人胎视网膜血管发生方式的研究

目的：观察人胎视网膜血管的形成方式。方法：收集13～38周胎儿视网膜86例，免疫组织化学染色(ABC)法，光镜观察。结果：在胎儿12—13周时，间充质的梭形细胞从视盘处进入视网膜，并向锯齿缘迁移，同时分化、增殖为内皮细胞索，此索经管道化和改建成为节细胞层内的血管。第26周，节细胞层内已生成血管“出芽”，朝神经纤维层和内核层生长，分别在神经纤维层内和内核层的内、外缘各形成一层毛细血管网。结论：足月胎儿视网膜基本具有四层血管，分别由两种不同方式生成。 （中华眼底病杂志，1996，12：88-90）     

血管内皮生长因子对人胚胎视网膜血管发生的调节作用

目的研究血管内皮生长因子(vascular endothelial growth factor, VEGF)对人胚胎视网膜血管发生的调节作用。方法收集54例9-40周龄胎儿眼球后壁标本,免疫组织化学染色,光镜观察。结果①VEGF在视网膜的表达呈波峰式分布,高峰在9-13周及26周左右。②节细胞层的梭形细胞（血管内皮细胞前提细胞）、血管内皮细胞呈增殖细胞核抗原(proliferation cell nuclear antigen,PCNA)免疫反应阳性,水平波动,高峰在9-13周及21周前后,此期间梭形细胞不断增殖、分化形成内皮细胞索,经改建形成视网膜内层血管,26、34周起见内核层内、外缘血管内皮细胞呈PCNA免疫反应阳性,并保持至足月。③视网膜VEGF表达量与梭形细胞、血管内皮细胞PCNA表达量呈显著正相关(r=0.736,p＜0.01)。结论VEGF对视网膜血管的发生有显著促进作用。(中华眼底病杂志,1999,15:12-15)     

Wistar鼠和RCS鼠视网膜神经递质的分布和比较

目的：观察正常Wistar大鼠和皇家外科学院鼠（RCS）的视网膜兴奋性神经递质谷氨酸（Glutamate)和抑制性神经递质γ-氨基丁酸（GABA）的分布和两者间的差异。方法：74天Wistar鼠和RCS鼠各6眼分为2组，免疫组织化学染色法，光镜下观察上述组织视网膜内谷氨酸和GABG的分布。结果：Wistar鼠视网膜AGBA阳性免疫反应分布于视网膜内丛状层的部分神经纤维、GABA阳性免疫反应的无长突细胞和节细胞层的部分纤维。RCS鼠则分布于视网膜内丛状层的少量神经纤维、极少量GABA阳性免疫反应的无长突细胞和节细胞层的部分纤维。Wintar鼠视网膜Glutamate阳性免疫反应主要分布于外丛状层、内丛状层及节细胞层的神经纤维。RCS鼠则主要分布于内丛状层及节细胞层的神经纤维。RCS鼠视网膜中Glutamate阳性免疫反应的神经纤维的相对密度和GABA阳性免疫反应的无长突细胞的相对密度减少。结论：74天时RCS鼠视网膜中的视细胞完全萎缩，使Glutamate和GABA的分布受到影响。     

生长抑素基因在出生后视网膜发育过程中的表达

目的：用分子生物学原位杂交组化法研究视网膜生后发育过程中生长抑素（somatostatin,SOM)基因的表达。方法：10只SD大鼠分为5组，每组2只，年龄分别为1、2、3、4、8周。原位杂交探针为地高辛标记SOM反义cRNA探针。结果：1周时，SOM mRNA杂交显色在神经母细胞层内侧及节细胞层，呈弱阳性；2周时，杂交信号逐渐增强，节细胞层呈中等强度染色，内核层为弱阳性；3-4周时，显色明显增加，节细胞层呈深紫色着色，而内核层仍为中等强度染色；8周时成年大鼠，节细胞层阳性细胞散在分布，染色较强，而内核层为弱阳性。结论：大鼠出生后，视网膜内SOM基因表达逐渐增加，于3-4周时达高峰，至成年时又下降，可能在视网膜生后发育过程中，对视网膜神经元的分化起重要作用。     

