Expression of growth factor mRNA in rabbit PVR model systems.

Proliferative vitreoretinopathy (PVR) involves the formation of intravitreal fibrocellular membranes which may lead to traction retinal detachment and blindness. The cellular component of epiretinal membranes originates from the proliferation and migration of cells within the eye. Several growth factors and other cytokines are plausible candidates for directing the processes leading to membrane formation. A reproducible animal model is needed for experimental studies of cytokine expression during PVR induction or treatment. We found that intravitreal injection of > 10(6) mixed mononuclear leukocytes or adherent monocytes along with a trans-scleral incision through the pars plana leads to the development of PVR-like disease in rabbit eyes. The severity of the disease was related to the number of monocytes injected. Typically, organized membranes extending from the incision toward the optic nerve formed within one week. Progression to extensive traction retinal detachment required 1 to 4 weeks. Injection of up to 5 x 10(6) lymphocytes or freeze-thaw killed monocytes was ineffective, and coinjecting 100 micrograms endotoxin with the monocytes did not result in enhanced disease. The histological appearance of the epiretinal membranes was similar to human PVR membranes. Macrophage, cytokeratin-positive (epithelial), and fibroblast-like cells were present. Northern blot analysis of RNA extracted from the rabbit membranes revealed the presence of mRNA for acidic fibroblast growth factor (aFGF). Acidic FGF mRNA was not expressed by the injected monocytes. A comparable level of aFGF mRNA and also mRNAs for basic FGF, platelet-derived growth factor-B, and transforming growth factor beta were found in epiretinal membranes induced by a scleral incision in association with cryopexy.(ABSTRACT TRUNCATED AT 250 WORDS)     

紫外线照射大鼠角膜胶原ⅠmRNA的表达

目的：观察紫外线照射大鼠角膜胶原ⅠmRNA的表达变化，探讨紫外线辐射对角膜的损伤机制。方法：用20mW、260～365nm的紫外线照射大鼠角膜，用RT-PCR方法检测照射后不同时间点角膜胶原ⅠmRNA的表达变化。结果：角膜经过紫外线辐射后30分钟，角膜胶原ⅠmRNA的表达量稍有上升，2小时下降最多，6小时回升超过正常，在24小时、48小时及7天、14天中角膜胶原ⅠmRNA的表达量呈持续缓慢上升。结论：紫外线辐射可影响大鼠角膜胶原ⅠmRNA的表达。     

血管内皮生长因子干扰RNA对鼠视网膜中血管内皮生长因子mRNA表达的抑制作用

目的 研究血管内皮生长因子（vascular endothelial cell growth factor,VEGF）小片段干扰RNA（small interference RNA,siRNA）对鼠视网膜VEGF mRNA的抑制作用,探讨其对视网膜新生血管治疗的可行性.方法 体外培养人鼻咽癌细胞（CNE-2Z）,分成正常氧培养组（20% O2）和低氧培养组（1% O2）.采用脂质体（LF 2000）将VEGF siRNA转染两组细胞,RT-PCR检测VEGF mRNA的表达,确立VEGF siRNA对VEGFmRNA的抑制效率.然后,建立高浓度氧（75%）诱导的C57BL./6J小鼠视网膜新生血管动物模型,以脂质体为载体,将VEGF siRNA重组质粒注射到鼠玻璃体腔内,RT-PCR检测视网膜组织中VEGF mRNA的表达水平.结果 正常氧培养的CNE-2Z细胞有VEGF mRNA表达,低氧状态下VEGFmRNA表达增多,两者之间差异有显著性（P＜0.01）;与未转染组和转染空载体组相比,在正常氧和低氧状态下,VEGFsiRNA均能明显抑制VEGFmRNA的表达（P＜0.01）;正常氧状态下VEGF siRNA的抑制效率比低氧状态高.高浓度氧诱导的C57BL/6J小鼠视网膜新生血管动物模型中,玻璃体腔注射VEGF siRNA组视网膜组织中VEGF mRNA表达明显下降（P＜0.01）.结论 VEGF特异的siRNA能有效地抑制人鼻咽癌细胞CNE-2Z和C57BL/6J 小鼠视网膜新生血管动物模型视网膜中VEGF mRNA的表达.     

