视网膜母细胞瘤的免疫组织化学研究

本文应用GFAP,S-100蛋白和NSE(PAP法)免疫组织化学染色,探讨视网膜母细胞瘤的组织发生问题。我们认为该肿瘤起源于神经外胚层,具有向神经元系统和神经胶质细胞系统双向分化的潜在趋势,其中更主要的是向神经元系统分化。     

bcl-2及p53在视网膜母细胞瘤中的表达及意义

目的 :为探讨bcl -2及p5 3在视网膜母细胞的表达及意义。方法 :本研究用免疫组织化学SP方法检测 45例视网膜母细胞瘤及 12例正常视网膜组织中bcl-2及 p5 3蛋白的表达。结果 :bcl-2在视网膜母细胞瘤中的表达 (2 70 2± 1 0 82 )与正常视网膜组织中 (0 167± 0 3 89)相比差异具非常显著性 (P <0 0 0 0 1) ,p5 3在视网膜母细胞瘤中的表达为 1 0 89± 0 848,而在正常视网膜组织中的表达为 0 1667± 0 3 89,差异有非常显著性 (P =0 0 0 0 6)。结论 :bcl-2及p5 3在视网膜母细胞瘤中均呈明显高表达 ,其明显抑制凋亡的发生 ,与肿瘤的发展关系密切。     

Hsp70在视网膜母细胞瘤组织芯片中的表达及其意义

目的检测Hsp70在视网膜母细胞瘤和正常视网膜组织中的表达,并讨论其意义。方法利用组织芯片,采用免疫组化SA法检测视网膜母细胞瘤和正常视网膜组织中Hsp70的表达。结果视网膜母细胞瘤组织芯片中Hsp70在胞浆有高表达(13/16),在正常视网膜组织中表达阳性率低(1/5),二者间存在显著性差异(P=0.0254<0.05)。结论Hsp70在视网膜母细胞瘤中高表达,它可能参与了视网膜母细胞瘤的发病。     

视网膜母细胞瘤的细胞来源与分化研究

对５０例视网膜母细胞瘤石蜡标本作免疫组织化学（神经元特异性烯醇化酶ｎｅｕｒｏｎ ｓｐｅｃｉｆｉｃ ｅｎｏｌａｓｅ，ＮＳＥ），神经胶质原纤维酸性蛋白及组织化学（胶性铁粘多糖５０例及核糖核酸２例）研究，１３例作透射电视观察。Ｒｂ细胞显示不同程度的ＮＳＥ阳性或阴性反应。大多数Ｆ－Ｗ及Ｈ－Ｗ菊花团呈阳性ＮＳＥ反应，花状饰呈阴性或弱阳性。ＧＦＡＰ阳性细胞主要位于肿瘤边缘及血管周围，少数掺杂于ＮＳＥ阳性细胞之     

视网膜母细胞瘤增殖细胞核抗原表达的研究

目的 :探讨增殖细胞核抗原 (proliferatingcellnuclearantigen ,PCNA)在视网膜母细胞瘤 (retinoblastoma ,Rb)中的表达及其与Rb病理类型和侵犯视神经情况的关系。方法 :用免疫组化技术检测PCNA在 3 7例Rb组织标本中的表达 ,并应用多功能真彩色病理图像分析系统 (CMIAS系列 )来分析PCNA染色的阳性细胞个数 ,采用积分光密度值定量分析其增殖指数 ,并将各指标进行SPSS统计软件相关性分析。结果 :本组 3 7例Rb标本中PCNA的阳性表达为 97 3 0 % ,而在 2例正常视网膜组织内 (对照组 )的表达均为阴性。PCNA的表达水平与Rb不同的分化、增殖程度有显著的统计学差异 (P <0 0 1) ,Rb侵犯视神经情况越严重、分化程度越差 ,PCNA的表达越强。结论 :Rb中PCNA的高表达可作为判断Rb恶性及其程度的新型标记物。PCNA与Rb分化类型、视神经侵犯情况密切相关 ,为PCNA表达水平反映Rb分化程度和增殖能力提供理论依据     

