Retinitis pigmentosa is a hereditary disease which is characterized by damage in retinal photoreceptor cells and retinal pigment epithelium. Its main clinical features include low vision with night blindness, progressive visual field defects, and abnormal electroretinograms. The development of gene sequencing, the diagnosis and treatment methods of retinitis pigmentosa update year by year, including gene therapy, stem cell therapy, optogenetic therapy, etc. However, there is still a big gap in these treatments from laboratory technology into effective clinical treatment drugs. Some problems which include immune response, potential mutagenesis and tumorigenesis of the inserted region, genetic toxicity, quality and stability of gene technology and stem cell technology, mass production and promotion of clinical grade drugs, and optimization of the effectiveness of drugs and surgery, etc, remain to be solved by researchers.
Objective To observe the morphological changes and gene expression during the transdifferentiation of adult retinal pigment epith elial(RPE) cells into neuronal phenotype in vitro induced by retrovirus and ciliary neurotrophic factor (CNTF). Meothds The adult RPE cells derived from CRL 2302 were infected by retrovirus with green fluoresence protein(GFP)and then were transfected further by liposome mediated CNTF expressing plasmid.The cellular ability of producing CNTG,and the expression of CNTF, CNTF receptor (CNTFR), and signal transduction molecule janus tyrosine kinases (JAK) were detected by enzyme linked immunosorbent assay, immunohistochemical stainin gand Western blotting method. Results After infected by retrovirus, the configuration of adult RPE cells didnrsquo;t change much, but expressions of neurons and some glial cells markers likeneurofilament (NF) protein and glial fibraillary acidic protein (GFAP) were detected. After further transfected by CNTF expressing plasmid, RPE cells which expressed CNTF highly and continuously had differential neurocytes; the expression of CNTFR didnrsquo;t change, but the distribution position changed to the cell membrane; expression of signal transduction molecule JAK increased obviously. Conclusion The adult RPE cells may transdifferentiate into neurons induced by retrvirus and CNTF. The transdifferentiation may relate to CNTF-CNTFR-JAK signal transduction pathway. (Chin J Ocul Fundus Dis, 2006, 22: 400-403)
ObjectiveTo identify the pathogenic mutation in a three generation Chinese family with low penetrance retinoblastoma (RB). Methods8 from 9 family members received complete ophthalmic examinations. DNA was extracted from 6 family members. Using exon combined target region capture sequencing chip to screen the candidate disease-causing mutations. Sanger sequencing were used to confirm the disease-causing mutation. ResultsAmong 9 family members, the proband (Ⅲ2) was bilateral RB, Ⅲ1 was unilateral RB, Ⅲ3 was dead for bilateral RB. Normal fundus were observed in the left eye ofⅢ1 and the eyes of other family members except the proband. Sequence analysis of RB1 gene revealed a missense mutation c.1981C > T (p.Arg661Trp) in the proband and two carriers (Ⅱ2, Ⅱ3), but not in the two normal subjects (Ⅱ1, Ⅱ4). We suspect that the RB penetrance in the family was 50%. ConclusionsThere is a missense mutation c.1981C > T in a Chinese family with low penetrance RB. The RB penetrance is 50%.