人胎视网膜发育过程中Fas、Fas-L、bax和 bcl-2蛋白的表达

目的研究人胎视网膜发育过程中细胞凋亡相关基因Fas、Fas-L 、bax和bcl-2的蛋白表达。方法收集12～38周（受精龄）胎儿视网膜共50例,石蜡包埋切片,免疫组织化学染色,光镜观察。结果发育第16周和第38周,视网膜节细胞层、内、外核层表达Fas蛋白。第26周,Fas-L阳性染色开始出现于视网膜各层细胞,到第32周,免疫阳性反应主要集中在节细胞层,第3 8周时,神经纤维层也为阳性反应。bax免疫阳性反应从第12周起出现,第16周,节细胞层和 外核层多数细胞核为阳性。第24周,内核层中的细胞为阳性反应,但到第26周时,仅Mülle r细胞内侧终足为bax免疫阳性染色。第26周以后,视网膜内各种成分都为bax免疫反应阴性 。bcl-2免疫阳性反应于第16周时出现在正在分化的神经母细胞层。第24周开始,bcl-2免 疫阳性反应集中于节细胞层的神经胶质细胞以及Müller细胞的内侧终足。结论发育中的视网膜的细胞凋亡可能不依赖Fas/Fas-L途径。bax可能参与介导胎儿视网膜细胞凋亡。(中华眼底病杂志,2001,17:55-57)     

人胎视网膜内调节血管形成因素的研究

目的：研究人胎视网膜内纤维连接蛋白(Fibronectin，Fn)，碱性纤维母细胞生长因子(basic—fibroblast growth factor，b-FGF)和星形胶质细胞对血管两种发生方式的调节作用。 方法：收集13—40周胎儿视网膜86侧，免疫组织化学染色，光镜现察。 结果：梭形细胞、血管内皮细胞均呈Fn免疫阳性反应，梭形细胞前方的基质中有Fn免疫反应物沉积，血管内皮细胞、节细胞和视锥细胞呈b+FGF阳性反应，后两种细胞的出现先于邻近区域血管的形成；星形腔质细胞紧密伴随梭形细胞向锯齿缘迁移，其突起包裹两种方式发生的血管。 结论：Fn、FGF和星形腔质细胞这三种因素对血管发生的两种方式均有促进作用。 （中华眼底病杂志，1996，12：180-182）     

大鼠骨髓间充质干细胞视网膜下移植观察

目的：鉴定骨髓间充质干细胞（mesenchymal stem cells,MSCs)在体外不诱导的条件下视网膜下移植后的定位。方法：体外培养雄性大鼠MSCs，直接作为细胞供体视网膜下移植于成年雄性大鼠视网膜下，4周后将动物处死，取眼球做石蜡切片，用Y染色体鉴定，为做进一步验证，将MSCs用重组腺相关病毒AAV－gfp感染后移植，分别于4、8周取动物眼球作冰冻切片，于荧光显微镜下做绿色荧光蛋白（green fluorescence protein，GFP）表达观察。结果：培养的MSCs集落生长迅速，均一性好；Y染色体原位杂交鉴定表明来源于MSCs的阳性细胞融合入了原来的视网膜结构，在光学显微镜下可分布于视锥，视杆细胞层，双极细胞层及节细胞层；在荧光显微镜下可见GFP标记的阳性细胞存在，分布于视网膜色素上皮层、视锥，视杆细胞层，双极细胞层及节细胞层，细胞形态与结构同周围的视网膜相似；2种标记方法检测到的视网膜结构完整，未见到玫瑰花结样结构。结论：MSCs可在视网膜下移植后4周与原视网膜结构相融合，2种方法检测到的阳性细胞分布于视网膜色素上皮层，视细胞层，双极细胞层及节细胞层。     