Nitric oxide synthase expression in ischemic rat retinas

PURPOSE: To investigate the expression of nitric oxide synthase (NOS) in the ischemic retina. METHODS: Retinal ischemia was induced in rats by bilateral common carotid artery occlusion (BCCAO) for various lengths of time. Using the retina after BCCAO, expression of neuronal NOS (nNOS) and inducible NOS (iNOS) and identification of their positive cells were studied by histological and immunohistochemical examinations. RESULTS: Histological examinations revealed significant reduction in the thickness of the inner plexiform layer and the outer plexiform layer of the retina. Expression of nNOS was detected in retinal ganglion cells, amacrine cells, and Müller cells after BCCAO. The expression of nNOS and iNOS detected in Müller cells became stronger and persisted long after BCCAO. CONCLUSIONS: In the ischemic retina, Müller cells and retinal ganglion cells expressed nNOS and iNOS. These phenomena may be involved in the ischemic damage to the retina.     

IL-1β在实验性增生性玻璃体视网膜病变胶原沉积中的作用

目的　检测实验性增生性玻璃体视网膜病变 (proliferative vitreoretinopathy,PVR)玻璃体内白介素 1β(interleukin1β,IL- 1β)和 型前胶原 (procollagen ,PC )的含量变化并分析二者的相关性。方法　用同种巨噬细胞诱发兔眼 PVR,在不同时间点收集玻璃体液 ,用双抗体夹心法酶联免疫吸附检测 (enzyme- linked im munosorbent assay,EL ISA)法测定 IL - 1β含量 ,用放射免疫法测量 PC 含量。做相关分析。结果　 IL - 1β的基线含量为4ng· L- 1 ,7d时明显增加 ,在 14～ 2 8d呈一较高水平 (171～ 175 ng· L- 1 ) ;2 1d达高峰值2 3 4ng· L- 1 。 PC 在对照眼中未能检出 ,在 7d时含量上升 (2 64 .73μg· L- 1 ) ,14d以后明显升高 (816.2 9～ 941.48μg· L- 1 ) ;2 1d达高峰 ,2 8d仍维持高水平。相关分析显示 IL- 1β与PC 有高度相关性 (r=0 .90 8,P=0 .0 46) ,并与 PVR病程的炎症期、增生期和瘢痕期相吻合。结论　实验性 PVR玻璃体中 IL- 1β和 PC 含量随病程明显增加。IL- 1β与 PC 的含量变化明显相关 ,提示 IL- 1β在 PVR胶原沉积中有调控作用     

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.     

Elevated level of protein phosphatase 2A activity in retinas of rd mice.

Phosphorylation of rhodopsin is not detectable in vitro in the retina of the rd mouse. We investigated the enzymatic system responsible for this abnormality by measuring the levels of rhodopsin kinase and protein phosphatase 2A in normal (rd/+) and diseased (rd/rd) mouse retinas of several ages. For each enzyme, we developed micro assays that were suitable for measuring enzyme activity in one-half mouse retina. Our results indicate that rhodopsin kinase activity is identical in rd/+ and rd/rd retinas until post-natal day 11, and it decreases thereafter in the rd/rd retina, correlating with the loss of rod photoreceptors that occurs in this tissue. Protein phosphatase 2A has a constant level of activity in rd/+ retinas from ages 5 to 32 days but it is higher than normal in rd/rd retinas from post-natal days 5 to 10. It then decreases to levels that are comparable to those in rd/+ retina. Although the rd/rd extract contains the elevated protein phosphatase 2A activity, when rd/rd and rd/+ retinal extracts are each subjected to gel filtration, the elution profiles of protein phosphatase 2A activity appear to be quantitatively identical. This apparent loss of rd/rd phosphatase activity suggests a difference in the regulatory behavior of the enzyme in the normal and degenerative retinas. Thus, the failure to detect in vitro phosphorylation of rhodopsin in the rd/rd retina seems to result from the elevated level of protein phosphatase 2A activity which could more rapidly remove the phosphate from phosphorylated rhodopsin. Since protein phosphatase 2A is a ubiquitous enzyme with broad specificity, an elevation in its activity also could affect other protein phosphorylations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