Objective To observe the regional expression of hypoxia inducible factor-1alpha; (HIF-1alpha;) in retinoblastoma and its relationships with vascular endothelial growth factor (VEGF), Bax and Ki-67. Methods Immunohistochemical study for HIF-1alpha;, VEGF, Bax and Ki-67 was performed in 39 paraffinaceous examples of retinoblastoma. Each pathological section was divided into five regions: the surface region, the central part, the bottom part, the choroidal region and seeding tumors. The expressions, correlations and distributional differences of these factors were all invested both integrally and regionally. Results In the 39 cases of retinoblastoma, 10 cases (25.6%) were negative for HIF-1alpha;; 29 cases (74.4%) were positive for HIF-1alpha;, including 17 cases (43.6%) (+), 12 cases (30.8%) (++). Regionally, HIF-1alpha; was positive in 71.1%, 36.8%, 84.2%, 54.5% and 82.1% of the cases in the surface region, the central part, the bottom part, the choroidal region and seeding tumors, respectively, which was statistically reliable (chi;2=24.55，P＜0.001). The positive rate of VEGF, Bax and Ki-67 was 53.8%, 66.7% and 59.0%, respectively. In different regions, the positive rates of VEGF and Bax were different (chi;2=26.77, 22.79; P＜0.001), but there was no regional distinctions in the expression of Ki-67 (chi;2=0.47, P=0.976). Both the expression of VEGF and Bax had a positive correlation with that of HIF-1alpha;(rs=0.48, 0.39; P=0.002, 0.021), but there was no relationship between the expressions of Ki-67 and that of HIF-1alpha; (rs=0.09, P=0.606). Regionally, the expressions of VEGF, Bax and HIF-1alpha; shared similar distributional features: positive rates were higher in the surface region, bottom part and seeding tumors, and were lower in the central part and choroidal region, which was different from the expression of Ki-67. Conclusion The anoxic zones are more likely to be located in the marginal parts in retinoblastoma, and the expressions of VEGF and Bax had a positive correlation with that of HIF-1alpha; in different regions in retinoblastoma.
Objective To investigate the feasibility of gene transfection into retinal pigment epithelial (RPE) cells and photoreceptors (PRs) in vivo electroporation. Methods A total of 147 Sprague-Dawley (SD) rats were divided into 5, 10, 15, 20, 25, 30 and 35 V group according to different voltage. The right eyes of rats underwent the injection of eukaryotic expressive plasmid of enhanced green fluorescent protein (EGFP) pEGFP-N1 into subretinal space as experimental eyes; the left eyes were injected with TE buffer as control eyes. Each group was divided into RPE and RP subgroups according to different transfection direction. There were same parameters of 99 ms pulse width, 0.5 s pulse interval and 5 consecutive pulses except different voltage in groups. With a negative charge in the electric field was transfected into RPE cell layer, reverse electrode set to be transfected into PR cell layer. Retina mounts were made on seven days after transfection and the fluorescence of EGFP was photographed by fluorescent microscope. The expression of EGFP mRNA and protein were detected by reverse transcription polymerase chain reaction technique (RT-PCR) and Western blot.Results On seven days after transfection, in RPE subgroups, there were no specific fluorescence expressions in RPE cell layer and retina mounts of control eyes, while there were fluorescence expressions in experimental eyes. Western blot showed that the grayscale ratio of EGFP protein and beta;actin protein bands rose with the increased voltage. RT-PCR showed that each group produced positive amplification bands, and the relative ratio of gray level of EGFP mRNA and GADPH mRNA amplified bands gradually increased with the increased voltage.Conclusion Electroporation is an effective method for gene delivery into RPE cells in vivo.
Retinitis pigmentosa (RP) is a group of hereditary blinding fundus diseases caused by abnormalities in photoreceptors of the retina. RP is highly heterogeneous in hereditary and cdinical phenotypes. It can be divided into simple type RP and syndrome type RP. The main inheritance patterns are autosomal dominant, autosomal recessive inheritance and X-linked inheritance. With the popularization and clinical application of gene sequencing technology, more and more disease-causing genes have been discovered, and these genes are mainly expressed in photoreceptor cells and retinal pigment epithelial cell. ln-depth understanding of RP pathogenic genes not only provides a theoretical basis for RP diagnosis and genetic counseling, but also provides guidance for RP gene therapy.
Stargardt disease (STGD) is one of the most prevalent inherited macular dystrophy, and most often occurs in child or adolescence. Irreversible vision loss is observed in almost all cases. Type 1 (STGD1) is one of the most common type. It is an autosomal recessive condition, caused by mutations in the Abca4 gene. In recent years, encouraging progress has been made in the treatment of STGD1. C20-D3-retinyl acetate (ALK-001), fenretinide and ICR-14967 (A1120) as visual cycle modulators, StarGen as gene supplementation therapies, and the stem cell transplantation of human embryonic stem cell-derived retinal pigment epithelium cells are the most promising therapies. With the development of studies and clinical trials, the clinical application of various treatments of STGD1 are expected in the near feature, which are expected to save the vision of most patients.