PTEN和p53在视网膜母细胞瘤中的表达及临床意义

目的检测视网膜母细胞瘤(retinoblastoma,RB)组织中PTEN基因和p53基因的蛋白表达水平并探讨二者在RB发生发展中的作用以及相关性。方法采用PV-9000免疫组织化学二步法,检测40例RB组织和16例正常视网膜组织中PTEN和p53蛋白的表达,统计分析不同临床和病理阶段RB表达PTEN和p53的差异及其相关性。结果(1)PTEN在正常视网膜组织中的阳性表达率为100%,显著高于RB55%(P<0.01);PTEN蛋白的阳性表达率与临床分期、病理分型以及是否伴有视神经浸润有关(P<0·05);与性别差异无关(P>0.05)。(2)p53在正常视网膜组织中不表达,在RB组织中阳性表达率为57.5%,差异有统计学意义(P<0·05);p53蛋白的阳性表达率与临床分期、病理分型以及是否伴有视神经浸润有关(P<0.05);与性别差异无关(P>0·05)。(3)PTEN和p53在RB中的蛋白表达呈负相关(r=-0.3710,P<0.05)。结论PTEN和p53可作为判断RB恶性程度和预后的指标。PTEN可作为RB早期诊断的分子标记物之一。     

视网膜母细胞瘤预后因素分析

目的 探讨影响视网膜母细胞瘤(RB)患者预后的因素.方法 前瞻性系列病例研究.采用免疫组织化学两步法检测31例手术摘除的RB患者标本神经元特异性烯醇化酶(NSE)、神经胶质原纤维酸性蛋白(GFAP)、S-100蛋白的表达,Spin-病理图像分析仪计数每例1000个肿瘤细胞中NSE阳性的细胞数,对GFAP、S-100蛋白染色阳性的细胞进行定量,全部随访5～17年,采用Cox回归模型分别将性别、年龄、临床分期、分化类型、NSE计数、GFAP、S-100蛋白染色定量、治疗方法与预后进行分析;Spearman法分析等级相关因素,t检验用于两均数的比较.结果 (1)NSE在31例RB标本中均有不同程度表达,GFAP染色阳性19例,阳性率61.29%(19/31),S-100蛋白染色阳性18例,阳性率58.06%(18/31);NSE阳性数值越高、GFAP及S-100蛋白染色定量分级越高,预后越好(P=0.0361,0.0430,0.0477).(2)术后生存时间≥5年患者的NSE平均计数(324/1000)高于术后生存时间<5年患者的NSE平均计数(182/1000),差异有统计学意义(t=3.602,P<0.01).(3)NSE计数与GFAP及S-100蛋白染色定量的结果密切相关(r=0.547,0.581).结论 (1)RB患者标本的NSE染色阳性瘤细胞计数值越高,生存率越高.(2)GFAP及S-100蛋白在RB中的表达与预后呈正相关.(3)NSE染色阳性细胞计数、GFAP、S-100蛋白染色分级可作为评估RB患者预后的组织学参数之一.     

Pax6基因在视网膜母细胞瘤中的表达及临床意义

目的:探讨Pax6基因在视网膜母细胞瘤(retinoblastoma,Rb)中的表达及临床意义。方法:选择我院2001-01/2012-12收存在眼科病理室的15例Rb组织切片设为观察组,再选取15例正常视网膜组织切片设为对照组。应用Western-Blot及RT-PCR(逆转录酶链反应)法分别对正常视网膜组织及Rb组织中的Pax6蛋白和Pax6 mRNA的表达进行检测,同时应用Western-Blot法对Pax6基因下游的BRN3b及MATH5促分化基因在蛋白水平的表达进行检测,最后进行组间比较,进而对Pax6基因在Rb中的表达及临床意义进行探讨。结果:观察组Pax6基因mRNA表达平均值为0.99±0.03,Pax6基因蛋白表达平均值为2.07±0.15,BRN3b蛋白表达平均值为0.195±0.016,MATH5蛋白表达平均值为0.190±0.031,均明显高于对照组,差异有统计学意义(P<0.05)。结论:异常表达的Pax6基因可能对Rb的出现起到促进作用。     

