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 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.
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 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)
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)
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 genetic predisposition of complement C5 gene polymorphisms in proliferative diabetic retinopathy (PDR) in Chongqing Han population. Methods 400 type 2 diabetes (T2D) patients (case group) and 600 age- and sex-matched healthy controls (control group) were enrolled in this study. There were 8 PDR patients in case group. All the subjects were Han ethnic people. The immune-related representative SNP locus of C5 gene including rs2269067, rs7040033, rs7027797 were screened by linkage disequilibrium analysis. Locus rs1017119 was selected by TagSNP and was around the above three loci. Subjects′ peripheral venous blood was collected and DNA was extracted. Genotyping was examined by PCR-restriction fragment length polymorphism method. The level of C5 plasma protein was measured by enzyme-linked immunoabsorbent assay. Results The frequency of GG genotype of rs2269067 was significantly increased in PDR patients in cases group compared with controls (Pc=3.4×10-5, OR=1.87, 95%CI=1.43 - 2.44；P=3.1×10-6). There was no differences in frequency of G, CC and CG genotype of rs2269067 between two groups (P=1.4×10-4, 1.000, 1.0×10-6). There were no differences in frequency of G, CC, CG, GG genotype of rs7040033, rs1017119, and rs7027797 between two groups (P > 0.05). The production of C5 plasma protein was significantly increased in case group as compare with control group (P=0.0004). An increased production of C5 plasma protein was observed in rs2269067 GG genotype cases compared to CG or CC cases (P=0.003, 0.001). Conclusion C5 rs2269067 GG genotype may be associated with the PDR of T2D in Chongqing Han population.
ObjectiveTo detect the level of serum melanoma-inbibiting activity (MIA) in patients with uveal melanomas, and investigate the value of MIA in diagnosing and inspecting uveal melanomas.MethodsEnzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of MIA in peripheral serum of 27 patients with uveal melanoma, 6 with melanocyte tumor, 7 with other ocular tumors and 16 healthy individuals, respectively.ResultsThe concentration of MIA in patients with uveal melanoma was significantly higher than that in the healthy ones (16 individuals) and the patients with adenoma of non pigmented ciliary epithelium (4 patients), retinoblastoma (2 patients), and retinal angioma 91 patient). The concnetration of MIA in patients with uveal melanoma without scleral infiltration or remote metastasis was obviously lower than that in the patients with scleral infiltration or remote metastasis, but didn′t differ much from which in the patients with melanocyte tumor. In the patients with uveal melanoma without infiltration or remote metastasis, there was no significant difference of MIA level between patients with spindle cell and mixed and epithelioid cell.ConclusionThe level of serum MIA may be an effective index in diagnosing uveal melanoma, which can monitor the metastasis of uveal melanoma.(Chin J Ocul Fundus Dis, 2005,21:153-155)
Objective To investigate the effect of apoptosis-related gene p53 on the apoptosis of retinal capillary cells in rats with spontaneously hypertensive (SHR) after ischemic reperfusion injury. Methods A total of 60 SHR rats were randomly divided into sham group (SHR-SH) and retinal ischemic reperfusion group (SHRRIR), which were subdivided into 5 subgroups according to the time after RIR: 2, 6, 24, and 72 hours and 7 days, with 6 rats in each subgroup. Another 60 Wistar-Kyoto (WKY) rats were divided into the same groups as the SHR rats as the control. The RIR model was set up. The apoptosis of retinal capillary cells was detected by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) methods and the expression of p53 was determined by streptavidin-perosidase (SP) immunohistochemistry. Results The apoptosis rate of retinal capillary cells in the 5 SHRRIR groups was (8.64plusmn;0.56)%, (14.92plusmn;0.99)%, (24.72plusmn;2.98)%, (16.53plusmn;1.80)%, and (7.12plusmn;1.10)%, respectively. The expression of p53 in SHRRIR groups increased at the 2nd hour after RIR, reached the peak at the 24th hour, kept the high level at the 72nd hour, and remained a little at the 7th day, which was significantly different from which in the SHRSH groups (Plt;0.01). The expression of p53 were higher in SHR-IR groups than that in the WKY-RIR groups (Plt;0.01). Conclusions p53 may play a part in RIR injury by inducing or promoting apoptosis. The apoptosis of retinal capillary cells after RIR is more severe under the hypertension, and reaches the peak at the 24 hour after RIR.
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)