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 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.
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.
PURPOSE:To investigate the status and detailed structure of Rb gene in primary tumors and somatic cells of patients with retinoblastoma. To identify the character, origin and transmission of oncogenie point mutations. METHODS:DNA hybridization,SSCP analysis and PCR-associated direct sequencing. RESULTS:Among 108 RB patients examined 80 cases were found to have subtle alterations affecting Rb locus,including 44 cases with homozygous Rb point mutations, 20 cases with two independent heterozygous Rb point mutations, 16 cases with heterozygous mutations involved in one allele of Rb gene. Majority of bilateral RB patients and a small fraction of unilateral RB patients were detected to have a germline mutation. In addition the higher frequency of new germline mutation and parental origin of mutation were observed. CONCLUSION :Rb gene is closely associated with retinoblastoma. Two mutation events and resulting inaetivations of both Rb alleles are required for RB tumorigenesis. Based on our own data,the first event is exclusively point mutation. As for the second event,LOH accounts for two third of cases,point mutation for one third of cases. （Chin J Ocul Fundus Dis,1997,13: 12- 16）
Objective To investigate the effect of subretinal fluid (SRF) with different grades of proliferative vitreoretinopathy (PVR) on bcl-2 oncoprotein expression in retinal pigment epithelium (RPE) cells and fib roblast (FB)． Methods Using immunohistological staining technique and Western-blotting method to detect the expression of bcl-2 protein in RPE cells and FB under the stimulation of SRF． Results The expression levels of bcl-2 increased in both types of cells to a certain extent compared with those of the control group 4 hours after the cells subjected to SRF； 36 hours later, the expression levels of bcl-2 of experimental groups decreased more quickly than those of the control group， and the control group showed relatively higher bcl-2 protein levels at the end of observation． Conclusions SRF may stimulate the e xpression of bcl-2 in RPE cells and FB under culture at early stage, but accel arate the declining of bcl-2 protein levels a certain time after subjected to SRF． (Chin J Ocul Fundus Dis, 2001,17:58-60)
The debate on the cell of origin of human retinoblastoma lasted for more than one century. In the recent issue of ldquo;cellrdquo;, David Cobrinikprime;s group shows that L/M cone precursors are the most likely answer as they have an intrinsic circuitry, including murine double minute 2 (MDM2), Nmyc, the nuclear receptors retinoid X receptor and thyroxine receptor 2, making them extremely sensitive to transformation following retinoblastoma gene inactivation.
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 analyze the BEST1 gene mutations and clinical features in patients with multifocal vitelliform retinopathy (MVR). Methods This is a retrospective case series study. Five MVR families with MVR, including 9 patients and 10 healthy family members were recruited. Clinical evaluations were performed in all MVR patients and their family members, including best-corrected visual acuity (BCVA), intraocular pressure (IOP), refraction, slit-lamp examination, 90 D preset lens examination, gonioscopy, color fundus photography, optical coherence tomography (OCT), fundus autofluorescence (AF), ultrasound biomicroscopy (UBM) and axial length measurement. Electro-oculogram (EOG) was performed in 12 eyes and visual field were performed in 13 eyes. Peripheral blood samples were collected in all subjects to extract genomic DNA. Coding exons and flanking intronic regions of BEST1 were amplified by polymerase chain reaction and analyzed by Sanger sequencing. Results Among the 5 MVR families, 3 probands from three families had family history, including 1 family had autosomal dominant inheritance pattern. Two patients from 2 families were sporadic cases. Screening of BEST1 gene identified four mutations, including three missense mutations (c.140G>T, p.R47L; c.232A>T, p.I78F; c.698C>T, p.P233L) and 1 deletion mutation (c.910_912del, p.D304del). Two mutations (p.R47L and p.I78F) were novel. The BCVA of affected eyes ranged from hand motion to 1.0. The mean IOP was (30.39±11.86) mmHg (1 mmHg=0.133 kPa). The mean refractive diopter was (-0.33±1.68) D. Twelve eyes had angle-closure glaucoma (ACG) and 4 eyes had angle closure (AC). EOG Arden ratio was below 1.55 in all patients. The mean anterior chamber depth was (2.17±0.29) mm. Visual field showed defects varied from paracentral scotoma to diffuse defects. The mean axial length was (21.87±0.63) mm. All MVR patients had multifocal vitelliform lesions in the posterior poles of retina. ACG eyes demonstrated pale optic disc with increased cup-to-disc ratio. OCT showed retinal edema, extensive serous retinal detachment and subretinal hyper-reflective deposits which had high autofluorescence in AF. The genetic testing and clinical examination were normal in 10 family members. Conclusions MVR patients harbored heterozygous mutation in the BEST1 gene. Two novel mutations (p.R47L and p.I78F) were identified. These patients had clinical features of multifocal vitelliform retinopathy and abnormal EOG. Most patients suffered from AC/ACG.
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.