兔视网膜中蛋白激酶C的定位研究

目的：证实兔视网膜中有α、β和γ三种蛋白激酶C(protein kinase C，PKC)亚型的分布。方法；采用免疫组织化学技术，以抗PKC同功酶I(α)、Ⅱ(β)、Ⅲ(γ)单克隆抗体对兔视网膜进行PKC同功酶定位研究。 结果：兔视网膜中有PKC-α、PKC-β和PKC-γ的阳性免疫反应。PKC-α免疫反应主要呈现在内核层的双极细胞；PKC-β免疫反应主要呈现在神经节细胞层的神经节细胞；PKC-γ则在神经节细胞层、内网状层以及光感受器的外节呈弥漫性弱阳性染色。 结论：作为中枢神经系统的一部分，视网膜可以同时存在PKC-α、PKC-β和PKC-γ三种亚型。 （中华眼底病杂志，1996，12：242-244）     

血管内皮生长因子在新生小鼠视网膜的表达

目的 研究血管内皮生长因子（vascular endothelial growth factor,VEGF)与新生小鼠视网膜血管系统形成的内在联系，方法 分别于小鼠出生后3d,1、2、4周以免疫组化观察VEGF在视网膜的表达，并与视网膜铺片和光镜结果相对照。结果 小鼠出生后视网膜血管系统由视盘处呈放射状穿出，逐渐由后极部向周边部，视网膜病理切片显示；出生后3d时，视网膜只有2层，即神经节细胞层和神经母细胞层，内层毛细血管仅限于视网膜后极及中周部，7d时外丛状层形成，出生后14d，视网膜外层毛细血管发育基本完成，出生后28d，视网膜与14d时大致相似，VEGF免疫组化结果显示；出生后3d，阳性信号位于神经节细胞层和神经母细胞层内，外缘，7d时，VEGF明显表达在神经节细胞层，外丛状层和几乎全层内核层，出生后14d及28d。当视网膜血管系统发育基本完成时，VEGF阳性细胞数量和强度都明显减少和减弱。结论 本研究提示，VEGF的表达在空间和时间上与视网膜血管形成密切相关，VEGF的存在对于小鼠视网膜血管系统的形成及维持正常生理功能可能至关重要。     

Somatostatin gene expression in the rat retina during postnatal development

目的用分子生物学原位杂交组化法研究视网膜生后发育过程中生长抑素（ｓｏｍａｔｏｓｔａｔｉｎ，ＳＯＭ）基因的表达。方法１０只ＳＤ大鼠分为５组，每组２只，年龄分别为１、２、３、４、８周。原位杂交探针为地高辛标记ＳＯＭ反义ｃＲＮＡ探针。结果１周时，ＳＯＭｍＲＮＡ杂交显色在神经母细胞层内侧及节细胞层，呈弱阳性；２周时，杂交信号逐渐增强，节细胞层呈中等强度染色，内核层为弱阳性；３～４周时，显色明显增加，节细胞层呈深紫色着色，而内核层仍为中等强度染色；８周时成年大鼠，节细胞层阳性细胞散在分布，染色较强，而内核层为弱阳性。结论大鼠出生后，视网膜内ＳＯＭ基因表达逐渐增加，于３～４周时达高峰，至成年时又下降，可能在视网膜生后发育过程中，对视网膜神经元的分化起重要作用。     

Induced expression of hematopoietic- and neurologic-expressed sequence 1 in retinal pigment epithelial cells during newt retina regeneration