p53和Bag-1在视网膜母细胞瘤组织芯片中的表达及意义

目的检测p53和Bag1在视网膜母细胞瘤和正常组织中的表达,并讨论其意义。方法利用组织芯片采用免疫组化SP法检测视网膜母细胞瘤和正常组织中p53和Bag1的表达。结果p53在视网膜母细胞瘤组中高表达(13/16),正常视网膜组无阳性表达(0/5),二者之间差异有显著性意义(P=0.00275<0.01)。Bag1在视网膜母细胞瘤组中呈高表达(14/16),正常视网膜组中低表达(1/5),二者之间差异有显著性意义(P=0.0114<0.05)。结论p53和Bag1可能参与了视网膜母细胞瘤的发生。     

P53蛋白及P21蛋白在视网膜母细胞瘤表达的研究

目的：探讨P53和P21在视网膜母细胞瘤发生中的作用,及其与预后的关系。方法：应用抗人P53,P21抗体,采用链霉素亲生物素蛋白一过氧化霉（SP）免疫组化方法。对30例RB常规石蜡标本进行P53和P21蛋白的测定,结果：P53和P21在RB中的阳性表达率分别为60％和80％,P53的表达与临床分期有关（P＜0．05）,P53和P21的表达均与生存时间有关,P53阳性组生存时间低于P53阴性组（P＜0．05）,P21阳性组生存时间高于P21阴性组（P＜0．01）,P53阳性组的2年生存率低于P53阴性组,而P21阳性组的2年生存率高于P21的阴性组,结论：在RB的发生中P53和P21起着重要的作用,P53和P21的表达可以作为临床预后的一个参考指标。     

双眼异时性视网膜母细胞瘤21例临床分析

目的　通过分析双眼异时性视网膜母细胞瘤（RB）患儿的临床资料,确定高危因素,制订合理的随访计划,从而使肿瘤得以早期发现、早期治疗。方法　回顾性病例分析。收集2014年12月至2019年3月北京儿童医院收治的第一眼确诊为RB后,第二眼发现新发RB的病例共21例,分析双眼确诊间隔时间、发病年龄、首发症状,以及第二眼的确诊方法与肿瘤分期（IIRC）关系等。眼底检查以全身麻醉下间接眼底镜联合RetCam作为常规检查。结果　第一眼发病时患儿的年龄为1~36个月,平均12.2个月,13例年龄≤12个月;第二眼发病年龄为3~84个月,平均25.7个月,19例年龄≤36个月;双眼发病间隔时间为1~48个月,平均13.7个月。第二眼有首发症状者4例,17例（81.0%）在随诊例行检查时发现,无临床症状。第二眼肿瘤分期:A期8例,B期4例,C期1例,D期6例,E期2例。第二眼肿瘤以全身麻醉下间接眼底镜联合RetCam检查发现为主,患者多为早期。结论　第一眼RB发病年龄≤12个月是第二眼发生RB的高危因素,双眼确诊的间隔时间多≤ 36个月;定期、规律随诊和全身麻醉下间接眼底镜联合RetCam检查是早期发现第二眼RB的有效方法。     

Immunohistochemical characterization of human retinoblastomas in situ with multiple markers

