05),Lamp-2的蛋白表达各组间没有明显差异,其中核性年龄相关性白内障组与透明晶状体对照组比较P值接近0.05。结论:年龄相关性白内障LECs的IGFBP-5和Lamp-2表达异常与该白内障的形成有关。     

Nerve growth factor (NGF) and lenses: effects of NGF in an in vitro rat model of cataract

Background The aims of this study are to investigate the presence and production of nerve growth factor (NGF) in the rat lens in basal conditions and to evaluate, in vitro, the role of NGF in a model of xylose-induced cataract.Methods Rat lenses were dissected and the expression of NGF, NGF mRNA and high-affinity NGF-receptor (TrkA) was evaluated by immunohistochemistry, immunoenzymatic assay (ELISA) and in-situ hybridization (ISH) techniques. To investigate the role of NGF in cataract formation we used an in vitro model of sugar-induced cataract by culturing rat lenses for 48 h in Eagle's minimum essential medium (MEM) supplemented with xylose. To evaluate the potential protective effect of NGF on xylose-induced cataract formation, exogenous NGF at different concentrations or antibodies neutralizing endogenous NGF (NGF-Ab) or aspecific antibodies were added to xylose-cultured lenses, and the following cataract-related parameters were evaluated and compared to xylose-treated lenses. Cataract formation was evaluated using three different parameters: staging of the cataract by lens photography, quantification of lens transparency in terms of gray level medium (GLM) and evaluation of the hydration percentage (H%) of the lens. To investigate the role of endogenous NGF in cataract onset, NGF levels were evaluated and compared in lenses cultured in xylose supplemented medium versus lenses cultured in control culture medium.Results The epithelium from fresh rat lenses expresses NGF-receptor, NGF protein and NGF-mRNA. NGF levels in fresh lens were 54.0±24.5 pg/g as quantified by ELISA. Xylose-cultured lenses develop cataract changes, including a decrease of GLM and an increase in hydration percentage, associated with a decrease in NGF levels when compared to lenses cultured in the control culture medium. The addition of NGF to xylose-cultured lenses reduces cataract formation, increasing GLM and decreasing the hydration percentage as compared to xylose-treated lenses. On the other hand, the addition of NGF-Ab induces an increase in cataract formation and lens hydration.Conclusions This study demonstrates that rat lens epithelium expresses and synthesizes NGF. Moreover, immunohistochemistry shows that lens epithelial cells also express the NGF receptor. Although the functional significance of TrkA on lens epithelium is at present not clear, the expression of NGF and its high-affinity receptor on the same cells together with our experimental results suggest that NGF is involved in supporting trophism and/or the function of the lens epithelium.     

miR-210靶向沉默Bcl-2诱导人晶状体上皮细胞凋亡

目的　观察ｍｉＲ-２１０在年龄相关性白内障晶状体组织中的表达,并探讨其对人晶状体上皮细胞凋亡的影响与调控机制。方法　收集年龄相关性白内障患者与新鲜人眼透明晶状体前囊膜（正常对照组）标本;培养人晶状体上皮细胞ＳＲＡ０１／０４,予或不予（正常对照组）紫外线照射,利用实时荧光定量ＰＣＲ检测上述组织或细胞中ｍｉＲ-２１０表达。此外,将遭受紫外线照射的ＳＲＡ０１／０４细胞分为空白对照组、阴性对照组、ｍｉＲ-２１０模拟物组和ｍｉＲ-２１０抑制物组,分别予培养液及ｍｉＲ-２１０阴性对照物、模拟物、抑制物处理４８ｈ,行实时荧光定量ＰＣＲ检测ｍｉＲ-２１０表达,以验证转染效率;Ｗｅｓｔｅｒｎｂｌｏｔ检测ｍｉＲ-２１０的靶基因Ｂｃｌ-２表达,流式细胞术分析细胞凋亡率。结果　与正常对照组相比,年龄相关性白内障患者晶状体前囊膜组织和紫外线诱导的ＳＲＡ０１／０４细胞凋亡模型中ｍｉＲ-２１０表达均显著上调（均为Ｐ＜０．０１）。相对于空白对照组,ｍｉＲ-２１０模拟物组ｍｉＲ-２１０水平和细胞凋亡率均增加（均为Ｐ＜０．０１）,Ｂｃｌ-２蛋白表达水平降低（Ｐ＜０．０１）;而ｍｉＲ-２１０抑制物组ｍｉＲ-２１０水平和细胞凋亡率均减少（均为Ｐ＜０．０５）,Ｂｃｌ-２蛋白表达水平升高（Ｐ＜０．０１）;阴性对照组上述指标与空白对照组比较,差异无统计学意义（Ｐ＞０．０５）。结论　ｍｉＲ-２１０在年龄相关性白内障患者晶状体组织中呈高表达,ｍｉＲ-２１０可能通过靶向沉默Ｂｃｌ-２促进人晶状体上皮细胞凋亡。     