PDR、PVR增生膜组织超微结构及生长因子受体表达生物学特点的比较研究

目的比较研究增生性玻璃体视网膜疾病PVR及PDR增生膜组织超微结构与生长因子受体表达的生物学特点。方法PVR、PDR增生膜组织标本29例,通过电镜与免疫组织化学方法比较研究PVR与PDR增生膜组织超微结构、细胞膜上TGFβR1和EGF受体表达的生物学特点。结果(1)PVR、PDR增生膜中细胞类型主要是RPE细胞、纤维细胞、纤维母细胞样细胞和巨噬细胞;PVR与PDR膜组织超微结构不同。(2)TGFβR1受体在PDR增生膜组织中的表达,其平均光强度(P<0.01)与平均阳性面积(P<0.05)均强于PVR增生膜组织,EGF受体在PDR增生膜组织中(13/14,92.8%)表达较PVR增生膜组织中(3/15,20%)明显(P<0.05)。(3)无论PDR、PVR增生膜组织TGFBRI受体在病程>6个月的增生膜组织中的表达,明显强于小于6个月的标本(P=0.002),而EGF受体在病程2-6个月的膜组织中表达平均面积(P=0.024)明显小于其他病程的膜组织。结论PDR、PVR增生膜组织,其超微结构与生长因子表达的生物学特点不同。     

Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) and developmental expression of IRBP mRNA in normal and rd mouse retinas.

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) was quantitatively assessed in retinas of normal and rd mutant mice using short-term organ culture with [35S]methionine. Retinas were studied at ages P9-P12, time points prior to and immediately after the onset of the degeneration of the rd retina. Soluble proteins of the retinal pellet and the incubation medium were subjected to SDS-polyacrylamide gel electrophoresis. Analysis of labeled protein bands utilized a radioactivity scanning system to quantify [35S]methionine incorporation into newly synthesized IRBP. The synthesis and secretion into the incubation medium of IRBP by rd mouse retinas was comparable to normal retinas at P9-P10 but decreased by more than 50% by P12. IRBP mRNA levels were evaluated in retinas of normal and rd mice ages P7-P14. Although IRBP mRNA expression increased in the rd mouse through P10, it decreased markedly thereafter. Previously reported immunocytochemical studies suggested that IRBP was not secreted in the rd mouse retina. The results of this study indicate, however, that rd mouse retinas, when removed from the eye, have the capacity to synthesize and secrete IRBP.     

糖尿病大鼠视网膜黏附连接蛋白表达的变化

目的:探讨链脲佐菌素(streptozotocin,STZ)诱导的糖尿病视网膜黏附连接VE-cadherin和β-catenin的表达。方法:SD大鼠50只,随机分为正常对照组(Con)、糖尿病组(DM)。腹腔注射STZ建立DM模型,2,5,8wk处死动物。用伊凡思蓝方法检测视网膜的渗透性;用免疫组织化学和Western blot方法检测各组大鼠视网膜VE-cadherin和β-catenin的表达情况。结果:造模后2,5,8wk大鼠视网膜血管渗透性增加。免疫组化分析证实DM大鼠视网膜VE-cadherin主要表达在视网膜血管上,β-catetin主要表达在视网膜外界膜、外核层、外网状层、内网状层和内界膜。Western blot分析证明随着DM视网膜病变的发生发展,视网膜VE-cadherin表达量显著减少,2,5,8wk组与Con组两两比较均有差异(P<0.05);而β-catetin表达量显著增加,2,5,8wk组与Con组两两比较差异有统计学意义(P<0.05)。结论:STZ诱导的DM大鼠VE-cadherin表达量减少而β-catetin表达量显著增加,两者成反比。     

Increased expression of angiotensin-converting enzyme in retinas of diabetic rats.

PURPOSE: The aim of this study was to examine the localization and the changes in the amount of angiotensin-converting enzyme (ACE) and the relationship between the renin-angiotensin (RA) system and vascular endothelial growth factor (VEGF)/VEGF-receptor system in the retinas of diabetic rats. METHODS: Immunohistochemical localization of ACE, VEGF, and VEGF-receptor fetal liver kinase-1 (Flk-1) was examined in cryosections of the retinas of streptozotocin-injected diabetic rats. A semi-quantitative comparison of diabetic rats with age-matched controls was also performed by counting the ACE- or Flk-1-positive vessels per microscopic field. RESULTS: ACE immunoreactivity was localized in the retinal vessel walls, and the percentages of ACE-positive vessels were significantly increased in the retinas of diabetic rats maintained 3 to 5 months. Both VEGF and Flk-1 signals increased simultaneously with the increment of ACE immunoreactivity. CONCLUSIONS: ACE, expressed in the retinal vessel walls, increases simultaneously with the increment of both VEGF and Flk-1 in the retinas of diabetic rats, suggesting that upregulation of ACE might play some role in the progression of diabetic retinopathy through the VEGF/VEGF receptor system.     