We studied paraffin-embedded specimens from 18 surgically enucleated eyes with retinoblastoma by peroxidase-antiperoxidase immunohistochemistry with antibodies against glial fibrillary acidic protein, S-100 protein, Leu 7 epitopes, neuron-specific enolase, the 200-kilodalton subunit of the neurofilament triplet polypeptide, and retinal S-antigen. We found that (1) glial fibrillary acidic protein, S-100 protein, and Leu 7 epitopes were detected only in well-differentiated glial cells that were interpreted as reactive and not neoplastic, (2) undifferentiated neoplastic cells expressed both neuron-specific enolase and retinal S-antigen immunoreactivity, and (3) differentiated cells forming Flexner-Wintersteiner rosettes were found to express neuron-specific enolase, retinal S-antigen, and, occasionally, neurofilament protein. These results support the view that retinoblastomas are composed of neuron-committed cells and favor the origin of these tumors from photoreceptor progenitor cells. We did not find any morphologic or immunohistochemical evidence of glial differentiation from tumor cells that would support the concept that retinoblastoma arises from a primitive neuroectodermal cell capable of divergent differentiation along neuronal and glial lines.     

MMP-2和MMP-9在视网膜母细胞瘤中的表达及意义

目的探讨视网膜母细胞瘤(retinoblastoma,Rb)中基质金属蛋白酶-2(matrixmetalloproteinase-2,MMP-2)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的表达及其与临床病程的关系。方法应用免疫组织化学SP法检测41例Rb组织中MMP-2和MMP-9的表达;用HMIAS-2000型全自动医学彩色图像分析系统测量其平均光密度值。统计学方法比较不同临床和病理阶段Rb表达MMP-2和MMP-9的差异。结果在41例Rb组织切片中均有MMP-2和MMP-9的表达。有视神经浸润的肿瘤MMP-2和MMP-9表达水平明显高于无视神经浸润的肿瘤(P<0.01),处于眼外期的肿瘤MMP-2和MMP-9的表达水平明显高于眼内期和青光眼期的肿瘤(P<0.01)。结论Rb细胞内有MMP-2和MMP-9的表达。MMP-2和MMP-9的表达水平与视神经浸润和临床分期存在密切的关系,提示其表达水平与该肿瘤的浸润与发展有关。     

Glutamine synthetase in the developing rat retina: an immunohistochemical study.

The distribution of glutamine synthetase (GS) was studied in rat retinas from the day of birth to adulthood by means of immunohistochemistry. GS was present in the pigment epithelium in the newborn rat, and over the next few days it was demonstrated around blood vessels and within small glial cells. The enzyme was first detected in perikarya and processes of Müller cells on day 5. The adult GS pattern was acquired by day 12, except for persistence of GS in the pigment epithelium. GS in the pigment epithelium gradually diminished to trace amounts between day 12 and 2 months. Additionally, GS was found in the inner epithelium of the ciliary body and posterior epithelium of the iris during the developmental period and in adults. Our results indicate that the dramatic rise in retinal GS biochemically demonstrated by other workers occurs exclusively in Müller cells. Moreover, the complete acquisition of GS in rat retina corresponds chronologically with certain maturational events including the onset of the electroretinographic response. The role of GS in the pigment epithelium and extraretinal structures is uncertain but it may denote their involvement in mucopolysaccharide metabolism.     

MMP-2和MMP-14在视网膜母细胞瘤中的表达及意义

目的:探讨视网膜母细胞瘤(retinoblastoma,Rb)中基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和MMP-14的表达及其与临床病程的关系。方法:应用免疫组织化学SP法检测50例Rb组织中MMP-2和MMP-14的表达;用HMIAS-2000型全自动医学彩色图像分析系统测量其平均光密度值。统计学方法比较不同临床和病理阶段Rb表达MMP-2和MMP-14的差异。结果:患者50例Rb组织切片中MMP-2阳性表达38例,占76%;MMP-14的阳性表达41例,占82%;有视神经浸润的肿瘤MMP-2和MMP-14表达水平明显高于无视神经浸润的肿瘤(P<0.01),处于眼外期的肿瘤MMP-2和MMP-14的表达水平明显高于眼内期和青光眼期的肿瘤(P<0.01);Rb中MMP-2和MMP-14的阳性表达水平呈密切相关(P<0.01)。结论:MMP-2和MMP-14的表达水平与视神经浸润和临床分期存在密切的关系,提示其表达水平与该肿瘤的浸润与发展有关。     

