ObjectiveTo investigate the expression of interleukin-18（IL-18）and signal transducers and activators of transcription 5（STAT5）in retina of 4-24-week-old diabetic rats, and explore the potential molecular mechanisms involved in diabetic retinopathy (DR).MethodsRetinal gene expression profile of healthy and 8-week-old diabetic rats was established with restriction fragment differential displaypolymerase chained reaction (RFDD-PCR), and the differences was analyzed by bioinformatics. IL-18 and STAT5 were filtrated as the candidate genes of DR. The expression of IL-18 and STAT5 in retina of diabetic rats with the age of 4, 8, and 24 weeks was observed by semi-quantitative reverse transcriptase-polymerase chain reaction（RT-PCR）.ResultsThe result of RFDD-PCR showed:expression of IL-18 was higher in healthy retina than that in diabetic one; expression of STAT5 was not found in healthy rats but in diabetic ones. The result of RT-PCR showed：compared with the normal, high expression of IL-18 was found in 4-week diabetic retina, reduced in 8-week one, and decreased to the lowest in 24-week one. The expression of STAT5 was not observed in healthy or 4week diabetic retina, but occurred in 8-week one, and increased in 24-week one. ConclusionThe expression of IL-18 and the activation of STAT5 may relate to the occurrance of DR. The expression of IL-18 doesn′t depend on the activation of STAT5. (Chin J Ocul Fundus Dis, 2005,21:258-260)
Objective To evaluate the potential of specific mRNA marker keratin 19(K19) to detect micrometastasis by reverse transcriptase polymerase chain reaction (RT-PCR) .Methods One hundred and ninty four regional lymph nodes harvested from 6 cases of benign diseases, 4 cases of breast carcinoma, 5 cases of gastric carcinoma and 12 cases of colorectal carcinoma patients were examined by conventional pathology and amplifying tissue specific K19 mRNA by RT-PCR separately, then the two methods were compared with each other. Results None of the 34 lymph nodes which were pathological metastasis-negative from benign diseases expressed K19 mRNA by RT-PCR, all of the 28 regional lymph nodes which were pathological metastasis-positive from malignant cases showed trains of K19 mRNA by RT-PCR. Of the 132 lymph nodes which were pathological metastasis-negative from malignant cases, 11 lymph nodes were detected with micrometastasis by genetic diagnosis.Conclusion Genetic diagnosis of lymph node micrometastasis is more sensitive than conventional pathology and has diagnostic value and merits further study.
Objectives To study the relationships between methylenetetrahydrofolate reductase(MTHFR ) gene polymorphism and diabetic retinopathy (DR) in Chinese Han race． Methods With polymerase chain reaction and restriction fragment length polymorphism (PCR-FLP), MTHFR gene 677 T mutation (cytosine is replaced by thymine in No. 677 site) was detected in 85 health controls, 62 with DR and 117 without DR of type 2 diabetics comfrimed by ophthalmoscope． Results The frequency of MTHFR variant genotypes and alleles of DR in Chinese Han race．patients were signigicantly higher than those without retinopathy and healthy controls (Plt;0．01)． Conclusions The results suggested that MTHFRgene C677T mutation was probably one of the genetic risk factors of diabetic retinopathy in Chinese Han rase． (Chin J Ocul Fundus Dis, 2001,17:198-200
Objective To investigate the genetic interaction of HLA-DQB1 promoter and coding alleles in the pathogenesis of Vogt-Koyanagi-Harada syndrome (VKH). Methods Eighty-eight Chinese Han patients with VKH and eighty-eight non-VKH normal controls were enrolled in this study. DNA was extracted from white blood cells of the subjects by phenolchloroform method. Thirteen alleles were genotyped by polymerase chain reaction-sequence-specific primers (PCR-SSP), polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and clone-sequencing was applied to determine the polymorphisms of the promoter and coding regions of HLA-DQB1 gene. Chromas and Bioedit software were used to analyze the sequences of the promoter of HLA-DQB1. Chi-square test and Fisher exact test were the statistical methods. Relationships among single nucleotide polymorphism (SNP) in the promoter and coding region were analyzed. Results Twelve of thirteen already known HLA-DQB1 alleles were genotyped by