The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E.coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired. Beclin1 mRNA and Beclin1 protein were detected by RT-PCR and Western blot analysis respectively. The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1. Two SH-SY5Y transfected cell lines were successfully selected. Compared with the control group, RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28%±1.45%(P<0.05)and 75.50%±2.63%(P<0.05), respectively. The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SY5Y cell lines with inhibited Beclin1 expression were established. It provides a useful cell model for studying the biological function of Beclin1.
Purpose To investigate bax expression and induction of apoptosis in normal cultured retinal pigment epithelium (RPE) cells ． Methods Cultured human RPE cells were transfected by PMDNA3-hbax，which incoded the whole bax gene and may be induced by Zn2+ under the MTII promoter， through lepofectin mediated protocol．The tested RPE cells were divided into three groups of A，PMDNA3-hbax transfected ;B，PMDNA3 (nude vector) transfected and C ，normal RPE cells．After transfection， DNA gel electrophoreses were perform ed ，the tested RPE cell cycles were analyzed with flow cytometry (FCM)． Results The gel electrophoretogram showed DNA ladder phenomenon，FCM confirmed the apoptosis of RPE cells PMDNA3-hbaxtransfected ， consisting of significant apoptotic peak sited before the G 1 phase and the apoptotic rate was 36%． Conclusion The foreign bax gene can be effectively conducted in to the RPE cell through lepofectinmediated protocol and induced expression ． The foreign bax overexpression may induce the cultured human RPE cell susceptibi lity to apoptosis． (Chin J Ocul Fundus Dis, 2001,17:132-134)
ObjectiveTo study the characteristics of the genotype and phenotypic in a family with X-linked retinoschisis (XLRS) due to RS1 mutation. MethodsA retrospective clinical study. An XLRS family of 4 generations of 26 people were included in the study. Among them, 8 participants were males and 7 participants were females. Routine ophthalmologic examination was performed on 3 patients in the family including the proband and 12 patients with normal phenotype. Optical coherence tomography was performed in 2 of the 3 patients. Peripheral venous blood was extracted from all participants, whole-genome DNA was extracted, and potential pathogenic genes were screened by Panel sequencing. Conservative analysis, pathogenicity analysis and protein structure prediction were carried out by software tools. The pathogenicity of gene mutations was analyzed according to the American Society of Medical Genetics and Genomics (ACMG) guidelines. ResultsThe proband was 3 years old. Optical coherence tomography (OCT) examination showed that the retinal core layer in the macular area of both eyes had a cystic change, which was segmented by vertical or oblique bridging tissue. The proband's uncle was 32 years old. OCT examination showed atrophy in the macular area of the left eye. The macular area of the right eye was cystoid, segmented by vertical or oblique bridging tissue. No abnormality was found in the fundus examination of the proband's parents and 10 members of his family. Panel sequencing showed that c.361C>T/ p.Q121X hemizygous mutation was found in the fifth exon of RS1 gene in the proband (Ⅳ3) and 2 patients (Ⅱ1, Ⅲ8). The mother was a heterozygous mutation carrier of the gene, while the father had no mutation. The mutant gene causes premature termination of RS1, a truncated protein encoding 224 amino acids to 120 amino acids. Of the 10 patients with normal fundus examination, 6 participants were normal. The mutation was carried by four people, which were women. Homology analysis of the protein sequence showed that the mutant site was highly conserved in 12 mammals. Three-dimensional structural analysis of RS1 protein showed that the c-terminal amino acid sequence of the mutant protein was more than 50% missing. Analysis of ACMG guidelines indicated that the mutation was pathogenic. ConclusionThe RS1 mutation site c.361C>T/p.Q121X is a new mutation site of XLRS.
ObjectiveTo observe and analyze the pathogenic gene types and clinical phenotypes of Leber congenital amaurosis (LCA).MethodsA retrospective clinical study. Six patients with LCA confirmed by genetic testing and 18 family members were included in the study. The patients came from six unrelated families. The family was investigated with a specific hereditary eye disease enrichment panel which contained 463 known pathogenic genes and based on targeted exome capture technology first to indentify the potential pathogenic genes and mutations. Then the TULP1, RPGRIP1, GUCY2D pathogenic mutations were conformed by Sanger sequencing. The pathogenicity of the gene variation was searched through relevant databases and PubMed literature, and its function was explained by protein prediction software.ResultsOf the 6 patients, 3 were males and 3 were females; the age was from 3 to 33 years. Nystagmus, finger pressing eyes, photophobia, and night blindness were seen in 5 cases; electroretinogram showed 3 cases of extinction or near extinction; and 4 cases of retinopathy. The results showed patients with compound heterozygous mutation of c.1318C＞T and c.1142T＞G, homozygous mutation ofc.1318C＞T and compound heterozygous mutation of c.1153G＞A and c.1561C＞T of TULP1 in Family 1, Family 2 and Family 5, respectively. There were compound heterozygous mutations of RPGRIP1 c.391delG and c.1468-2A＞G in Family 3 and c.715delA and c.1765C＞T in Family 6, respectively. Homozygous mutation of c.3177_3178delAC of GUCY2D was found in Family 4.The parents of all six patients were carriers of corresponding heterozygous mutations.TULP1 gene c.1142T＞G, RPGRIP1 gene c.391delG, c.715delA and c.1765C＞T and GUCY2D gene c.3177_3178delAC mutations were novel mutations and unreported. The 381th amino acid locus of product protein of TULP1 gene was highly conserved among species. The protein prediction software predicted that the mutation pathogenic. The c.391delG, c.715delA and c.1765C＞T mutations of RPGRIP1 gene and c.3177_3178delAC mutation of GUCY2D gene can lead to early translation termination of their product proteins, which are pathogenic variants.ConclusionThe pathogenic mutations of TULP1, RPGRIP1 and GUCY2D genes led to LCA 15, LCA 6 and LCA 1 in six families.
