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Age-related macular degeneration ( AMD ) is a kind of age-related blinding degenerative fundus lesions, totally about 30 million patients suffering from AMD all over the world, with about 500 000 people blind for it yearly. As the development of economy and the aging of the population intensified, incidence of AMD indicates a trend of rising year by year, being the third major cause of blindness in our country. At present, the pathogenesis of AMD is not fully clear, as reported it may be related to oxidative stress, inflammatory immune response, VEGF and genetic manipulation. Clinical treatments mainly include photodynamic therapy, drug therapy, radiation therapy, laser photocoagulaory operation, the pupil warm treatments, Chinese medicine and intravitreous injection VEGF antagonists such as Ranibizumab, Conbercept and so on. ln this issue, we mainly expound on the progress in the epidemiological studies of AMD, especially elaborate the progress made on genetic manipulation in recent years.
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Background Researches showed that wild-type p53(wtp53)and Rb94 genes inhibit the growth of retinoblastoma(RB),and these genes are involved in signal pathway in the induction and maintenance of cellular senescence.Thus the combination of two genes to inhibit growth of RB is concerned.Objective This study was to observe the inhibitory effect of the co-transfection of Rb94 and wtp53 gcnc into subretina on RB with ultrasound microbubble.Methods HXO-Rb44 suspension was subretinally injected to establish the RB model in 40 SPF female Balb/c nude mice.The RB models were randomized into model control group,wtp53 group,Rb94 group and wtp53+Rb94 group,and 0.1 ml relevant gene microvesicle suspension was injected via caudal vein in the different groups,but no any gene was used in the model control group.Seven days after modeling,followed by 0.5 W/cm2ultrasonic wave irradiated the eyeballs immediately for 4 seconds ×2 times and interrupted for 24 seconds.Eyeballs were extracted 7 days after gene transfection,and the expressions of wtp53 mRNA and Rb94 mRNA in tumor tissuc were detected by RT-PCR,and wtp53 and Rb94 protein in tumor tissue were assayed using Western blot.Immunochemistry was used to exam the VEGF expression,and TUNEL was used to evaluate the apoptosis of the tumor cells.Results The model successful rate after HXO-Rb44 suspension was 80％ (32/40)and obvious malformation cells were seen under the light microscope.In 7 days after gene transfection,no response band for wtp53 mRNA and Rb94 mRNA were found.The relative expression valuc of wtp53 mRNA was 0.65±0.07 in the wtp53 group,and that in wtp53+Rb94 group was 0.32±0.02,showing a significant difference between them (t =11.743,P =0.000).Rb94mRNA relative value was 0.42 ±0.03 in Rb94 group,and that in the wtp53 + Rb94 group was 0.23 ± 0.03,with a significant difference(t=5.041,P=0.001).The response bands of wtp53 and Rb94 proteins were seen in wtp53group,Rb94 group and wtp53+Rb94 group.Immunochemistry showed that the positive reactive intensity for VEGF in tumor tissue was obviously weaker in wtp53+Rb94 group than that in the wtp53 group,Rb94 group and model control group.Apoptotic index(Al) was 37.35±2.14 in the wtp53+Rb94 group,showing a significant increase in comparison with model control group (0.46 ± 0.05),wtp53 group (5.05 ± 0.80) and Rb94 group (6.43 ± 1.02) (t =-34.395,-28.206,-26.006,P＜0.01).Conclusions Ultrasound microvesicle enable double gene transfecting into RB tumor tissue,and Rb94 gene cooperation with wtp53 gene enhance the inhibitory effect on RB by promoting the apoptosis of RB cells.
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Background Cell immunologic therapy for retinoblastoma(RB)is becoming a hot research topic.Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors.However,its effect on RB has not been investigated.Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes(CTL)induced by the cancer-testis antigen,NY-ESO-1-sensitized dendritic cells(DCs),on human RB.Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI.Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1.The expression of NY-ESO-1 protein in human RB cells strain,HXO-RB44,was detected by immunofluorescence and Western blot.Monocytes were isolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 × 107/ml.DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor(rhGM-CSF)and recombinant human interleukin-4(rhIL-4).The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes.The resultant CTL were used as effector cells.The growth of the CTL was detected by MTT assay.The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay.Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-1 was conforms with the sequence of the NY-ESO-1 gene.The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot.DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC.The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs.These DCs had high expression of surface molecules such as HLA-DR(42.1％),CD80(54.2％),CD83(39.7％)and CD86 (94.8％).The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells.CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs,as shown by MTT asssay(P＜0.05).The anti-tumor activity was highest when the ratio of effector to target was 75∶1(P＜0.05).Conclusions DCs transfected by the recombinant plasmid pDC316/NY-ESO-1 can induce the proliferation of allogenic CTLs,which showed a specific anti-tumor effect against HXO-RB44 cells.These results present a new type of immunotherapy for the treatment of RB.
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<p><b>BACKGROUND</b>Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization (CNV), and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor (PEDF) could inhibit CNV.</p><p><b>METHODS</b>Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDF gene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure (intravitreal injection); infusing liposomes with the naked plasmid DNA of PEDF into the vitreous (lipofectamine + PEDF); infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately (retrobular injection); infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately (vein injection). The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.</p><p><b>RESULTS</b>In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively.</p><p><b>CONCLUSIONS</b>Ultrasound-microbubble technique could increase PEDF gene transfer into rats' retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.</p>