ObjectiveTo investigate the expressions and clinical significance of human telomerase reverse transcriptase (hTERT) mRNA and γglutamyl transpeptidase mRNA-H (GGT mRNA-H) in the peripheral blood of small hepatocellular carcinoma (HCC) patients. MethodsThe expressions of hTERT mRNA and GGT mRNA-H were detected in the peripheral blood of thirty patients with small HCC by RT-PCR, eighteen patients with benign liver diseases, and twelve normal volunteers. ResultsThe positive rate of hTERT mRNA and GGT mRNA-H expression in patients with small HCC were 80.0% (24/30) and 46.7%(14/30), respectively. In patients with hepatitic cirrhosis the positive rate of hTERT mRNA expression was 33.3% (6/18), while the expression of GGT mRNA was not detected. Both the expressions of hTERT mRNA and GGT mRNA-H were negative in all normal volunteers. The combination analysis of hTERT mRNA and GGT mRNA-H expression achieved positive rate of 86.7% in the diagnosis of small HCC, which was significantly higher than the positive rate of AFP (26.7%)， Plt;0.05. ConclusionThe hTERT mRNA and GGT mRNA-H are significantly expressed in small HCC patients, the combination analysis of hTERT mRNA and GGT mRNA-H seems to be useful in the early diagnosis of small HCC.
Objective To investigate the role of β-catenin gene in breast tumorigenesis by detecting mutation and expression of β-catenin gene in breast hyperplasia and breast cancer. Methods Mutation and expression of β-catenin gene in 42 breast cancer, 15 simple hyperplasia and 15 atypical hyperplasia were detected by polymerase chain reaction-single strand conformation polymorphism and immunohistochemistry. Results Normal expression of β-catenin occurred in tissue of breast simple hyperplasia. The rate of abnormal expression of β-catenin in tissue of breast atypical hyperplasia and breast cancer were 26.7% （4/15） and 59.5% （25/42）, respectively, which were higher than that of simple hyherplasia tissue (P＜0.05). And there was a markedly difference between the atypical hyperplasia tissue and breast cancer tissue (P＜0.05). Mutation of β-catenin gene wasn’t detected in this three kinds of tissues. Conclusion Abnormal expression of β-catenin plays an important role in human breast tumorigenesis, reason of abnormal expression of β-catenin isn’t mutation of β-catenin gene. Expression of β-catenin can be regulated by other mechanisms.
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.
To investigate the mRNA expression of nm23-H1 gene in human liver tumor. In tumor and corresponding nontumoral liver specimens from 20 patients, nm23-H1 mRNA were examined by reverse transcriptionpolymerase chain reaction (RT-PCR) method with specific primers. Results: The primers designed in this study could amplified nearly entire coding sequence of nm23-H1 gene. All the samples showed positive expression of nm23-H1 mRNA, indicating there was no expression loss or obvious alteration. Conclusions: The achievement of RT-PCR method lays foundation for quantitative gaugement of nm23-H1 mRNA in liver tumor.
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.
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 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 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 observe the clinical features of acute retinal necrosis syndrome (ARN).Methods The clinical data of 84 patients (98 eyes) with ARN were retrospective analyzed. The patietns had undergone the examinations of best visual acuity, intraocular pressure, Bscanning, slitlamp biomicroscope, preset lens, direct and (or) indirect ophthalmolscope,and trihedral reflector; fundus fluorecein angiography had been performed on the patients with clear refracting media. Some of the patients had undergone polymerase chain reaction (PCR) to dectet the types of the causative virus.Medication,laser photocoagulation,and vitreous surgery had been performed on the patients after the diagnosis was confirmed. The visual acuity and the change of ocular fundus had been followed up; the average followup was 24.1 months. Results The average age of the patients at the onset was 42.8 years with the bilateraleye rate of 16.6% and retinaldetachment rate of 57.1%. There were 53.5% and 35.5% patients had the final visual acuity of gt;0.02 after 6 and 12 months, respectively. Better prognosis was found in patients diagnosed within 2 weeks and second involved eye. Varicella zoster virus DNA was identified in 15 patients and herpes simplex virus 1 was found in 3.Conclusions ARN is an acute disease with high incidence of retinal detachment.Serious retinal vasculopathy always happens at the late stage, and the prognosis is poor. Diagnosis in early stage is important and application of PCR will do contribution to the right diagnosis.
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)