Despite its low incidence, retinoblastoma and its rela ted gene (Rb gene) have attracted some of the most brilliant minds in medicine and biology fi elds over the past years. Great advances have been achieved in the tumoregenesis mechanism and clinical management of retinoblastoma recently. However as always , more questions arise from those results. In order to improve retinoblastoma re search in China, we need to strengthen the communication and cooperation with di ffe rent countries, different institutes and disciplines, and utilize the great reso urces of retinoblastoma patients in China.
Objective To study the effect of hypoxia on proliferation of cultured bovine retinal pigment epithelium (RPE) cells and expression of the antiapoptotic protein bcl-2. Methods The bovine RPE cells were cultured under normal and hypoxic chamber respectively. After 24 hours, the proliferation of RPE cells was evaluated by［3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide, MTT］test. At the same time, anti-bcl-2 protein antibody was examined by immuno-histochemistry method. Results The A value in the hypoxia group was higher than that in the normal group after 24 hours (P＜0.05 )in MTT-test. Positive staining for anti-bcl-2 protein antibody was seen in 72.6% cells in hypoxia group and 38.64% in normal group. The positive staining was more obvious near the nucleus, and fine granules scattered in cytoplasm of some cells. Conclusion Hypoxia can stimulate the proliferation of RPE cells and expression of antiapoptotic protein bcl-2. The results indicate that bcl-2 may play an important role in mediating the proliferation activity of RPE cells. (Chin J Ocul Fundus Dis, 2002, 18: 293-295)
ObjectiveTo analyze the characteristics and prognosis of visual field of Leber hereditary optic neuropathy (LHON) with G11778A mutation.MethodsA retrospective clinical study. Twenty-two (44 eyes) of LHON patients diagnosed with G11778A site mutation by mt-DNA examination from May 2008 to February 2018 in Ophthalmology Department of Dongfang Hospital of Beijing University of Chinese Medicine, were enrolled in this study. All patients underwent best corrected visual acuity (BCVA), visual field and optical coherence tomography (OCT). The BCVA examination was performed using the international standard visual acuity chart, which was converted into logarithm of the minimum angle of resolution (logMAR) BCVA for record. The thickness of the retinal nerve fiber layer (RNFL) in the 200μm×200μm annular region 1.73 mm outside the optic disc was measured by OCT. At least 7 visual field examinations were performed within one month before and after 2, 4, 8, 12, 18, 24 and 30 months of the course of disease by using Octopus 101 perimetry. Among 44 eyes, 27 eyes were detected with G2 procedure (G2 group) and 17 eyes were detected with LVC procedure (LVC group). The mean field defect (MD) and mean optical sensitivity (MS) were used as the main outcome indexes. According to the onset age, the patients were further divided into the ≤14 years old group and＞14 years old group. There was a significant difference in initial logMAR BCVA between the G2 group and LVC group (t=4.994, P=0.000), but there was no significant difference in gender (χ2=1.896, P=0.169) and age (t=0.337, P=0.708) between the two groups. Independent sample t test was used for comparison between groups, paired t test was used for comparison within groups, and one-way analysis of variance was used for comparison between groups. The statistical data were compared by χ2 test.ResultsIn the G2 group, the MD value of the subgroup of children (≤14 years old) decreased gradually during the follow-up period, and the MD value since 18 months after onset was significantly lower than the value of 2 months after onset (t=3.813, 4.590, 5.033; P=0.002, 0.001, 0.000). No obvious visual field index changes were seen in other subgroups (P＞0.05). The central scotoma was the most common type of visual field defect in the early stage, and the diffuse defect was the most common type of visual field defect in the late stage. There was a significant difference in the types of visual field distribution between the early and late stage in G2 group (χ2=17.414, P=0.015). There was no significant difference in the type of visual field distribution between the early and late stage in LVC group (χ2=4.541, P=0.474). The MD value in the G2 group remained stable within 8 months after onset, but significantly improved after 18 months after onset (t=2.100, 3.217, 3.566; P=0.046, 0.003, 0.001). The MS in the LVC group did not significantly improve during follow-up (P＞0.05). The average visual acuity of the G2 group was significantly improved from 12 months (t=3.039, 3.678, 4.264, 5.078; P=0.008, 0.002, 0.001, 0.000). The visual acuity of the eyes in the G2 group was better than that of the LVC group during all follow-up periods (P≤0.05). The RNFL thickness of all patients continued to decrease after onset, but the RNFL thickness was significantly higher at 4, 8, 18, 24, 30 months in the G2 group than those in the LVC group (t=2.471, 2.269, 2.474, 2.509, 2.782; P=0.018, 0.028, 0.017, 0.016, 0.008).ConclusionsThe main types of visual field defect of LHON with G11778A mutation are the central scotoma in the early stage, while the diffuse defect and central scotoma are both very common in the later stage. The visual field of LHON patients examined by G2 procedure is significantly improved during the follow-up, as well as the visual acuity improved significantly, and the visual field improvement in younger cases (≤14 years old) is better than that of older cases (＞14 years old), but the visual field of the LVC procedure cases did not improve during follow-up.
