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.
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.
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)
Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
ObjectiveTo conduct a systematic review of clinical manifestations, treatment, and associated genotyping of Sorsby fundus dystrophy (SFD). MethodsAn evidence-based medicine study. Sorsby fundus dystrophy, anti-vascular endothelial growth factor therapy, choroidal neovascularization, macular neovascularization, and TIMP3 gene were hereby used as search terms. Relevant literature was searched in CNKI, Wanfang, PubMed of the National Library of Medicine, and Embase of the Netherlands. The time span for literature searching ranged from the establishment of the database to April 2022, and two reviewers independently screened the literature and extracted relevant data, with duplicates, incomplete or irrelevant articles, and review articles excluded. SPSS26.0 software was used for analysis. The 95% confidence interval (CI) was used as an estimate of the effect size. The clinical manifestations, treatment and related pathogenic genes of SFD were counted and recorded. ResultsAccording to the search strategy, 157 pieces of literature were initially retrieved, and 49 eyes of 35 patients from 16 articles were finally included for analysis, among which, 17 patients were male, 13 patients were female, and 5 patients were unknown gender; 16 involved left eyes, 19 involved right eyes, and 14 involved unidentified eyes. The age of the disease onset was 42.33±2.19 years (28-59) years old. There were 19 cases with a positive family history, and the total positive rate was 54.3% (19/35, 95%CI 36%-72%). There were 31 cases of gene mutation, all of which were TIMP3. In the included literature, there were 2 and 2 cases with no mutation and unreported loci, respectively, with a total positive rate of 93.9% (31/33, 95%CI 85%-100%). Among the 31 cases with gene mutation, 22, 4, 1, and 4 cases were in the UK, Germany, Switzerland, and Chinese, respectively, and the detection rates were all 100% (22/22, 4/4, 1/1, 4/4). The clinical manifestations of SFD were mainly yellow-white deposits in the fundus and choroidal neovascularization (CNV) in the macula, thereby leading to a decrease in central vision, followed by the expansion of the deposits to the periphery, the further development of CNV, and a severe decline in vision caused by peripheral retinal and choroidal atrophy. The treatment methods for SFD include photodymatic therapy, anti-VEGF drugs, glucocorticoids, vitamin A, etc., among which, anti-VEGF drugs were considered the first-line treatment, and the combined treatment was provided with a better prognosis than a single treatment. ConclusionsVariations in the TIMP3 gene cause SFD, the fundus characteristic manifestations of which, are yellowish-white deposits and CNV, which develop from the center to the periphery, thus resulting in progressive decline of visual acuity. Current studies have shown that combined therapy presents a better prognosis than monotherapy.
Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1 (Rac1-siRNA) on rat retinal neovascularization in retinae. Methods Retinal vein occlusion was induced by retinal photodynamic medthod in 25 Sprague-Dawley rats. Rac1-siRNA vector DNA was injected into the vitrous of one eye of those rats (gene intervention group), and empty vector DNA was injected into the fellow eye (blank control group). Rac1-siRNA vector was injected in other 25 SD rats without retinal vein occlusion (blank intervention group). Two weeks after injection, fluorescein isothiocyanate (FITC)-dextran was perfused into the hearts of all the rats, and the retinal wholemount was made to observe the neovascularization. The numbers of endothelial cells which break through the internal limiting membrane were counted after hematoxylin-eosin staining. Results A massive of neovascularization and FITC leakage were found in blank control group. Small part of neovascularization and a little FITC leakage were observed in the gene intervention group. Retinal vessels were normal in blank intervention group. Compared with blank contrast group and blank intervention group, the difference of the mean numbers of endothelial cells which broke through the internal limiting membrane in the gene intervention group was significant（t=? P=0.000??lt;0.05）. Conclusion Rac1-siRNA can inhibit retinal neovascularization induced by retinal vein occlusion in rats.
It is clear that genetic background contributes to the development and progression of diabetic retinopathy (DR). However, the identification of susceptibility loci through candidate gene approaches, linkage disequilibrium analysis of case-control data and genome wide association study is still in its infancy and faces many challenges due to the complexity of the disease itself. China has rich resources of clinical samples. In order to facilitate elucidating the susceptibility genes of DR in China, we look forward multi-disciplinary, multi-regional collaboration studies integrating novel technologies, such as proteomics, metabolomics and next-generation sequencing to analyze gene-gene and gene-environment interaction factors comprehensively.
