Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.
Objective To analyze the variation of intestinal microflora in patients with colorectal cancer by SYBR GreenⅠreal-time fluorescence quantitative PCR and reveal the role and significance of intestinal microflora in the colorectal cancer-associated molecular pathogenesis. Methods A set of 16S rRNA gene group of species-specific primers for Bifidobacterium spp., Lactobacillus group, Escherichia coli, and ddl gene-targeted species-specific primers for Enterococcus faecalis and feces Enterococcus were designed. Patients with colorectal cancer (colorectal cancer group, n=30) and healthy volunteers (normal control group, n=30) were included and whose feces were collected to extract bacterial genome DNA. SYBR GreenⅠ real-time fluorescence quantitative PCR was used to analyze the five mentioned bacterial amounts. Results Level of Bifidobacterium spp. (4.52±0.49) and Lactobacillus group (5.46±0.12) in colorectal cancer group were significantly lower than those （9.25±0.83 and 7.45±0.37） of normal control group (Plt;0.05), whereas levels of Escherichia coli (5.82±0.47), Enterococcus faecalis (10.6±0.30) and feces Enterococcus (5.74±0.16) in colorectal cancer group were significantly higher than those （4.68±0.32, 4.95±0.24, and 5.03±0.43） of normal control group (Plt;0.05). Conclusions The fecal microflora composition of patients with colorectal cancer is significantly decreased in Bifidobacterium spp. and Lactobacillus group, whereas increased in Escherichia coli, Enterococcus faecalis, and feces Enterococcus. These data underline that the occurrence and progress of colorectal cancer may be related to intestinal microflora.
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 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 explore the influence of patients who accepted chemotherapy of Folfox4 scheme before operation for the expression of miR-196 and HoxB8 in colorectal cancer, and illustrating the differences between the miR-196 and HoxB8 expressions in colorectal cancer tissues and sensitivity to chemotherapy with Folfox4 scheme and its corre-lation and significance.
MethodsFluorescence quantitative PCR (RT-PCR) and immunohistochemistry were used to determine the expressions of miR-196 and HoxB8 in 50 specimens of neoadjuvant chemotherapy group (chemotherapy sensitive group and chemotherapy insensitive group) and 30 specimens which received the surgery directly (no-chemo-therapy group), and analyzing the relationship and discrepancy between miR-196 and HoxB8 in these groups.
ResultsThe RT-PCR examination showed that the relative expression levels of miR-196 and HoxB8 in the neoadjuvant chemo-therapy group were lower than the no-chemotherapy group (0.646 8±0.683 9 vs.1.000 0±0.000 0, P < 0.01;0.607 6±0.418 9 vs.1.000 0±0.000 0, P < 0.01).Expression of miR-196 in the chemotherapy sensitive group was higher than the chemotherapy insensitive group (0.948 9±0.691 0 vs.0.344 7±0.536 1, P < 0.01), however, the expression of HoxB8 mRNA in the chemotherapy sensitive group was lower than the chemotherapy insensitive group (0.489 9±0.371 5 vs.0.725 3±0.437 5, P < 0.05).Expression positive rate of HoxB8 protein in chemotherapy sensitive group was lower than the chemotherapy insensitive group (Z=-2.396, P=0.017).The expressions of miR-196 and HoxB8 in the neoadjuvant chemotherapy group had negative relationship (r=-0.595, P < 0.01), which was also exist in the no-chemo-therapy group (r=-0.435, P < 0.01).
ConclusionsThe neoadjuvant chemotherapy with Folfox4 scheme before oper-ation can reduce the expression levels of miR-196 and HoxB8 in colorectal cancer tisssues.The different expression levels of miR-196 and HoxB8 could influence the sensitivity of neoadjuvant chemotherapy with Folfox4 scheme in colorectal cancer.The high level expression of miR-196 could restrain the expression of HoxB8, and then increase the sensitivity of chemotherapy with Folfox4 scheme in colorectal cancer.
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.
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 speci?c method to detect lymph node micrometastasis in gastric cancer patients，and it has importantly clinical significance in evaluating clinical staging，prognosis and treatment prescription.
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.
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 difference of minichromosome maintenance protein 2 (MCM2) mRNA expression among colonic normal mucosa, colonic adenoma and carcinoma. Methods The expressions of MCM2 mRNA were determined by real-time RT-PCR in 12 colonic normal mucosa, 33 colonic adenomas and 12 colonic carcinomas. Data were evaluated by relative expression software tool (REST-XL). Results The expressions of MCM2 mRNA elevated in turn by colonic normal mucosa, adenoma and carcinoma. The expression of MCM2 mRNA in colonic carcinomas was significantly higher than that in adenomas， as well as in normal mucosa (P=0.001), while the difference of MCM2 mRNA expressions between adenoma and normal mucosa was insignificant (P=0.184). Conclusion The difference of MCM2 mRNA expressions between adenoma and carcinoma indicated the potential value on early diagnosis for colonic carcinoma.