四种细胞因子对牛晶状体上皮细胞胶原合成的影响及对前胶原mRNA表达的调控

目的 探讨表皮生长因子(EGF)、白细胞介素1(IL-1)、γ干扰素(IFN-γ)和生长抑素8肽(SS-8肽)在晶状体后囊膜混浊形成中的作用及影响胶原合成的机制。方法 细胞接种于96孔培养板贴壁后分别加入含有浓度为10^-2～10^2ng／ml的EGF、10～10^5ng／ml的IL-1、10^-11～10^-7mol／L的SS．8肽及10～10^5U／ml的IFN-γ细胞培养液,采用^3H-脯氨酸掺人法检测对胶原合成的影响;用Northern blot技术检测对Ⅰ、Ⅲ型前胶原mRNA表达的调控。结果 EGF 10^-1～10^2ng／ml、IL-1 10^2～10^5ng／ml显著促进胶原合成,IFN-γ 10^3～10^5U／ml、SS-8肽 10^-10～10^-7mol／L显著抑制胶原合成;1ng／ml的EGF及10^3ng／ml的IL-1可增加Ⅰ、Ⅲ型前胶原mRNA表达,而10^3U／ml的IFN-γ及10^-9mol／L的SS-8肽可减少Ⅰ、Ⅲ型前胶原mRNA表达。结论 EGF、IL-1参与了晶状体后囊膜混浊形成中的胶原合成,且对胶原合成的影响是作用于转录水平;IFN-γ、SS-8肽的作用提示其可用于防治晶状体后囊膜混浊。（中华眼科杂志,2004,40：545-548)     

光化学损伤下大鼠视网膜超氧化物歧化酶基因mRNA表达的实验研究

通过建立大鼠视网膜光化学损伤模型，应用原位杂交技术对正常及光化学损伤大鼠视风膜超氧化物歧化酶基因ｍＲＮＡ表达情况进行动态观察。结果表明，视网膜组织各层均可见超氧化物歧化酶基因ｍＲＮＡ的表达；连续光照可使其表达水平迅速下降，原位杂交为研究视网膜基因表达状况的一种良好方法。     

前部PVR睫状体上皮损伤修复过程中Cx43表达变化及意义

目的研究Cx43在前部增生性玻璃体视网膜病变（PVR）睫状体上皮损伤修复过程中表达的变化并探讨其机制和意义。方法利用苏木精一伊红染色、荧光免疫组织化学、透射电镜和Westernblot技术对外伤性前部PVR模型不同时间点睫状体上皮组织病理学变化及Cx43蛋白表达分布和表达量变化分别进行观察和检测。结果伤后6h睫状突表面有纤维蛋白渗出，1周时纤维增生膜基本形成。24hCx43在睫状体色素上皮（PE）细胞间出现表达，且蛋白含量略增；造模1周时，无色素上皮（NPE）细胞间Cx43表达增加，蛋白含量检测显著增高；伤后24h之前和1周之后Cx43表达于NPE—PE交界，其表达部位和表达的量的变化也无显著性差异。结论外伤性前部PVR睫状体上皮损伤修复过程中Cx43表达变化活跃，表明Cx43缝隙连接通道蛋白参与了该病理状态下睫状体上皮的组织损伤修复与功能调节。     

A high-resolution RNA expression atlas of retinitis pigmentosa genes in human and mouse retinas

PURPOSE: Retinitis pigmentosa (RP) is one of the leading causes of visual handicap in the world population and is characterized by high genetic heterogeneity. The study of the disease mechanisms and the development of efficient therapeutic approaches have mostly relied on the availability of animal models for this condition, so far. Nevertheless, little information is available about the RNA expression profiles of RP genes in the human retina. An expression atlas of 34 known RP genes in human and murine retinas was generated to overcome this lack of information. METHODS: Appropriate templates were retrieved for 34 RP genes that were used to perform RNA in situ hybridization studies on human and murine adult eyes. RESULTS: Most of the genes displayed similar patterns between human and mouse retina. Different expression patterns were observed for the CNGB1, USH2A, and FSCN2 genes, compared with those in previously reported profiles. In addition, different expression profiles were detected for the RPGR, CA4, PAP1, RGR, and RLBP1 genes in human and mouse retinas. CONCLUSIONS: The first gene expression atlas has been generated of RP genes in human and murine retinas. Differences observed in the expression patterns of some genes in humans and mice, will open new perspectives on the function of these genes and their putative roles in disease pathogenesis.