原代视网膜母细胞瘤耐药基因及耐药蛋白表达的研究

目的 研究视网膜母细胞瘤(Rb)瘤细胞多药耐药基因(MDR1)和多药耐药相关蛋白(MRP)基因以及相应耐药蛋白P-gp和MRP的表达水平.方法 共35例Rb患儿,行眼球摘除术时取新鲜的肿瘤细胞,用RT-PCR的方法 检测Rb瘤细胞是否有耐药基因MDR1和MRP基因的表达,用免疫组织化学的方法 检测相应耐药蛋白P-gp和MRP的表达.结果 34例Rb瘤细胞进行了耐药基因的检测,MDR1基因平均表达水平为0.41±0.38,MRP基因平均表达水平为0.73±0.92;35例Rb进行了耐药蛋白的检测,P-gp有15例阳性(42.9%),MRP有23例阳性(65.7%);34例Rb两种基因和蛋白两种方法 的检验结果 呈一致性;两种耐药基因及两种耐药蛋白的表达相互间有一定的联系.结论 原代Rb瘤细胞中一部分个体有不同程度的耐药基因MDR1和MRP基因及其产物耐药蛋白P-gp和MRP的表达,提示Rb可能存在原发耐药性.     

反义Rb基因促进细胞生长

视网膜母细胞瘤易感基因（Rb基因）是一个搞癌基因，在多种肿瘤组织中存在缺失、突变或失活。我们成功构建了Rb基因的反义表达载体DLORBAS并用电穿孔技术将DOLRBAS导入人胚肺纤维细胞（HEL细胞），转染后72小时测定发现HEL细胞的Rb蛋白表达降低，同时生长速度加快，但不能在软琼脂上形成克隆。这表明正常的Rb基因可以抑制HEL细胞的分裂，但单纯的Rb基因失活还不足以导致HEL细胞的恶变。 （中华眼底病杂志，1993，9：198-201）     

视网膜母细胞瘤组织直接免疫制备抗人视网膜母细胞瘤单克隆抗体及其抗原特性的实验研究

目的 研究新鲜视网膜母细胞瘤（retinoblastoma,Rb)细胞制备的抗人Rb单克隆抗体（monoclonal antibody,McAb)的抗原特性。方法 利用手术中获得的新鲜Rb组织细胞制备成细胞悬液,并将其注射入3呈BALB－C小鼠腹腔中,免疫小鼠。取其脾淋巴细胞与SP2／0骨瘤细胞融合,以抗原的性质进行检测,观察McAb相应抗原蛋白在Rb组织中的表达。结果 经反复单细胞克隆化操作及抗体特异性筛选,共获得3株能持续,稳定分泌抗人Rb McAb的杂交瘤细胞系。其中3C6株杂交瘤细胞在体外经≥2年反复冻存,传代培养,仍能稳定分泌特异性强,效价高的抗体,为IgG1亚类。免疫荧光及免疫组化检测结果显示,McAb3C6相应抗原蛋白在Rb组织中呈强阳性表达,而在其他肿瘤及正常组织中无表达。免疫印迹法检测表明,McAb3C6相应抗原蛋白相分子质量约25000。结论 采用新鲜的实体瘤细胞制备的抗人Rb McAb 3C6在Rb组织中呈高度特异性表达,与其他肿瘤及正常眼组织无交叉反应,其所针对的抗原蛋白相对分子质量约为25000,提示可能有新的基因参与Rb肿瘤的发生。     

HXO-Rb44视网膜母细胞瘤株多药耐药现象的基因研究

目的 研究视网膜母细胞瘤（retinoblastoma,Rb)细胞系HXO－Rb44细胞多药耐药基因（multidrug resistance gene,MDR1)和多药耐药相关蛋白（multidrug associated protein,MRP）基因的表达,探讨Rb多药耐药现象的发生机制。方法 用逆转聚合酶链反应检测HXO－Rb44细胞的MDR1和MRP基因的表达,并用免疫组织化学方法对其蛋白产物P－gp和P190进行检测。结果 HXO－Rb44系同时存在MDR1和MRP基因,并呈现P-gp和P190的过度表达（96％和97％）。结论 MDR1和MDRP基因的过度表达是Rb多药耐药现象发生的重要机制。     

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。     

视网膜母细胞瘤基因多效性的分子生物学研究

视网膜母细胞瘤(Rb)基因的发现,导致“抗癌基因”理论的建立。1986年国外学者成功地分离丁Rb4.7kb cDNA克隆,推测其蛋白产物是一个约105kd大小的具有DNA结合能力的核磷蛋白质,它能同一些病毒癌基因产物如EIA、SV40大T抗原等结合,可能具有控制正常细胞生长发育的功能。利用Rb cDNA探针及其表达产物制备的多克隆抗体,不仅在视网膜母细胞瘤中发现有Rb基因结构,转录和蛋白产物的异常,而且先后在成骨肉瘤,小细胞肺癌,乳腺癌、滑膜肉瘤以及膀胱癌组织或细胞株中发现Rb基因相似的异常情况。本文就Rb抗癌基因蛋白产物的功能以及与其它恶性肿瘤发生发展的关系作一综述。     

