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.
ObjectiveTo detect the expression of Prox1 (prospero-related homeobox 1) gene in primary hepatocellular carcinoma (HCC), and to analyze the correlation of Prox1 gene expression with pathological grade and clinical stage of HCC. MethodsThe expressions of Prox1 gene in carcinoma tissues and adjacent cancerous tissues in HCC as well as normal liver tissues were detected by semi-quantitative RT-PCR, then the correlation of Prox1 gene expression with HCC pathological grade and clinical stage were analyzed. ResultsThe expression of Prox1 gene in carcinoma tissues (0.243±0.102) and adjacent cancerous liver tissues (0.537±0.235) was significantly lower than that in normal liver tissue (0.812±0.372), respectively （ Plt;0.01 or Plt;0.05）. Furthermore, the expression of Prox1 gene in carcinoma tissues was significantly lower than that adjacent cancerous liver tissues (Plt;0.05). The expressions of Prox1 gene in different pathological grade (F=97.950, Plt;0.001) and clinical stage were significantly different (F=228.300, Plt;0.001), and when compared with each other, the differences of pathological grade and clinical stage were also significant (Plt;0.001 or Plt;0.01). The expressions of Prox1 gene in HCC carcinoma tissue were negatively correlated with pathological grade (r=-0.930, Plt;0.01) and clinical stage (r=-0.980, Plt;0.01) of HCC. ConclusionsExpression of Prox1 gene may be related to the initiation and development of HCC, however, that whether Prox1 gene functions as tumor suppressor in HCC needs further investigation.
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 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 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.
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 analyze the cost and performance of five methods for detection of carbapenemase-producing Enterobacteriaceae (CPE), including PCR (method A), Carba NP test (method B), ultraviolet spectrophotometry (method C), modified carbapenem inactivation method (mCIM, method D), and loop-mediated isothermal amplification (LAMP, method E).MethodsPubMed, EMbase, The Cochrane Library, CNKI, WanFang Data and CBM databases were searched using the computer regarding literature on detection of CPE with the same or similar designs, same objectives, and independent results. The search was limited between May 2009 and May 2019. Data on the cost and detection performance of all five methods were extracted, and the four special indexes for laboratory tests, such as sensitivity, specificity, simplicity, and rapidity in the utility were quantified as specific values; subsequently, the cost-effective analysis (CEA), cost-utility analysis (CUA), and multi-attribute utility theory (MAUT) in the detection economic analysis were used to conduct health economics evaluation of five detection methods for CPE.ResultsThe cost of methods A, B, C, D and E were 210.00 yuan, 22.00 yuan, 10.50 yuan, 6.00 yuan, and 60.00 yuan, respectively. The C/E of CEA for the above five methods were 210.00, 22.96, 10.66, 6.14, and 60.00, respectively. The C/U of CUA for the above five methods were 302.16, 32.13, 19.30, 11.13, and 80.00, respectively. The MAUT value of the above five methods were 42.56, 5.00, 2.54, 1.63, and 12.56, respectively.ConclusionIn terms of CEA, CUA, and MAUT, the method D was the highest in economic value, which usually can be used as a routine method for detecting CPE, but it needs a long procedure time; thus, the method E can be used for rapid detection when clinical severe infection occurred, which is superior in both cost-effectiveness and rapidity.
Objective To research the expressions of miR-196b and HoxB8 mRNA in colorectal cancer and theircorrelation with clinicopathologic features，and to explore the relationship between miR-196b and HoxB8 in vivo. Methods Expressions of RNA （including miR-196b and HoxB8 mRNA） and HoxB8 protein were detected respectively by using quantitative real-time reverse transcriptase PCR and Western blot in 30 cases of colorectal cancer and corresponding normalmucous membrane tissues. Results In colorectal cancer tissues，expressions of miR-196b and HoxB8 mRNA were higher than those of the corresponding normal mucous membrane tissues （P＜0.05）. Expression of miR196b mRNA was assoc-iated with lymph node metastasis，neoplasm stages （Ⅰ＋ⅡandⅢ＋Ⅳ），and distant metastasis （P＜0.05），on the otherhand，no significant differences were observed regarding tumor site，size，gross type，depth of invasion，tissue differentiation，age，and sex （P＞0.05）. Expression of HoxB8 mRNA was no significant differences concerning lymph node metastasis，tumor stages （Ⅰ＋Ⅱ，Ⅲ＋Ⅳ），distant metastasis，tumor site，size，gross type，depth of invasion，tissue differentiation，age，and sex （P＞0.05）. The expression of miR-196b mRNA was negatively correlated with HoxB8 mRNA expression （r=-0.458，P＜0.05），and HoxB8 protein expression with no obvious correlation （r=-0.236，P＞0.05） in colorectal cancer tissues. Conclusions The expressions of miR-196b and HoxB8 mRNA in colorectal cancer tissues are higher，the high expression of miR-196b mRNA is related to the tumorigenesis and progression of colorectal cancer as well as correlated with prognosis in colorectal cancer. The miR-196b inhibits the expression of HoxB8 mRNA by binding to the3′-UTR of target HoxB8 mRNA.
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.
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.