Objective To explore the expression and function of NDRG2 gene in human primary hepatocellular carcinoma and normal hepatic tissues. Methods The immunohistochemical ABC method， Western blot， and Real-time PCR were used to investigate the expression and content of NDRG2 in human hepatocellular carcinoma and hepatic normal biopsies. Results The NDRG2 protein located in cytoplasm. The positive rate was 16.67%（5/30） and 100%（30/30） in hepatocellular carcinoma and normal hepatic tissues, respectively. The relative content of NDRG2 protein in hepatocellular carcinoma and normal hepatic tissues were 0.029 0±0.005 9 and 0.109 2±0.002 8. There were significant differences between human hepatocellular carcinoma and hepatic normal biopsies both in staining positive rates and relative content（P＜0.05）. The Western blot also agreed with the result，the expression level of NDRG2 protein in hepatocellular carcinoma and normal hepatic tissues was 1.13±0.15 and 1.57±0.18， respectively, there was significant difference（P＜0.05）. Also, compared with normal hepatic tissues, the expression level of NDRG2 mRNA in carcinoma tissues was reduced significantly (0.89±0.15 vs. 1.48±0.17, P＜0.05). However, there were no significant differences in NDRG2mRNA expression between Edmondson-Steiner grades. Conclusions There possibly have difference in NDRG2 expression between human primary hepatocellular carcinoma and normal hepatic tissue. NDRG2 gene may take part in the pathogenesis of human primary hepatocellular carcinoma. Futher study will be needed to study its mechanism and function.
One of the major clinical characteristics of congenital stationary night blindness(CSNB)is dysfunction of rod photoreceptors of the retina.Rhodopsin,the photosensitive pigment of the rods,is essential for maintaining the normal function of rod photoreceptors.It is resonable to hypothesize that mutations or deletions of rhodopsin gene may be involved in the molecular defect of CSNB.To test this hypothesis,we are searching for rhodopsin gene mutations in patients with autosomal dominant CSNB.In this study,DNA fragments containing the coding sequences in exon 5 of rhodopsin gene were amplified by polymerase chain reaction(PCR)in 15 patients and 5 unaffected members from a large family with autosomal dominant CSNB.RFLP analysis of these DNA fragments demonstrated that in comparison with a control group of 12 normal persons,there is no obvious deletion in exon 5 of rhodopsin gene,and that mutations or deletions do not exist in codon 314,codon 347,and the third base of codon 313 as well as the first base of codon 348 of the rhodopsin gene in these CSNB patients,which suggest the molecular pathogenesis of autosomal dominant CSNB not involve mutations or deletions of these codons of the rhodopsin gene. （Chin J Ocul Fundus Dis,1993,9:66-69）
Objective To investigate the feasibility of detection of epidermal growth factor receptor ( EGFR) exon 19 deletions and exon 21 L858R mutations in pleural effusion fromnon-small-cell lung cancer ( NSCLC) patients by mutant enriched PCR assay. Methods The mutations of exon 19 and 21 of EGFR gene in pleural samples fromthirty NSCLC patients were analyzed using both the mutant-enriched PCR assay and the non-enriched PCR assay. Results Ten ( 33. 3% , 10/ 30) exon 19 deletions and five ( 16. 7% , 5/30) exon 21 L858R mutation were detected by the mutant-enriched PCR assay, while only 6 cases ( 20. 0% ) and 1 case ( 3. 3% ) were detected by the non-enriched PCR assay respectively. The difference of mutation detection rate of EGFR gene between the two methods was statistically significant ( P = 0. 032) . Mutations were detected in all of partial responders ( 2 /4) among the four patients who received gefitinib therapy. Conclusions Mutant-enriched PCR assay can detect EGFR exon 19 deletions and exon 21 L858R mutation in pleural effusion from NSCLC patients effectively, economically and accurately. It may be a valuable biomarker for gefitinib therapy in advanced NSCLC.
