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.
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)
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.
Objective To compare the differences of chromosome aberration and Rb 1 gene mutation among 3 cloned cells of SO-Rb50 cell line of retinoblastoma. Methods 1.Three cell cloned strains named MC2, MC3, MC4 were isolated from SO-Rb50. 2. Gbanding and karyotype analysis were performed on the llth passage cells of the 3 cell strains.3.All exons and the promoter region of the Rb gene were detected by PCR-SSCP analysis in tumor cell DNA extracted from the 3 cell strains. Results 1.Both numerical and structural chromosomal aberrations could be observed in these 3 cell strains.Several kinds of structural chromosomal aberrations were observed.The chromosome aberrations in the same passage of different cell strains were different.Aberration of chromosome 13 was rare and the aberration feature was different in the 3 cell strains.Five marker chromosomes were identified.M1,t(1;1)qter-p35∷q24-ter could befound in all cell strains.Two of them M4 and M5,have not been reported in SO-Rb50 cell line previously.2.SSCP analysis of exon 24 showed that MC411 and MC3138 had abnormal band. Conclusions The characteristics of heterogeneity of the original tumor cell line SO-Rb50are still kept during a long-term culture in vitro and the cloned strains had dynamic changes during this period.Aberration of chromosome 13 is not the only cause of RB;aberration of chromosome 1,a commom event in some neoplasias as well as in SO-Rb50, plays a meaningful role in the immortalization of this cell line. (Chin J Ocul Fundus Dis, 1999, 15: 146-148)
Objective To investigate the mutations of the gene in Chinese patients with X linked juvenile retinoschisis (XLRS), and to provide the genetic diagnosis and consultation of heredity for the patients and their families. Methods Genomic DNA was isolated from leukocytes of 29 male patients with XLRS, 38 female carriers and 100 normal controls (the patients and the carriers were from 12 families). All 6 exons of XLRS1 gene were amplified by polhism (SSCP) assay. The positions and types of XLRS1 gene mutations were determined by direct sequencing. Results Eleven different XLRS1 mutations were identified in these 12 families, including one frameshift mutation due to base loss of the first exon: c.22delT（L9CfsX20）, one nonsense mutation due to base loss of the first exon (Trp163X), one splice donor site mutation(c.52+2 Trarr;C; IVS1+2T to C), and eight missense mutation due to base replacement（Ser73Pro, Arg102Gln, Asp145His, Arg156Gly, Arg200Cys, Arg209His, Arg213Gln, and Cys223Arg). No gene mutation was detected in the control group. Four new mutations included frmaeshift mutation（L9CfsX20）and mutations of Asp145His, Arg156Gly, and Trp163X at the fifth exon. A newly discovered non-disease-related polymorphism (NSP) was the c.576C to T (Pro192Pro) change at the sixth exon. Conclusion Eleven different XLRS1 mutations were detected, which is the cause of XLRS in Chinese people. The detection of gene mutations may provide the guidance of genetic diagnosis and the consultation of family heredity for the patients and their families. (Chin J Ocul Fundus Dis, 2006, 22: 77-81)
Objective To observe the mutation frequency and the characteristics of rentinitis pigmentosa (RP)1 gene in the Chinese patients with autosomal dominant (AD) RP or sporadic RP (SRP), and to evaluate their potential effects on the pathogenesis of RP. Methods Fifty-five members from 7 Chinese families with ADRP, 30 patients with SRP, and 75 healthy adults were recruited. Polymerase chain reaction (PCR) and direct DNA sequencing were used to detect the sequence mutation in the entire coding region and splice sites of RP1 gene. Univariate analysis and multivariate analysis were used to detect the effect of RP1 gene mutation sites on RP. Results Four coding sequence variants were detected in the codes of 852，872，921 and 939 at the exon 4 of RP1 gene. The R872H alteration, which was found in both ADRP families and patients with SRP, showed positive correlation with RP confirmed by the multivariate logistic regression analysis. The P903L alteration was only found in ADRP families but not in the patients with SRP or the healthy adults. Conclusions The R872H alteration in the RP1 gene is likely to increase the risk of RP, and may be a susceptible gene of RP. Whether the P903L alteration is a diseasecausing factor needs to be further studied.
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.
ObjectiveTo observe the expression and mechanism of hypoxia-inducible factor-1α (HIF-1α) and p53 protein at the altitude of 5000 meter plateau hypoxia environment in rats, as well as the effect of Astragalus injection. MethodsSixty Sprague Dawley rats were randomly divided into the Astragalus injection intervention group and normal saline control group, 30 rats in each group. Astragalus injection group rats were intraperitoneal injected of Astragalus injection (15 ml/kg) before 30 minutes into the plateau environment simulation cabin, normal saline group rats were intraperitoneal injected with the same volume of saline. 30 minutes after injection, rats in each group were reared in the plateau experiment cabin which simulated altitude of 5000 m (oxygen partial pressure 11.3 kPa) for 2, 6, 8, 12, 24 hours, each time period of 6 rats. When get out, the rats were executed immediately and eyes were harvested. Retinal sections were studied by hematoxylin eosin stain, and immunohistochemical method for HIF-1α and p53 expression. ResultsFor control rats, after 2 hours in the cabin, there was edema in retinal layers. HIF-1α and p53 were expressed mainly in the cytoplasm of retinal layers. When the periods in cabin extended, there was atrophy of retinal nerve fiber layer, swelling and degeneration of ganglion cells. The expression of HIF-1α and p53 was increased. Compared with the control group, the intervention group rat had similar but less severe retinal changes, and the expression of HIF-1α and p53 was significantly decreased (P<0.05). ConclusionAstragalus injection can reduce pathological retinal damage in rats at high altitude environment, and its mechanism may be associated with reduced HIF-1α, p53 expression.
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.