Newts can regenerate their organs even as adults. For instance, when their neural retinas are completely removed by operation, the remaining retinal pigment epithelial (RPE) cells dedifferentiate to reconstruct neural retinas. To elucidate the molecular mechanisms of newt retina regeneration, we investigated genes upregulated in dedifferentiating RPE cells using differential display methods. We observed that a cDNA fragment of hematopoietic- and neurologic-expressed sequence 1 (Hn1) appeared to be induced within a few days of surgical removal of newt neural retina. Using an anti-HN1 antiserum against the recombinant HN1 protein, we carried out immunohistochemical analyses. The anti-HN1 antiserum recognized the plexiform layers and ganglion cell layer (GCL) but not the RPE cell layer in unoperated (normal) newt retinas. Using a glial fibrillary acidic protein antibody, Hn1 was shown to be possibly expressed in glial cells in normal neural retina. During retina regeneration, immunoreactivity for HN1 appeared in dedifferentiating RPE cells 10 days post-operation, and in retinal progenitor cells 18 days post-operation. Twenty seven days post-operation, HN1 immunoreactivity was localized in the plexiform layers and GCL as in the normal retina. Therefore, HN1 possibly plays an undefined role in dedifferentiating RPE cells and retinal progenitor cells during newt retina regeneration.     

Diabetes induces expression of aquaporin-0 in the retinal nerve fibers of spontaneously diabetic Torii rats

Diabetes redistributes the expression of glial aquaporin (AQP) water channels in the retina. However, it is not known whether diabetes also affects retinal AQP-0 expression. This study examined the effects of the development of diabetes on the expression of retinal AQP-0 in spontaneously diabetic Torii (SDT) rats. Male SDT rats at 10 and 40 weeks of age and age-matched male Sprague-Dawley (SD) rats were used. The localization of AQP-0 was assessed immunohistochemically using sagittal cryosections of the rats’ retinas and optic nerves. Fold changes in AQP-0 gene expression relative to controls were assessed by real-time RT-PCR. All SDT rats spontaneously developed diabetes by 40 weeks of age (the mean hemoglobin (Hb) A1c levels were 2.8 ± 0.2% and 11.2 ± 1.0% at 10 and 40 weeks, respectively). SD rats did not develop diabetes (the HbA1c levels were 2.7 ± 0.2% and 2.6 ± 0.3% at 10 and 40 weeks, respectively). In the retinas of SD rats and in those of SDT rats at 10 weeks of age, immunoreactivity for AQP-0 was confined predominantly to the inner nuclear layer and to the border between the inner plexiform layer and the ganglion cell layer (GCL), where AQP-0 colocalized with protein kinase C-α. AQP-0 immunoreactivity was also observed in the GCL to a lesser degree, which colocalized with the neuronal nuclei. In the 40-week-old SDT rat retinas, additional AQP-0 immunoreactivity was observed in the GCL and colocalized with neurofilaments, indicating expression of AQP-0 in ganglion cell axons. However, the axonal AQP-0 immunoreactivity was restricted to the retinal nerve fibers, whereas the optic nerve axons were devoid of AQP-0. Retinal blood vessels did not express AQP-0. AQP-0 gene expression was 3.4-fold higher in SDT rat retinas than in SD rat retinas at 40 weeks of age.AQP-0 was predominantly expressed in the bipolar cells of the non-diabetic rat retinas, whereas it was also expressed in the retinal nerve fibers of diabetic rat retinas. The disrupted water transport between astrocytes and retinal nerve fibers may be associated with the known accelerated apoptosis of retinal ganglion cells induced by diabetes.     