特殊表型先天性白内障小鼠晶状体中细胞缝隙连接蛋白的表达

目的观察γD-晶状体蛋白点突变导致的小鼠先天性白内障表型,检测该特殊表型先天性白内障小鼠晶状体中细胞缝隙连接蛋白（Cx）的表达。方法观察突变小鼠出生后不同时间晶状体的形态学变化;应用免疫荧光染色法分析晶状体内Cx46和Cx50的表达和分布。结果突变小鼠模型呈稳定一致的显性遗传,出生后7 d即表现为明显的核性白内障,出生后21 d纯合子小鼠晶状体混浊严重,后囊膜自然破裂;免疫荧光染色分析发现突变小鼠晶状体内Cx46和Cx50的表达均出现下降,越靠近晶状体中心部下降越明显。结论γD-晶状体蛋白点突变可导致晶状体后囊膜破裂这种特殊表型的先天性白内障,白内障的形成和后囊膜破裂的产生与晶状体内Cx46和Cx50的表达下降有关。     

色素上皮衍生因子在人晶状体上皮细胞表达的意义

目的:观察色素上皮衍生因子(PEDF)在人晶状体内的分布,探讨人晶状体上皮细胞(LEC)中PEDF表达水平与年龄及白内障发生发展的关系。方法:收集老年性和先天性白内障患者术中所取前囊膜、新鲜眼库眼透明晶状体样品,冰冻切片标本用间接免疫荧光组织化学法检测PEDF蛋白在人晶状体中的分布,前囊膜标本分别用蛋白质免疫印迹(Western-blot)及逆转录聚合酶链反应(RT-PCR)技术检测LEC中PEDF蛋白和基因的表达水平。结果:人晶状体中存在PEDF蛋白,主要分布于前囊下LEC胞浆中。按透明晶状体、轻度白内障、重度白内障及年龄分组分析,人LEC内PEDF基因及蛋白的表达水平在晶状体由透明变混浊、混浊由轻变重的过程中呈下调趋势(P<0.01),且随着机体的衰老显著下降(P<0.01)。结论:人LEC中PEDF表达随晶状体衰老和白内障发生发展显著下调。     

Development of Retinal Pigment Epithelium from Human Parthenogenetic Embryonic Stem Cells and MicroRNA Signature

Purpose. We investigated the potential of human parthenogenetic embryonic stem cells (hPESCs) to differentiate into RPE cells, and identified development-regulating microRNAs (miRNAs). Methods. RPE cells were derived from hPESCs. The expression of markers and miRNA expression profiles during differentiation were studied by immunocytochemistry, real-time RT-PCR, and miRNA expression array at three time points. Human fetal RPE (hfRPE) cells also were analyzed. The target genes of candidate miRNAs then were validated. Results. hPESC-derived RPE cells exhibited similar morphology and pigmentation to hfRPE cells. The expression of markers during differentiation indicated that the hPESC-derived RPE cells were immature. Most specific miRNAs had a role at some time point during the differentiation and maturation of RPE from hPESCs, except for two miRNAs (miR-204 and the miR-302 family). The miR-204 was upregulated and miR-302 was down-regulated throughout the process. Subsequently, pigmented clusters and RPE signature gene expression increased significantly in the miR-204 overexpression group and miR-302 inhibition group compared to the control groups. CTNNBIP1 and TGFBR2 were confirmed to be the target genes of miR-204 and miR-302, respectively. Conclusions. hPESCs can develop into RPE-like cells and, thus, can be additional promising sources of RPE cells in cell therapy. The miR-204, miR-302s, and their targets are involved in regulating directed differentiation during the full course, thereby contributing to the search for a new method of improving differentiation efficiency using miRNAs.     

p53和 bcl-2在老年性白内障晶状体上皮细胞中的蛋白表达

目的：研究p53,bcl-2基因在老年性白内障晶状体上皮细胞中的蛋白表达，探讨p53,bcl-2在老年性白内障晶状体上皮细胞凋亡中的作用。方法：应用免疫组织化学方法，对30例老年性白内障及10例正常透明晶状体上皮细胞进行p53,bcl-2基因蛋白表达的检测，计算p53,bcl-2基因蛋白表达阳性细胞百分率，结果：p53在正常晶状体上皮细胞中无蛋白表达，在老年性白内障晶状体上皮细胞中蛋白表达率为16．9％－19．1％，bcl-2在2组中均无蛋白表达，结论：p53在老年性白内障晶状体上皮细胞中蛋白高表达，p53可能促进了老年性白内障中晶状体上皮细胞的凋亡。     

老年性白内障晶状体上皮细胞凋亡及相关基因蛋白的表达

目的 探讨老年性白内障与晶状体上皮细胞凋亡的关系。方法透射电镜下观察老年性白内障晶状体上皮细胞的超微结构；Tunel法检测凋亡细胞百分率；并对其晶状体上皮细胞DNA进行琼脂糖凝胶电泳；免疫组化法检测P53、bcl-2在老年性白内障晶状体上皮细胞中的蛋白表达。结果 透射电镜下发现老年性白内障晶状体上皮细胞中有凋亡细胞；凋亡细胞百分率为8．4％～37．8％，琼脂糖凝胶电泳出现梯状条带；P53在老年白内障晶状体上皮细胞中蛋白表达率为16．9％～19．1％，bcl-2无蛋白表达。结论 老年性白内障的发生可能与其晶状体上皮细胞凋亡有关。     