ObjectiveTo detect expressions of PTEN and Ki-67 in primary thyroid cancer tissues and explore its clinical significances. MethodsThe expressions of PTEN protein and Ki-67 protein in 40 cases of paraffin-embedded tissues of primary thyroid cancer and the corresponding paracancerous tissues were detected by immunohistochemical method. The expressions of PTEN mRNA and Ki-67 mRNA in 14 cases of resected fresh tissues of primary thyroid cancer and the corresponding paracancerous tissues were detected by RT-PCR method. The relations between clinicopathologic characteristics and expression of PTEN protein or Ki-67 protein in the primary thyroid cancer tissues were analyzed. Results① The PTEN protein positive expression rate and the PTEN mRNA in the primary thyroid cancer tissues were significantly lower than those in the corresponding paracancerous tissues[35.0% (14/40) versus 60.0% (24/40), P<0.05; 0.225 7±0.036 3 versus 0.503 6±0.037 5, P<0.05], the Ki-67 protein positive expression rate and Ki-67 mRNA in the primary thyroid cancer tissues were significantly higher than those in the corresponding paracancerous tissues [72.5% (29/40) versus 42.5% (17/40), P<0.05; 1.212 1±0.042 1 versus 0.293 6±0.027 4, P<0.05]. ② The expressions of PTEN protein and Ki-67 protein were associated with the histological grading, pathological type, tumor stage, and presence of regional lymph node metastasis (P<0.05), which not associated with the patient's gender, age and integrity of tumor capsule or not (P>0.05). ③ The PTEN and Ki-67 protein expressions in the primary thyroid cancer tissues had a significantly negative correlation (rs=-0.605, P=0.000), which in the corresponding paracancerous tissues had no correlation (rs=-0.021, P=0.899). ConclusionPTEN and Ki-67 genes abnormally express in thyroid cancer tissue, which might be related with occurrence and development and its mechanism of primary thyroid cancer. Combination of two genes might contribute to identification of pathologic type, judge of biological behavior, and tumor stage of primary thyroid cancer, which might serve as a new target for diagnosis and treatment of it.
Objective To explore the relationship between the HBsAg positive patients suffering from hepatocellular carcinoma (HCC) and HBV DNA genotype. Methods By using PCR type-specific primers combined with sequencing of genotype, we analyzed the genotype of HBV DNA in the serum of 500 patients with positive HBsAg in our hospital. Among them, 150 cases suffered from HCC. Results Genotype B and C were both predominant genotypes in HBsAg positive patients. But in HCC group, the rate of genotype C was 65.33% （98/150）, which was significantly higher than that in non-HCC group (88/350, 25.14%), while genotype B, in contrast, was 28.67% （43/150） and 68.86% （241/350）， χ2=75.45， Plt;0.05. The distribution of HBV DNA genotype B or genotype C in different gender or different age groups were not statistically significantly different in cases of HCC (Pgt;0.05). Conclusion Genotype C of HBV DNA is more common in patients with HCC, and maybe there is relationship between genotype C and the occurrence of HCC.
Objective To study the effect of chitosan (CS) mediated insul in-l ike growth factor 1 gene (igf-1) transfection on the repair of articular cartilage defect. Methods Twelve 3-month-old healthy male rabbits weighting 2.0-2.5 kg were randomly divided into 2 primary groups, control and intervention groups (n=6 per group). Control group was further divided into normal control (left knee) and normal saline (NS) control (right knee) groups. While, intervention group was divided into CS (left knee) and CS/igf-1 intervention (right knee) groups. Cartilage defects were created in the knee joints except normalcontrol. Intra-articular injections of CS/igf-1 complex was administrated 2 times a week for 4 weeks in CS/igf-1 interventiongroup, 0.5 mL CS in CS intervention group, and 0.5 mL sal ine solution in normal control and sal ine control groups. At 28days after treatments, the cartilage samples were collected for histological observation and collagen type II and aggrecan mRNA evaluation. Results HE staining and toluidine blue staining revealed that CS/igf-1 and CS intervention could significantly stimulated cartilage regeneration accompanied with fibrosis and inflammatory cell infiltration, however, CS/igf-1 treatment resulted in the best repair of cartilage defect. In contrast, sal ine control group only showed fibrous tissue prol iferation and inflammatory cell infiltration without significant cartilage repairing. In terms of collagen type II and aggrecan gene expression, significant differences were observed in each pairwised comparison among 4 groups in the order of CS/igf-1 gt; CS gt; NS gt; normal control (P lt; 0.05). Conclusion In situ CS/ifg-1 complex transfection can enhance the formation of mesochondrium by upregulating collagen type II or aggrecan expression, which might enhance the repair of articular cartilage defect.