Endothelin-1 and endothelin receptors in light-induced retinal degeneration

We have studied the distribution of endothelinergic molecules: prepro-endothelin-1 (PPET-1), endothelin-1 (ET-1), and receptors A and B (ET-A) and (ET-B) in the retina of mice. The localization of these molecules in normal mice was compared to their localization in retinas from animals submitted to continuous illumination during 1, 6, 9 or 18 days. We also evaluated the distribution of smooth muscle actin (SMA) and glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthase (GS). PPET-1 immunoreactivity mainly appeared in retinal pigment epithelium (RPE) and cells of the ganglion cell layer (GCL), whereas ET-1 immunoreactivity was present in the RPE, outer plexiform layer (OPL) and astrocytes. Astrocytes exhibited the strongest immunostaining in the retina. ET-A immunoreactivity was observed in endothelium, RPE, OPL and cells of the GCL. By contrast, ET-B immunoreactivity could be detected in endothelial cells, horizontal cells and astrocytes. Astrocytes of the optic nerve also exhibited ET-1, ET-A, and ET-B immunoreactivities. After light-induced degeneration, there was an increase of RPE immunostaining. Degeneration of photoreceptors was accompanied by disappearance of immunoreactivity in the OPL. However, ET-A immunoreactivity appeared in the amacrine sublayer of the INL. There was an enormous increase in astrocytes and its cell processes. The increase of astrocytic immunoreactivities for ET-1 and ET-B was confirmed by quantitative image analysis. Growth of astrocytic cell processes was most marked around retinal blood vessels. Our findings indicate that there are at least three endothelinergic pathways in the normal retina: (1) between the RPE and choriocapillaris, (2) at the OPL, and (3) between blood vessels, astrocytes and cells of the GCL. After light-induced degeneration of photoreceptors, endothelinergic molecules were overexpressed at the RPE and astrocytes, but mostly disappeared from the OPL.     

玻璃体内注射先锋霉素Ⅵ视网膜酶组织化学的实验研究

目的：研究先锋霉素Ⅵ玻璃体腔内注射的安全剂量。 方法：不同浓度的先锋霉素Ⅵ（100、200、250、300、400μg)注入不同组别的兔眼玻璃体腔内，以酶组织化学染色法确定注射后不同时间(1、3、7天)视网膜能量代谢酶琥珀酸脱氢酶(succinic dehydrogenase,SDH)和乳酸脱氢酶(lactic dehydrogenase,LDH)活性的变化并观察视网膜的组织结构及超微结构损害。 结果：随用药剂量增大，视网膜内SDH、LDH活性均逐渐降低；各剂址组活性分别于术后3天和术后1天降至最低，然后开始恢复，至术后7天100、200μg组恢复至正常，其余剂量组仍低于正常.100、200μg剂量组视网膜内仅出现水肿，而250、300、400μg组均出现细胞变性坏死、杆锥体脱失等改变。 结论：先锋霉索Ⅵ玻璃体腔内注射的安全剂量为200μg。 （中华眼底病杂志，1997，13：139-142）     

血管内皮生长因子在糖尿病大鼠视网膜中的表达

目的 :观察血管内皮生长因子 (VEGF)蛋白在链脲佐菌素 (STZ)诱导的糖尿病大鼠视网膜中的表达情况 ,以探讨其在早期糖尿病视网膜病变 (DR)中的作用。方法 :选择健康成年Wistar大鼠 4 0只 ,随机分成正常对照组 (CON)、糖尿病 1个月组 (DM1)、糖尿病 3个月组 (DM3)及糖尿病 6个月 (DM6 )组 ,每组 10只。大鼠腹腔内注射STZ诱发大鼠糖尿病。制备大鼠视网膜石蜡切片行免疫组化ABC法染色 ,光镜观察。结果 :VEGF于CON和DM1组只表达于内核层及节细胞层部分细胞 ;病程 3个月时 ,尚可见视网膜血管的阳性反应 ;6个月时 ,阳性反应范围又扩大至视杆内节和色素上皮细胞。结论 :随病程进展 ,VEGF在视网膜中的表达呈逐渐增强的趋势 ,提示它在早期DR的发生、发展中起重要作用     

Engraftment of adult neural progenitor cells transplanted to rat retina injured by transient ischemia