Objective To observe the expression and relationship of high-mobility group A(HMGA)1, HMGA2, MIB-1 labeling index (LI) and let-7 in retinoblastoma (RB). Methods Forty-four RB samples were studied, including 11 poorly-differentiated samples, 33 well-differentiated samples; eight invasive and 36 non-invasive samples. The expression of HMGA1, HMGA2 and MIB-1 LI in RB were analyzed by immunohistochemitry. The HMGA1, HMGA2 were scored on a scale of 0 to high expression. 0: no expression; low: 1%-10%; medium: 11%-50%; high: ＞50%. The MIB LI were scored on a scale of 0 to high expression. 0: no expression; low: 1%-40%; high: ＞40%. Semiquantitative reverse transcription-polymerase chain reaction was used to assay the let-7 expression level: ge;80% showed no significantly decreased expression; 60%-79% showed medium decrease in expression; ＜60% highly decreased in expression. ResultsIn 44 RB samples, there were 14 cases with no HMGA1 expression (32%), 11 cases with low expression (25%), 10 cases with medium expression (23%), and nine cases with high expression (20%). Expression level of HMGA1 was significantly higher in poorly differentiated RB than in well-differentiated RB (chi;2=11.3,P＜0.01); however, no statistically significant difference was found between invasive tumors and noninvasive tumors (chi;2=5.9,P＞0.05). There were 11 cases with no HMGA2 expression (25%), 11 cases with low expression (25%), nine cases with medium expression (20%), and 13 cases with high expression (30%). Expression level of HMGA2 was significantly higher in poorly differentiated and invasive RB than in well-differentiated and noninvasive RB respectively (chi;2=20.9, 8.7;P＜0.05). There were 4 cases with no MIB-1 LI expression (9%), 18 cases with low expression (41%), and 22 cases with high expression (50%). Expression level of MIB-1 LI was significantly higher in poorly differentiated RB than in well-differentiated RB (t=5.2,P＜0.05). Higher expression of MIB-1 LI was found in invasive tumors than in noninvasive tumors, with no significant difference (t=-1.1,P＞0.05). Twenty-seven cases had no significantly decreased expression of let-7 (61%). There were eight cases with medium decreased expression (18%) and nine cases with highly decreased expression (21%). Correlation analyses revealed that MIB-1 LI expression significantly correlated with HMGA1and HMGA2 proteins (r=0.327, 0.602;P＜0.05). A significantly inverse correlation existed between let-7 expression and HMGA1, HMGA2 proteins and MIB-1 LI respectively (r=-0.247,-0.310,-0.392;P＜0.05). Conclusions Overexpression of HMGA1, HMGA2 and MIB-1 LI and down regulation of let-7 were demonstrated in RB. Supplying let-7 to RB cells can possibly inhibit HMGA1 and HMGA2 expression.
ObjectiveTo investigate the expression of transcription factor SOX11 in retinoblastoma (RB) and the relation with the genes and cellular pathways.MethodsA public data set gse59983 containing the full mRNA expression profile of cancer tissues in 76 RB patients was downloaded from the GEO Database at the National Center for Biotechnology Information. According to the expression of 15 marker genes, these genes were divided into cell cycle marker genome (group 1, 26 patients), vertebral photoreceptor marker genome (group 2, 4 patients) and rod photoreceptor marker gene (group 3, 46 patients). R2 bioinformatics platform (http://r2.amc.NL) was used to analyze the gse59983 public data set. The SOX11 expression in cancer tissues of patients in 3 groups were observed and the SOX11-related genes were identified. According to the gene correlation, the clustering heat map was drawn, and the related genes and pathways of SOX11 were preliminarily analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis tool and Gene Annotation (GO) analysis method.ResultsSOX11 expression in cancer tissues of patients in group 1 was significantly higher than that of groups 2 and 3 (P＜0.001). SOX11 expression in cancer tissues of patients in group 3 was significantly higher than that in group 2 (P＜0.001). A total of 4524 genes significantly related to SOX11 expression were detected. Among them, there were 2139 positively correlated genes and 2385 negatively correlated genes. SOX11 and the 16 genes with the strongest correlation were screened and the clustering heat map was drawn. The clustering form of SOX11 and SOX11 significantly correlated genes was roughly the same as the original form. KEGG and GO analysis showed that SOX11 related genes were involved in regulating mitotic cell cycle, cell apoptosis, photoreceptor cell development, photoreceptor cell protection, etc.ConclusionThe expression of SOX11 in RB is significantly different in different types or periods. Its expression is significantly correlated with genes involved in the regulation of cell cycle.