PCR-SSP in VKH patients. The most frequent allele in VKH patients was HLA-DQB10401 (0.318, 56∶176) which was significantly higher in patients than that in normal controls (0.045, 8∶176) (chi;2＝44.00, P＝0.000, OR＝9.8). So was for HLA-DQB10303 (0.068 vs. 0.006, chi;2＝9.67, P＝0.002, OR＝12.81). In contrast, the frequency of HLA-DQB10601 (0.017 vs.0.096, chi;2＝10.39, P＝0.001, OR＝0.16) and HLA-DQB10302 (0.062 vs. 0.193,chi;2＝13.48, P＝0.000, OR＝0.28) in VKH patients were significantly lower than normal controls. Twelve SNP were found in all subjects. The frequency of C allele at position -189C/A in VKH patients was significantly higher than that in controls (0.324 vs. 0.074, chi;2＝45.92, P＝0.000). However, the frequency of G allele at position -227G/A in VKH patients was significantly lower than that in the normal controls (0.011 vs. 0.108, chi;2＝15.63,P＝0.000). The frequency of combination of susceptible alleles in promoter and coding area （-189C and HLA-DQB10401） in VKH patients was statistically higher than that in controls, the frequency of combination of resistant alleles in control (-227G and HLA-DQB10601) was higher than that in VKH patients. Conclusions The specific interactions of SNP in the promoter and coding alleles of HLA-DQB1 are associated with the pathogenesis of VKH.
Objective To investigate the expression of T cell receptor (TCR) Vβ8.3 gene on CD4+ T lymphocytes in the rats with experimental autoimmune uveoretinitis (EAU). Methods Eighteen Lewis rats were divided into EAU, complete Freund′s adjuvant, and the control group. Inter photoreceptor retinoid-binding protein (IRBP) R16 peptide was synthesized using Fmoc procedure for induction of EAU. Magnetic absorption cell sorting (MACS) me thod was used to isolate the CD4+T lymphocytes from the spleen of the rats. Flow cytometry was used to monitor the efficiency of isolation. The expression of TCR Vβ8.3 gene segment on CD4+T lymphocytes was determined by fluorescent quantitative polymerase chain reaction. Results EAU was successfully induced in the Lewis rats immunized with IRBP R16 peptide. The proportion of CD4+T lymphocytes isolated by means of MACS was statistically higher than that before isolation (P＜0.001). The expression of TCR Vβ8.3 gene segment on CD4+ T lymphocytes in EAU rats was significantly higher than that in the control (P＜0.05). Conclusions There is a predominant usage of antigen-specific TCR Vβ 8.3 gene in EAU rats induced by IR BP R16 peptide, which may serve as a target for immunotherapy of EAU. (Chin J Ocul Fundus Dis，2004，20：165-167)
ObjectiveTo analyze the sensitivity and specificity of polymerase chain reaction (PCR) tests in the detection of cytomegalovirus (CMV) in the diagnosis of patients with acquired immune deficiency syndrome (AIDS), using aqueous humor samples. Methods25 AIDS patients (including 21 men and 4 women) were studied. The age of the patients varied from 24 to 59 years, with an average of (39.2±9.3) years. The CD4+ T cell count was from 1 to 523 cells/μl, with a medium of 40 cells/μl. They were infected with human immunodeficiency virus(HIV)for a period from 15 days to 9 years with a median of 10 months. They were divided into three groups according to the fundus and treatment, including untreated cytomegalovirus retinitis (CMVR), treated CMVR and control group. There were 10 patients without anti-CMV treatment and 7 patients treated previously with foscarnet or ganciclovir whose eyes were diagnosed CMVR. Control group has 8 patients who had normal fundus or minor retinopathy excluded from CMVR. Approximately 100 μl of aqueous humor was obtained by anterior-chamber paracentesis and PCR was performed in all cases. ResultsThere were CMV DNA in 9 of 10 eyes with untreated CMVR (90.0% sensitivity). Of 7 specimens from eyes with treated CMVR, 3 were CMV PCR positive (42.9% sensitivity). All 8 samples of the control group were negative for CMV DNA, indicating the clinical specificity of our PCR was greater than 99.9% for CMVR. The anterior chamber paracentesis did not cause any complications in our patients except for a patient with subconjunctival hemorrhage. ConclusionsThe assay had an estimated sensitivity of 90.0% in detecting untreated CMVR and a sensitivity of 42.9% in detecting CMVR that had been treated. The specificity of this assay was greater than 99.9%.