PURPOSE: To optimize delivery parameters for achieving engraftment, migration, and differentiation of adult neural progenitor cells transplanted to the retinas of rats after transient retinal ischemia. METHODS: Retinal ischemia was induced by transiently raising the intraocular pressure. Some animals then received transplantation of green fluorescent protein (GFP)-expressing cells derived from the adult rat hippocampus and were allowed to recover for 6 hours to 9 weeks. Retinal cryosections were prepared for TUNEL analysis to determine the time course of ischemia-induced cell death, and some sections were prepared for immunohistochemistry for retinal neuronal antigens. RESULTS: TUNEL analysis revealed that ischemia-induced cell death peaked at 24 hours. By 96 hours, the inner nuclear (INL) and ganglion cell (GCL) layers were largely obliterated in the central retina, sparing peripheral regions. By 2 weeks after transplantation, numerous GFP-expressing cells had engrafted into the host retina, migrated to the inner retina, and extended processes. At 4 weeks, many GFP-labeled cells were present throughout the INL and displayed horizontal-, bipolar-, and amacrine cell-like morphologies. GFP-expressing cells were also present in the GCL with fibers extending into the nerve fiber layer. At 5 weeks, many GFP-expressing cells were present at the optic nerve head, and some GFP-labeled fibers were present in the optic nerve, occasionally passing through the full extent of the lamina cribrosa. Only rarely were GFP-expressing cells found that coexpressed retinal phenotypic markers at any time point examined. CONCLUSIONS: Adult hippocampus-derived neural progenitor cells transplanted to the subretinal space readily engraft into a host retina that has undergone ischemic injury. Many cells migrate to specific retinal cellular layers and undergo limited morphologic differentiation reminiscent of retinal neurons, including extension of processes into the optic nerve. Concurrent control studies demonstrate that optimal engraftment is achieved by subretinal delivery within a specific temporal window. These results imply that certain inductive cues may be regulated after injury, and they demonstrate the potential for adult neural progenitor cell transplantation for the treatment of retinal neurodegenerative diseases.     

Suppressed expression of tubedown-1 in retinal neovascularization of proliferative diabetic retinopathy.

PURPOSE: Retinal neovascularization occurring as a complication of diabetes mellitus can cause vision loss and blindness. The identification and study of novel genes involved in retinal angiogenesis may define new targets to suppress retinal neovascularization in diabetes and other ocular diseases. A novel acetyltransferase subunit, tubedown-1 (tbdn-1), has been isolated, the expression of which is regulated during blood vessel development. Tbdn-1 is not detected in most adult vascular beds but persists at high levels in the adult ocular vasculature. The purpose of this study was to gain insight into the possible role of tbdn-1 in retinal blood vessels by characterizing its expression patterns in adult homeostasis and in retinal neovascularization associated with diabetes. METHODS: Western blot analysis and immunohistochemistry were performed to study the expression patterns of tbdn-1 during adult homeostasis in normal human retinas, in a model of choroid-retina endothelial capillary outgrowth in vitro, and in retinas showing neovascularization in patients with proliferative diabetic retinopathy (PDR). RESULTS: In adults during homeostasis, tbdn-1 was expressed highly in normal endothelium of retinal and limbic blood vessels. Tbdn-1 was also expressed in RF/6A, a rhesus macaque choroid-retina-derived endothelial cell line. In an in vitro model system using the RF/6A cell line, tbdn-1 expression was downregulated during the outgrowth of these cells into capillary-like structures on a reconstituted basement membrane matrix. Similar to this in vitro model, tbdn-1 expression is specifically suppressed in the endothelial cells of blood vessels and capillary fronds in vivo in both the neural retinal tissue and in preretinal membranes in eyes of patients with PDR. CONCLUSIONS: High levels of expression of tbdn-1 are associated with ocular endothelial homeostasis in adults. Conversely, low levels of tbdn-1 expression are associated with endothelial capillary outgrowth in vitro and retinal neovascularization in vivo. Because the tbdn-1 acetyltransferase subunit is a member of a family of regulatory enzymes that are known to control a range of processes, including cell growth and differentiation, through posttranslational modification, the current results support a hypothesis that tbdn-1 may be involved in maintaining homeostasis and preventing retinal neovascularization.