ObjectiveTo clone full-length cDNA of rat galectin-9 and construct recombinant adenovirus granule containing rat galectin-9 gene. MethodsThe galectin-9 gene was amplified by RT-PCR from rat liver tissue and inserted orientationally into plasmid pDC316-GFP digested by restriction endonucleases NotⅠ and HindⅢ. The recombinant pDC316-GFP-galectin-9 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then co-transfected with rescue plasmid pBHGlox△E1.3Cre into HEK-293 cells by liposome reagent. Recombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) was generated by sitespecific recombination and confirmed by PCR, and then Ad5-galectin-9 was propagated in HEK-293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay. ResultsConstruction of pDC316-GFP-galectin-9 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR. The infective titer of Ad5-galectin-9 was 1.4×109 U/ml. ConclusionRecombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) is successfully constructed, which provides the foundation of further research on the function of galectin-9 gene.
Objective To identify micrometastasis in regional lymph nodes of gastric cancer by quantitative real-time reverse transcription-PCR (qRT-PCR) assay and to evaluate the clinical significance of micrometastasis. Methods To study 320 lymph nodes collected from January 2010 to June 2010， 281 of which were from 40 patients with gastric cancer who had undergone a standard gastrectomy with lymphadenectomy， and other 39 of which were from 10 patients with gastroduodenal ulcer. Made CEA， CK-19， and CK-20 as primers， and used qRT-PCR assay in addition to hematoxylin and eosin staining to detect the micrometastasis， and to analyze the clinicopathologic characteristics.Results Totally， micrometastasis were detected by qRT-PCR assay in 31 (15.34%，31/202) lymph nodes of 28 (70.00%， 28/40) patients. Thirty-nine lymph nodes from 10 patients with gastroduodenal ulcer were negative by qRT-PCR and HE staining. The degree of differentiation， depth of gastric mural invasion， and clinical stage had statistically significant correlation with the incidence of lymph node micrometastasis (P＜0.05). Conclusions qRT-PCR assay is a sensitive and specific method to detect lymph node micrometastasis in gastric cancer patients，and it has importantly clinical significance in evaluating clinical staging，prognosis and treatment prescription.
Objectives To analyze and assess the status of detection of human immune-deficiency virus (HIV) by PCR, and to find a new screening test of HIV. Methods Using the following keywords "diagnosis tests", "AIDS", "PCR" and "HIV", we searched the Medline and CBM from 1991 to 2001. Then we assess each of diagnosis test according to the international standards. Results 567 articles were searched, in which 53 articles were chosen to assess. In these 53 articles, it was found that 47% applied comparison with Golden Standard, 25% calculated sensitivity, 23% calculated specificity, and 23% calculated predictive value, no likelihood ratio was calculated in these articles. Conclusions It was still a kind of pilot-study to apply PCR to screening detection of HIV. The design methods of study should be improved.
Objective To investigate the gene expression of beta-defensin-4 (mBD-4) and mBD-6 in acute lung injury (ALI) mouse.Methods Sixty adult mice were randomly divided into a control group and a ALI group.ALI was induced by intraperitoneal injection of lipopolysaccharide (LPS) in the ALI group.The control group was treated with same dose of normal saline.The lung tissues were harvested at different time point after stimulation.The expression of mBD-4 and mBD-6 mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction.DNA sequencing was used to confirm the specificity of mBD-4 and mBD-6 cDNA fragment.Results There were no obvious mBD-4 and mBD-6 mRNA expression in mouse lung in the control group at all time points and ALI 6 h group.In the ALI group a marked increasing expression was found on 12 h，1 d and 3 d after LPS stimulation.The mBD-4 mRNA expression was significant higher in the ALI groups of 1 d and 3 d points than that of ALI 12 h group with no obvious difference between each other.There were no significant differences of mBD-6 mRNA expression between ALI groups of 12 h，1 d and 3 d points Conclusion mBD-4 and mBD-6 mRNA is not constitutive expressed in mouse lung and show a up-regulative expression pattern after ALI.