Objective To investigate whether mutations exist in codon 58 and codon 347 of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa(ADRP)． Methods Point mutations at codons 58 and 347 were detected by restriction endonuclease digestion of exons 1 and 5 amplified by polymerase chain reaction(PCR)．This method was applied to screen genomic DNAs from 57 patients of 38 families with ADRP and 60 normal controls． Results Four patients from one family of ADRP were confirmed to have a point mutation at the second nucleotide of codon 58,and 6 patients from two families of ADRP were found to have a mutation at codon 347．None of these mutations were found in 60 normal subjects． Conclusion It is suggested that molecular genetic heterogeneity exists within ADRP and some subtypes of ADRP are caused by points mutations of the rhodopsin gene． (Chin J Ocul Fundus Dis,1998,14:108-110)
Objective To analyze the association between histocompatibility leukocyte antigen （HLA-A/B,HLA-DRB/DQB） alleles and Eales disease, and to explore susceptible genes and protective genes associated with Eales disease in a population of Han from ZUN YI city in Guizhou province. Methods The subjects were analyzed by casecontrol study consisted of two groups such as normal control group and Eales disease group. DNA samples from 100 healthy people and 26 patients with Eales disease were detected by polymeriase chain reaction with sequencespecific primers (PCR-SSP). The alleles of HLA-A/B and HLA-DRB/DQB from Eales disease group were compared with those from control group by SPSS 13.0. Results The distribution frequency in Eales disease was HLAA01(P=0.041, OR=20.5), A02(P=0.000, OR=54.667, OR 95%CI:11.837-252.473), B55 (P=0.047, OR=4.524; OR 95%CI:1.200-17.047), HLA-DRB01(P=0.048, OR=5.879, OR95%CI:1.227-28.169). DQB05 (P=0.021, OR=2.769, OR95%CI=1.145-6.692) alleles, and obviously higher than control, but the frequency of HLAA11 (P=0.031, OR=0.383, OR95%CI=0.158-0.930) was obviously lower than control (P＜0.05). Conclusion The results showed that the alleles of HLAA01, A02, B55, DRB01, DQB05 may associate with an antagonist effect in Eales disease, but HLAA11 may be a protective gene of this disease.
Objective To investigate the polymorphism of the vitamin D receptor gene (VDR)TaqⅠin relation to diabetic retinopathy. Method Fragment length discrepant allele specific PCR(FLDAS-PCR) were used to determine VDR genetypes in 158 patients with diabetic retinopathy and in 198 normal subjects. Results The frequency distribution of VDR genotypes in diabetic retinopathy patients was 106 (67.1%) in TT, 33(20.9%) in Tt, 19(12.0%) in tt; and in normal persons was 165 (83.3%) in TT, 23(11.6%) in Tt, 10 (5.1%) in tt. There was a significant difference between diabetic retinopathy patients and normal persons in distribution of VDR gene TaqⅠgenotypes(Plt;0.05). Conclusions There is some distribution alterations of VDR gene polymorphism in diabetic retinopathy patients. (Chin J Ocul Fundus Dis, 2006, 22: 94-96)
The aim of the this study was to search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture by NP-PCR technique. Bacterial gene fragments were amplified in vitro from DNA which were extracted from cholesterol gallstones in gallbladder for identifying the existence of bacteria. The gallbladder gallstones of 30 patients were analysed. Bacterial DNA was found in the stones of 26 patients, indicating that most cholesterol gallstones harbor bacterial DNA.