TRPM3/miR-204复合位点调控眼部疾病的研究进展

微小RNA(micro RNA,miRNA)是最主要的基因表达调控因子之一,涉及多种细胞、组织和器官的生长、发育、分化及凋亡等过程。TRPM3基因位于人类9号染色体的长臂,是钙通透性离子通道TRP家族M亚家族中成员之一。miR-204位于TRPM3内含子6上,通过对靶mRNAs的切割或抑制其翻译参与转录后基因表达的调控。研究显示TRPM3/miR-204复合位点在白内障、青光眼、角膜新生血管、角膜损伤愈合、视网膜疾病、视神经疾病等眼部疾病的发生发展中起着重要的调控作用。本文从TRPM3/miR-204分子通路的生物学功能、在眼部的表达与调控以及其与多种眼部疾病的相关性研究进展进行综述。     

miR-181调控人晶状体上皮细胞凋亡的机制研究

目的：探讨 miR-181在白内障晶状体组织中的表达情况,及其对人晶状体上皮细胞凋亡的调控机制。
　　方法：利用Real time q-PCR方法,检测年龄相关性白内障患者晶状体前囊膜和人晶状体上皮细胞凋亡模型中miR-181的表达情况;利用Lipofectamine 2000瞬时转染miR-181 mimic 和 inhibitor 调节人晶状体上皮细胞中miR-181的表达,利用Real time q-PCR方法验证转染效率,利用流式细胞仪检测细胞凋亡率的变化。
　　结果：与对照组相比,年龄相关性白内障患者晶状体前囊膜组和人晶状体上皮细胞凋亡模型组,miR-181的表达显著升高;miR-181 mimic转染组,miR-181的表达显著升高,细胞凋亡率显著升高;miR-181 inhibitor转染组, miR-181的表达显著降低,细胞凋亡率显著降低,差异均有统计学意义(P<0.01)。
　　结论：miR-181在年龄相关性白内障晶状体组织中呈高表达,miR-181能够促进人晶状体上皮细胞凋亡,从而可能在年龄相关性白内障的发病过程中发挥一定作用,miR-181可能成为白内障非手术治疗的一种新途径,但具体机制有待进一步研究。     

波形纤维蛋白在老年性白内障晶状体上皮细胞的表达

目的观察波形纤维蛋白在老年性白内障晶状体上皮细胞的变化.方法用卵白素-生物素过氧化物酶法对22例老年性白内障患者晶状体前囊膜上皮细胞进行波形纤维蛋白染色,采用包埋前免疫酶电镜技术处理6个晶状体前囊膜标本,并观察其超微结构;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳及Westemnblot法分析4个晶状体表层组织(前囊膜、上皮细胞和表层皮质)中的波形纤维蛋白.结果老年性白内障晶状体上皮细胞的波形纤维蛋白表达减弱,与对照组比较差异有显著性(t=2.0948,P<     

Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in the human lens: implications for cortical cataract formation

PURPOSE: To characterize the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in human cortical cataract and to determine whether there is a correlation with the localization of cortical cataract. To evaluate the expression and activity of MMPs and TIMPs after cytokine and UV-B exposure in a human lens epithelial cell line. METHODS: Twenty-eight human donor eyes with cortical cataract and 21 normal human donor eyes were photographed. Thirteen cortical cataract and six normal lenses were immunohistochemically analyzed for MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3. Twelve fresh cortical cataract and 12 normal lenses were divided into quadrants to quantify, by ELISA, the expression of MMP-1, -2, -3, and -9 and TIMP-1. Three fresh cortical cataract and three control lenses were assessed for MMP-1, -2, and -9 activity by SDS-PAGE zymography. Human lens epithelial cells (HLE-SRA-01/04) were exposed to proinflammatory cytokines and UV-B radiation to determine the protein expression profiles of MMP-1, -2, -3, and -9 and TIMP-1 and -2. RESULTS: Immunohistochemical analysis revealed specific localization of MMP-1 within lens epithelium and cortical lens fibers of cortical cataract. Normal lenses had equally low MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3 immunoreactivity, expression, and activity in all lens quadrants. IL-1 and TNF-alpha upregulated the expression of MMP-2, -3, and -9, and UV-B upregulated the expression of MMP-1 in the SRA-01/04 HLE cell line. CONCLUSIONS: This is the first study to localize the expression of MMP-1 in cataracts with clinically observed opacification in vivo and to examine the expression induced by UV-B, in vitro.     

Cold cataract formation in fish lenses.

Cold cataract formation in fish lenses occurs at temperatures, 10 to 40°C below that for mammals. It is partially an irreversible cataract: while the cortex and nucleus become transparent upon warming, a spherical layer at the interface between these two regions remains turbid.On the basis of light scattering and isoelectric focusing data, it is concluded that cold cataract formation in fish lenses is not due to any specific lens protein alone but to the specific supermolecular organization of lens protein in the different parts of the lens.