PURPOSE: To produce monoclonal antibodies directed against tumor-associated antigens expressed of retinoblastoma-derived tissue culture cell line SO-RBS0. METHODS:Hybridization was performed and the specificity of the antibody was tested by immunofluorescent and immunohistochemical methods. RESULTS:Two hybridomas secreted specific monoclonal antibody against retinoblastoma cells were produced ,and the isotype of the monoclonal antibody was IgG2a CONCLUION:The monoclonal antibodies were specific and highly active against retinoblastoma cells and might be used as immunoconjugate.
The Chinese human hepatocellular carcinoma cell SMMC7721 has been analysed by flow cytometry, Southern blotting and Western blotting. The results indicated that SMMC7721 cell is a hypoploid cell with a 0.81 DNA index, and that SMMC7721 cell has internal deletion in the 5'-end of its Rb gene and has no Rb gene product (Rb protein). The normal Rb cDNA has been inserted into a retrovirus vector DOL and introduced into SMMC7721 cells by electrporation transfection technique.About 75% of transfected SMMC7721 cells have been killed by Rb gene product. The remain 25% cells are alive as exogenous Rb gene has been mutationally inactivated. （Chin J Ocul Fundus Dis,1994,10:21-24）
Objective To observe the effect of resveratrol on multidrug resistance (MDR) in human retinoblastoma cells treated. Methods RB cells in logarithmic growth phase were divided into experimental group and control group. RB cells in experimental group were cultured with different concentrations of resveratrol (6.25, 12.50, 25.00, 50.00, 100.00 mu;mol/L) for 24 and 48 hours. The proliferation (absorbance value) was assayed using methyl thiazolyl tetrazolium (MTT). RB cells were cultured with 50.00 mu;mol/L resveratrol for 48 hours. The expressions of MDR-1, cyclooxygenase-2 (COX-2)、multidrug resistance-associated protein-1 (MRP-1), glutathione-S-transferases-pi; (GST-pi;) were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The RB cells of the control group were cultured with 0.5% dimethyl sulfoxide. Results Compared with the control group, the absorbance value decreased in experimental groups (6.25, 12.50, 25.00, 50.00 mu;mol/L) in a dose dependent manner (F=4.782，P＜0.05). The difference of absorbance value between 50.00 and 100.00 mu;mol/L experimental groups was not significant (F=6.351,P＞0.05). Compared with the control group, the mRNA (t=9.170, 5.758, 4.152, 4.638) and protein (t=3.848, 5.955, 4.541, 3.514) expression levels of MDR-1, MRP1, COX-2, and GST-pi; decreased in the experimental group (P＜0.05). Conclusion Resveratrol can down-regulate the expression of MDR in RB cells.
ObjectiveTo explore the presence of common genetic alterations in retinoblastoma and to localize the altered genomic regions.MethodsGenetic instability of chromosome 19, 20, 21, 22 and X of 15 microdissected retinoblastoma tumors were analyzed by the loss of heterozygosity (LOH) and microsatellite instability (MSI).ResultsAmong the 15 patients with retinoblastoma, genome instability ［LOH and(or) MSI］ at one or more loci on the 5 chromosomes in 10 (67%), in which the loss of a single allele was more frequent in chromosomes 19 (33%) and 20 (27%) than in the other 3 chromosomes. Highfrequency LOH between D19S902 and D19S571 suggested gene loci in the 19q13 region might be associated with tumor development in retina. According to the result of MSI, MSI occured at least in one subset of retinoblastoma.ConclusionsOur results provide first evidence of LOH in chromosomes 19 and 20 in retinoblastoma and further support the presence of genome instability in retinoblastoma that may play an important role in the tumorigenesis or progression of retinoblastoma.(Chin J Ocul Fundus Dis, 2005,21:28-31)
Synthesized Rb peptide was used as an antigen to immunize the BALB/C mice. After fusing and screening with ELISA and Western Blot, we got 3 hybridomas which secreted specific monoclonal antibodies against product of Rb gene. All of three kinds of antibodies were fourld to be IgG 1 through the appraisement. In addition,since we mixed three kinds of peptides as the antigens to immunize the mice,we have got three different hinds of monoclonal antibodies including one against Rb product and another two in the Same procedure. （Chin J Ocul Fundus Dis,1993,9:2-4）
Objective To investigate the correlation of expression of Fas/Fas ligand (FasL) and apoptosis in retinoblastoma (RB)． Methods The expression and distribution of Fas/FasL were detected by using immunohistochemical staining in 32 cases of RB． Light microsc opy (32 cases), electron microscopy (4 cases) and TdT mediated biotin-d UTP nick-end labeling (TUNEL) (12 cases) were used to study apoptosis in RB． Results Apoptotic RB cells mostly located at RB regress area． Chromatin margination and apoptotic bodies were found in RB． TUNEL posi tive labeling cells especially located in tumor regress area． Positive immunola beling for Fas and FasL was found in all RB specimens． There was a highly signi ficant and positive correlation between the expression of Fas/FasL and apoptotic indices (AI) (Plt;0.01 or 0.001)． Conclusion The results suggest that apoptotic cell death is prevalent in RB and it may be one type of the most dominant cell death． Fas system may play an important role in oncogenesis and progression of RB, and the up-regulation of Fas system expression might induce RB cell apoptosis． (Chin J Ocul Fundus Dis, 2001,17:21-23)
Microparticles are small vesicles that are released by budding of the plasma membrane during cellular activation and apoptotic cell breakdown. A spectrum of cell types can release microparticles including endothelial cells, platelets, macrophages, lymphocytes and tumor cells. Biological effects of microparticles mainly include procoagulant activity, inhibition of inflammation and cancer progression. The present study shows that vitreous microparticles isolated from proliferative diabetic retinopathy (PDR) stimulated endothelial cell proliferation and increased new vessel formation, promoting the pathological neovascularization in PDR patients. Oxidative stress induces the formation of retina pigment epithelium-derived microparticles carrying membrane complement regulatory proteins, which is associated with drusen formation and age related macular degeneration. Microparticles from lymphocyte (LMP) play an important role in anti-angiogenesis by altering the gene expression pattern of angiogenesis-related factors in macrophages. Besides, LMP are important proapoptotic regulators for retinoblastoma cells through reduction of spleen tyrosine kinase expression and upregulation of the p53-p21 pathway which ultimately activates caspase-3. However, how to apply the microparticles in the prevention and treatment of retinal diseases is a major challenge, because the study of the microparticles in the fundus diseases is still limited. Further studies conducted would certainly enhance the application of microparticles in the fundus diseases.
With the method of PCR-SSCP combined sequencing,we analysed the mutations in the RB1 gene of 10 retinoblastoma samples. All 27 exons of RB1 gene have been examined. The PCR products were digested with some restriction endonuclease to the size of about 200 bp and then denatured into single strands. Loading the single strands of DNA into the 6% polyacrilamide gel,we followed the technology named SSCP to f/nd the abnormal bands in the SSCP gel. After that, the samples found with the abnormal bands in the SSCP gel were amplified again and sequenced to confirm the types and locations of the mutations in the RB1 gene. We have fonnd some kinds of mutations in the RB1 gene. （Chin J Ocul Fundus Dis,1994,10:17-20）