Rb基因调节细胞周期抑制乳腺癌细胞生长及其肿瘤特性

视网膜母细胞瘤易感基因（Rb基因）在多种肿瘤组织中有缺失或失活。我们构建了含正常人Rb cDNA全长序互列的逆转录病毒载体DOLRB,用电穿孔转染DOLRB引入Rb基因几乎完全缺失的乳腺癌细胞的MDAMB468.Rb基因在该细胞内得到表达，使其生长速度下降约50%，软琼脂克隆形成能力完全受抑，裸鼠体内致癌性部分受抑。同时流式细胞学检查显示该细胞在G1期比例明显增加，S期比例亦有增加。增殖商数下降约50%.证实Rb基因可阻止G1期向S期转变。 （中华眼底病杂志，1993，9：135-140）     

人类视网膜母细胞瘤的细胞起源

人类视网膜母细胞瘤(RB)是起源于神经原,还是神经胶质细胞或视网膜祖细胞,是一个长期存在争论的问题.随着电子显微镜的广泛应用,以及对视网膜母细胞瘤基因(Rb基因)和蛋白功能研究的不断深入,大量证据表明人类RB很可能起源于红绿视锥前体细胞.若能选择性地在人视锥前体细胞中剔除Rb基因,就能确切地知道视锥前体细胞是否为人RB的细胞起源.因此,针对视锥细胞信号传导通路的研究将是今后RB研究的新热点.     

视网膜母细胞瘤多药耐药相关蛋白的研究

目的 检测P-糖蛋白(P-Gp)、多药耐药基因相关蛋白(MRP)及肺耐药相关蛋白(LRP)在视网膜母细胞瘤(Rb)中的表达及临床意义;初步分析Rb患者临床病理指标与MRP间的关系;探讨Rb多药耐药现象的可能机制.方法 实验研究.应用免疫组织化学染色方法检测P-gp、MRP、LRP在75例Rb肿瘤标本中的表达情况.分析3种蛋白表达的相关性及其与患者年龄、性别、眼别、临床表现、组织病理分化程度等临床病理指标的相关性.各蛋白表达情况与一般临床特点、组织病理学特征的比较采用卡方检验,各蛋白间的相关性采用多元相关分析.结果 P-Gp、LRP、MRP蛋自在Rb中阳性表达率分别为64.0%、25.3%、36.0%.P-gp与LRP、P-gp与MRP、LRP与MRP的共表达阳性率分别为18.7%、32.0%、20.0%.P-gp、LRP、MRP在分化型Rb组织中的阳性表达率均高于杀分化型,且组间差异具有统计学意义(χ2=8.002,χ2=17.327,χ2=28.421;P<0.05).3种蛋白的表达均与年龄、性别、眼别无关(χ2=0.003～3.385,P>0.05).P-gp、LRP的表达分别与MRP的表达其有相关性(r=0.389,r=0.521;P<0.05).结论 Rb原发性多药耐药的形成是一个多因素、多步弱的复杂过程,与包括P-gp、LRP、MRP等在内的多项因素的参与有关.P-gp、LRP、MRP蛋白可以作为反映Rb耐药的分子基础,耐药相关标志的联合检测更有利于准确判断Rb的多药耐药状态,为Rb化学治疗提供科学的理论依据.     

108例视网膜母细胞瘤Rb基因突变的特征

目的；研究RB患者肿瘤及体细胞内Rb基因的存在状态、细微结构、Rb基因突变的特征、起源与传递。 方法；DNA分子杂交、SSCP分析、DNA序列测定。结果：108例RB肿瘤标本中80例(?4％)存在Rb基因点突变，其中44例为纯合型，20例有二个独立发生的杂合型点突变，16例只检出一个杂合型点突变。通过对比分析RB患者肿瘤与白细胞DNA、RB患者家庭成员白细胞DNARlo基因的结构，揭示了Rb基因点突变的起源与遗传特征。 结论：Rb基因是与RB肿瘤形成关系最为密切的基因。RB肿瘤发生需二次突变事件导致Rb基因的二个等位基因失活，第一次突变事件突出表现为点突变；第二次突变事件主要是LOH，其次是点突变。 （中华眼底病杂志，1997，13：12-16）     