Objective To explore the expression of survivin gene in retinoblastoma (RB) and its relationship with the stages and histodifferentiation degree of RB and the expression of p53、bcl-2 proteins. Methods Expression of survivin, p53 and bcl-2 proteins in 38 RB conventional paraffin samples were detected with survivin, p53 and bcl-2 monoclonal antibodies respectively by immunohistochemical assay. The expression of survivin of normal retina in 6 control samples was observed. Results In 38 cases of RB, positive expression of survivin was found in 20 (52.6%); while none of the 6 normal retinal tissue expressed survivin, which had significant difference between the two group (P＜0.05). The positive expression of survivin did not correlate with sex of patient, disease stages and histological type (P＞0.05). In 38 RB cases, positive expression of p53 was in 25 with the rate of 65.8%, and of bcl-2 in 18 with the rate of 47.4%. The positive-expressed rates were much higher in positive-expressed p53 and bcl-2 group than those in the negative-expressed p53 and bcl-2 group(P＜0.05). Conclusion The increase of the expression of survivin implies that it may take part in the occurrence and development of RB; the interaction among survivin, p53 and bcl-2 may participate in the access and the course of RB. (Chin J Ocul Fundus Dis，2004，20：215-217)
ObjectiveTo identify 3 the disease-causing genes and mutations of Leber congenital amaurosis (LCA), and to study the correlation of phenotype and genotype. MethodsA retrospective study. Four LCA patients and seven family members who were diagnosed by eye examination in Ning Xia Eye Hospital of People's Hospital of Ningxia Hui Autonomous Region from January to December 2021 were included in the study. Four patients were from 3 unrelated families. Detailed collection of medical history and family history were received. Related ophthalmologic examination were collected and genomic DNA was extracted from peripheral blood. Whole-exome sequencing method was used for genetic diagnosis. The identified variant was confirmed with Sanger sequencing. Potential pathogenic mutation was analyzed using software and conserved domain analysis and performed co-separated analysis between the family member and the proband. ResultsOf the 4 patients, 1 patient was males and 3 patients were females; the age was from 4 to 18 years. Nystagmus were seen in 3 cases, finger pressing eyes and night blindness was seen in 1 cases; electroretinogram showed 4 cases of extinction or near extinction. The foveal reflection was visible in all eyes, and there was no obvious abnormality in the peripheral retina. One eye had strong reflection signal with raised ellipsoid in macular area; two eyes had weak reflection signal faintly visible between retinal layers; 1 eye had increased blood vessel branches, peripheral retinal non-perfusion area with capillary leakage; annular strong autofluorescence in macular area 4 eyes. No obvious abnormality was found in the phenotypes of family members. Genetic testing showed that the proband of pedigree 1 (Ⅱ-1) was found a homozygous missense mutation in c.640A>T (p.C214S) (M1) of PRPH2 gene. The proband of pedigree 2 (Ⅱ-2) was found compound heterozygous mutation in c.1256G>A(p.R419Q) (M2) and c.1A>C (p.M1L) (M3) of TULP1 gene. The proband 3 (Ⅱ-1) and her sister (Ⅱ-2) were both found compound heterozygous mutation in c.1943T>C (p.L648P) (M4) and c.380C>T (p.P127L) (M5) of GUCY2D gene. The parents and sister (Ⅱ-1) of the proband in family 2 and the parents of the proband in family 3 were all carriers of the corresponding heterozygous variant. M1, M3, M4, M5 were novel mutations and unreported. The genotype and disease phenotype were co-segregated within the family. According to the analysis of pedigree and genetic testing results, all 3 families were autosomal recessive inheritance. The amino acid conservation analysis found that M1, M2, M3, M4, and M5 were highly conserved among species. The results of bioinformatics analysis were all pathogenic variants. ConclusionsPRPH2 gene M1, TULP1 gene M3, and GUCY2D gene M4, M5 were novel mutations and not been reported in the literature and database. This research expanded the gene mutation spectrum of LCA. The patients with LCA have available characterristics, including onset age, varying ocular fundus and severe visual impairment.