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.
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)
Objective To detect and analyse the mutations in rhodopsin gene of members in a family affected by autosomal dominant retinitis pigmentosa (ADRP). Methods Using the polymerase chain reaction (PCR), we amplified exon 1-5 of rhodopsin gene in patients with ADRP,and analyzed it with direct sequence measuement. Results The Gly-182-Asp mutation in the rhodopsin gene was detected in most of affected members of this ADRP family, but no mutation was detected in two affected members and the control ones. Conclusion We cannot regard the Gly-182-Asp mutation in the rhodopsin gene as the pathagenic factor of the ADRP family. It is likely there is a new gene next to the rhodopsin gene. (Chin J Ocul Fundus Dis, 2002, 18: 256-258)
OBJECTIVE: Porcine stress syndrome (PSS) is one kind of molecular genetics defect diseases of pig which will cause malignant hyperthermia syndrome (MHS) and is the first index should be excluded in screening of a pig species for xenotransplantation. It was reported that mutation of pig rynodine receptor(RYR1) gene is the main reason for PSS. In this study, RYR1 genotypes of the Chinese Banna mini pig inbred line and inbreeding closed colony Wuzhishan pig were investigated with polymerase chain reaction-restriction endonuclease fragment length polymorphism (PCR-RFLP) technique. METHODS: Antevenocaval whole blood samples were collected from 50 Banna mini-pig inbred-line(BMI), 15 inbreeding Wuzhishan pig (WZSP) and 25 Neijiang pigs (NJP) as negative control, the primer were designed and synthesized, PCR reaction was conducted following the sequence of 94 degrees C (1 min), 58 degrees C (1 min) and 72 degrees C (1 min) for 30 cycles. The PCR products were digested with restriction endonuclease HhaI and then electrophoresis check. RESULTS: A 659 bp DNA fragment was amplified with these two primers, the HALNN sample fragment was cut into fragments as 493 bp and 166 bp individually after the digestion, indicates no point mutation at site 1,843 in RYR1 gene in all tested BMI pig and WZSP. Namely, the RYR1 genotype of 50 cases of BMI and 15 cases of WZSP were HALNN, therefore their phenotype is PSS negative. CONCLUSION: It indicates that the genotype of Banna mini pig inbred line and inbreeding Wuzhishan pig are HALNN therefore PSS absolutely negative, the group penetrance is 0. This is consistent with experimental observation. It suggests that Banna mini pig inbred line and inbreeding Wuzhishan pig may be the alternative donor for xenotransplantation.
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.
Optic atrophy,hereditary／diagnosis; Polymerase chain reaction; DNA,mitochondrial; Point mutation; Sequence analysis
Objective To validate the different expressions of human fxyd6 gene between normal bile duct tissues and malignant tumor tissues, and to observe the subcellular localization of human fxyd6 gene in human cholangiocarcinoma cells. MethodsThe different expressions between normal bile duct tissues and malignant tumor tissues were identified by RT-PCR. In situ polymerase chain reaction （IS-RT-PCR） was applied to detect the subcellular localization of fxyd6 gene in paraffin sections of human cholangiocarcinoma cells. Image analysis software was used to semiquantitatively determine the difference between normal and malignant tissues. ResultsHuman fxyd6 gene was highly expressed in cholangiocarcinoma tissues and lowly expressed in normal ones. There was a significant difference between the expressions of carcinoma cells and normal cells （P＜0.05）. IS-RT-PCR showed that fxyd6 gene localized in the kytoplasma of epithelial cells of human cholangiocarcinoma. ConclusionHuman fxyd6 gene may act as an essential component of the malignant transformation process in human cholangiocarcinoma.
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.
PURPOSE:To investigate mitochondrial DNA(mtDNA) of Leber's hereditary optic neuropathy(LHON). METHODS:Polymerase chain reaction(PCR)method was used to analyse mtDNA of 11 patients in a pedigree with LHON and 4 control subjects from none LHON pedigree. RESULTS:There was a loss of a restriction site for the restriction endonuclease SfaN.Ⅰin Ihe Patients with LHON. In this pedigree,maternal lineage was regarded a carrier of the pathogenic gene. CONCLUSIONS:The patients with Leber's hereditary optic neuropathy have a point mutation in mtDNA,which results in loss ol SfaN I endonuclease restriction site .and this change is one of mechanisms inducing this disaese. （Chin J Ocul Fundus Dis,1997,13: 27-29）