Rhodopsin's amino terminus is a principal antigenic site

Antisera and monoclonal antibodies to rhodopsin were examined for their binding specificity to rhodopsin by using peptides from the rhodopsin sequence as competitors for antibody binding to rhodopsin in an enzyme-linked immunoassay. Monoclonal antibodies tested were raised in mice against bovine and rat rhodopsin. Antisera tested were raised in sheep against bovine rhodopsin and in rabbits against human rhodopsin. Peptides were synthesized from the bovine rhodopsin sequences 2-32, 1-12, 13-23, 24-34, 5-11, 231-252 and 331-348 for use as competitors in the immunoassay. A mixture of soluble CNBr peptides, and the purified CNBr peptide representing the sequence 2-39 were also employed. The monoclonal antibodies were all anti-amino-terminal in their binding specificity, although each recognized slightly different regions of the amino terminus. Each of the three antisera was predominantly directed against rhodopsin's amino terminus. We conclude that the amino-terminal 30 or more amino acids, and particularly the amino-terminal 15 amino acids, represent a principal antigenic region of the rhodopsin molecule.     

晶体上皮细胞单克隆抗体的动物实验研究──第二部分 晶体上皮细胞膜单克隆抗体的制备

通过将免疫小鼠的脾细胞与HGPRT缺陷的NS－1骨髓瘤细胞进行细胞融合，并经HAT培养液对融合的杂交瘤细胞进行选择培养，再经二次限界稀释法对此杂交瘤细胞进行克隆化。采用无血清细胞培养技术，得到较高浓度而且较纯化的抗家兔晶体上皮细胞膜单克隆抗体。为进一步研究晶体上皮细胞的特性，以及利用免疫学方法抑制白内障术后晶体上皮细胞增殖奠定了基础。     

Hybridoma antibodies to insoluble antigens of the corneal epithelium

Human corneal epithelial water insoluble proteins were used to immunize mice. Immune splenocytes were fused with Sp 2/0-Ag14 mouse myeloma cells by 40% PEG. Hybridoma antibodies obtained by somatic cell fusion were tested by radioimmunoassay. Supernatants from antibody positive hybrid colonies were used in immunofluorescence and crossreaction assays to locate and characterize corneal epithelial antigens. At least three distinct epithelial cell antigens were detected, one of which cross-reacts with rabbit corneal epithelial cells.     

Monoclonal IgA antibodies protect against Acanthamoeba keratitis.

Acanthamoeba keratitis is a rare, yet sight-threatening corneal infection. Ocular infection does not appear to induce protective immunity as repeated corneal infections occur in both humans and experimental animals. However, we have recently demonstrated that activation of the common mucosal immune system by oral immunization with Acanthamoeba antigens protects both Chinese hamsters and pigs against ocular infection with A. castellanii. Protection correlates closely with the appearance of anti- Acanthamoeba antibodies in the tears. To test the hypothesis that oral immunization induces specific protective IgA antibodies, two monoclonal IgA antibodies specific for Acanthamoeba antigens were generated. Both antibodies detected epitopes on the surface of fixed Acanthamoeba trophozoites. When delivered intraperitoneally, one monoclonal antibody (14E4) was detected in stool and tear samples. This clone also protected naive animals against ocular challenge with Acanthamoeba trophozoites (43% infection rate compared to a 91% infection rate in animals receiving control IgA). In vitro functional studies showed that neither antibody induced encystment or directly killed Acanthamoeba trophozoites. However, both monoclonal anti- Acanthamoeba IgA antibodies produced a three-fold inhibition in the adherence of trophozoites to corneal epithelial cells in vitro. These data show that monoclonal anti- Acanthamoeba IgA antibodies can protect against Acanthamoeba keratitis and suggest that this occurs by inhibiting adhesion of the parasite to the corneal epithelium.     

Cytoskeletal filament typing of human corneal endothelial cells.

Flat mounts of human corneal endothelial cells (HCECs) were examined immunohistochemically by using a wide assortment of monoclonal antibodies against the five classes of intermediate filaments (IFs) and actin and myosin. HCECs showed uniform immunostaining with monoclonal antibodies against the 40-kD (CK 19) and 45-kD (CK 18) cytokeratin (CK). Only part of the endothelial cells reacted with monoclonal antibodies against the 52-kD (CK 8) and 54-kD (CK 7) cytokeratin polypeptides and with monoclonal antibodies against vimentin. Monoclonal antibodies against the low- and middle-molecular-mass neurofilament proteins produced positive staining of all HCECs. No positivity was obtained with antibodies against desmin or glial fibrillary acidic protein. In addition, positive immunostaining with monoclonal antibodies against actin and slow myosin demonstrate that these proteins form part of the cytoskeleton of HCECs. The results of this study show that immunostaining of flat cell preparations is very useful for studies on HCECs. HCECs display an unusual combination of cytokeratin IFs and neurofilaments, together with vimentin, and are heterogeneous with respect to their IF makeup. These findings are discussed in relation to the presumed origin of HCECs.     