Characterization of semenogelin proteins in the human retina

Semenogelin I and II are the major proteins present in semen coagulum. In the present study, semenogelin I and II were detected in human RPE lysates by proteomic analysis. We further analyzed the expression of these proteins in the retinal cells in vivo and in vitro. Western blots detected semenogelin I and II in both RPE and neural retina while the vitreous contained only SgII. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with an antibody that recognizes both forms of semenogelin proteins. Retina and RPE total lysates were evaluated for the presence of these proteins and in a human RPE cell line (D407). Both proteins were detected by western blot in human RPE and in D407 cell lysates. Immunoreactivity was detected in the ganglion cell and photoreceptor layer of the retina. Our data support the expression of semenogelin I and II in the human retina in several different compartments. Further studies towards addressing the function of these proteins in the retina are in progress.     

Transforming growth factor-beta in the chicken fundal layers: An immunohistochemical study

In the chicken model of myopia, it has first been shown that imposing defocus to the retina results in active remodelling of the sclera which, in turn, results in axial length changes of the eye. Transforming growth factor-beta (TGF-β) is one of the scleral growth modulators but its cellular localization in the fundal layers, colocalization and function are not well known. The aim of the current study was to investigate the cellular distribution of the three isoforms TGF-β1, 2 and 3 by immunohistochemical labelling. Furthermore, the effects of visual experience that induces refractive errors on TGF-β2 labelling were examined. Transversal cryostat sections of the fundal layers were analyzed by indirect immunofluorescent labelling and cell counts. Visual experience was changed by having the chicks wear either diffusers, or positive or negative lenses of 7D power in front of the right eyes for various periods of time. Left eyes served as uncovered controls. All TGF-β isoforms were localized in both scleral layers. In choroid, diffuse labelling of all isoforms was found. In retina, TGF-β1 and 3 were detected in bipolar, amacrine and ganglion cells and TGF-β2 in amacrine and ganglion cells. To further characterize these cells, double-labelling with known amacrine and bipolar cell markers was performed (calbindin, cellular retinoic acid binding protein (CRABP), Islet1, Lim3 and protein kinase C (PKC)). TGF-β1, 2 and 3 could be colocalized with calbindin and CRABP in single amacrine cells. TGF-β1-positive bipolar cells were immunoreactive to Lim3. TGF-β1 and 3 were never colocalized with PKC in bipolar cells. Also, colocalization with peptides known to be involved in myopia development in chicks, such as glucagon, or vasointestinal polypeptide and the key enzyme for dopamine synthesis, tyrosine hydroxylase, was not observed. Lenses or diffusers, worn by the chicks for various periods of time, had no effect on TGF-β2 immunoreactivity in choroid or sclera, or on the number of TGF-β2 (active and latent form) expressing amacrine cells. This result did not change when the two identified populations of TGF-β2 expressing amacrine cells (one calbindin-positive and the other CRABP-positive) were separately considered. Also no modulation was seen in choroid, although an earlier study had found changes in TGF-β2 mRNA after lens treatment. The lack of any visually-induced changes in retina or choroid suggests that TGF-β may not represent a key molecule in the retino-choroidal signalling cascade although it has previously been shown to have a primary role in scleral remodelling.     

Aldose reductase activity in a cultured human retinal cell line

The action of the enzyme aldose reductase has been shown to initiate cataract formation in diabetic animals. This enzyme may also play a role in other complications of diabetes such as retinopathy. In order to investigate aldose reductase from a human retinal source, the retinoblastoma cell line Y-79 was studied. Using a variety of substrates, these cells have enzyme activity consistent with aldose reductase. Immunologically, the aldose reductase from these cells cross-react with antibody to human placental aldose reductase. By immunohistochemistry, the enzyme is present in the cytoplasm of the Y-79 cells. Incubation of these cells with 30 mm-galactose resulted in considerable accumulation of dolcitol. This accumulation could be reduced when sorbinil, an aldose reductase inhibitor, was present in the culture medium. This report is the first to demonstrate the presence of human aldose reductase in a continuous cell line.