Monoclonal antibodies to chick crystallins

Several monoclonal antibodies against chick crystallins were obtained. Immunoblot analysis indicated that the monoclonal antibodies distinguish not only the three classes of crystallin (alpha, beta and delta), but also their subclasses. Monoclonal antibodies alpha 2 and alpha 1 reacted exclusively with alpha A and alpha B, respectively, demonstrating that alpha-crystallin subclasses of the chicken are antigenically distinct. An immunohistological study utilizing these monoclonal antibodies showed that the two alpha-crystallin subclasses, alpha A and alpha B, are co-expressed in the same cells and are more concentrated in the epithelium than the fibers in 14-day-old chick embryo lenses. However, immunofluorescence suggested different distribution of alpha-crystallin subclasses within an epithelial cell. alpha A is distributed evenly in the cytoplasm, but alpha B is more concentrated in the fiber-proximal side. Using anti-beta monoclonal antibodies, it was shown that beta-crystallins are divided into three distinct subclasses according to their antigenicity: 27-kDa and 25-kDa beta-crystallins are recognized by beta 1 antibody, 26-kDa and 19-kDa beta-crystallins by beta 2 antibody, and 35-kDa beta-crystallins by beta 3 antibody. All of the anti-delta-crystallin monoclonal antibodies (delta 1 to delta 3) obtained here bound to all delta-crystallin molecular species separable by isoelectrofocusing.     

Detection of surface antigens in uveal melanoma using monoclonal antibodies

Monoclonal antibodies (MA) were obtained by fusion of a myeloma line from mice with spleen cells of mice previously immunized by live human cells of uveal melanoma of an epithelioid type (UMEL-H). From the cell fusion hybridomas were obtained which produced antibodies against immunized uveal melanoma cells. The binding capacity of MA was assessed by the sandwich radio-immuno (RIA) test using indirect immunofluorescence. Extensive specific tests revealed that MA are strongly bound to uveal melanoma, the cells of which were used to immunize the mice, but also fresh allogenic uveal melanoma cells UMEL-K which were also of the epithelioid type. A smaller binding activity was observed with the cell line of the uveal melanoma VUP-1 which was started 16 years ago and is formed by 92% by epithelioid cells. A smaller binding activity of MA was observed with the line of skin melanoma cells (HMB-2, B-HM8). The bond of MA with carcinomatous cells, fibroblasts, uveal and retinal cells from eyes of healthy humans was negative. The preliminary investigations indicate an antigenic homogeneity of tumour antigens of melanoma cells of the same histological type and the antigenic variability of uveal and skin melanomas.     

Herpes simplex virus glycoprotein D. Protective immunity against murine herpetic keratitis

%$%%protective effect of glycoprotein D (gD) immunization against murine herpetic keratitis was investigated. gD was purified by affinity chromatography using anti-gD monoclonal antibodies. Prior immunization with gD was shown to be effective in protecting mice from both the development of stromal keratitis and the spread of the virus to the central nervous system. The level of serum antibodies for virus neutralization, as well as for complement-dependent cytolysis (CDC), was significantly elevated in gD-immunized animals. Cellular immunity, however, was not detected. These results indicate that two antibody-mediated defense mechanisms--virus neutralization and CDC--were responsible for the protective effect observed in our study.     

Immunoanalysis of keratan sulfate proteoglycan from corneal scars

Corneal keratan sulfate proteoglycan (KSPG) from scar tissue of experimental penetrating corneal wounds in rabbits was analyzed 2-8 weeks after injury using three previously characterized antibodies. Keratan sulfate (KS) was identified in 2 week scars and normal corneal tissue by indirect immunofluorescence using a monoclonal antibody against sulfated KS epitopes. KSPG was measured in unfractionated extracts of scar and of normal corneal tissue using a "sandwich" enzyme-linked immunosorbent assay (ELISA). In extracts of 2 week scars, KSPG molecules reacting with two different anti-KS monoclonal antibodies were 55% and 82% as abundant as in normal tissue extracts. Ion exchange high performance liquid chromatography (HPLC) of tissue extracts found qualitatively similar elution profiles of KSPG antigens from both scar and normal tissues. Direct ELISA of the HPLC-purified KSPG showed identical quantitative binding of antibodies against core protein and KS from normal and scar tissue. KS in the HPLC-purified extracts was sensitive to digestion with endo-beta-galactosidase, whereas core protein antigens were not affected by this enzyme, as expected. Alteration of the antigenic characteristics of the KSPG of scars was detected with a competitive immunoassay using immobilized monoclonal antibodies against KS. KS in extracts from 2, 6, and 8 week scars competed only 5-11% as effectively as KS from normal cornea, although core protein antigens in the scar extracts competed 61-80% as well as those of normal cornea.(ABSTRACT TRUNCATED AT 250 WORDS)