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Objective:To construct four retroviral plasmids for differential expression of green fluorescent protein (GFP) based on different terminal sequences of internal ribosome entry site (IRES) and provide reference for subsequent flow analysis or imaging.Methods:Based on the fact that the transcription efficiency of encephalomyocarditis virus IRES depends on its terminal sequence, IRES and enhanced GFP (EGFP) were fused into four fragments with different connection modes by overlapping PCR, and then cloned into retroviral plasmid pMSCV-NGFR. NGFR fragment was amplified by PCR and inserted in front of the retroviral plasmids pMSCV-IRES(1-4)-EGFP. These retrovirus plasmids pMSCV-NGFR-IRES(1-4)-EGFP were transfected into 293T cells. The expression ratio and mean fluorescence intensity (MFI) of EGFP were analyzed, and the expression of NGFR was also detected.Results:Four retroviral plasmids pMSCV-NGFR-IRES(1-4)-EGFP were successfully constructed. No significant difference in the expression efficiency of EGFP at 24 or 48 h was observed in 293T cells transfected with the four different retroviral plasmids, but there was significant difference in fluorescence intensity. Moreover, the expression of NGFR was not significantly different, indicating that the addition of different nucleotide sequences between IRES and EGFP would make a significant difference in the fluorescence intensity of EGFP.Conclusions:The expression intensity of EGFP was affected by the sequence between IRES and EGFP. Retroviral plasmids expressing EGFP of different intensity could meet different experimental requirements.
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Objective@#To assess the association of single nucleotide polymorphisms (SNPs) in SLCO1B3 gene with prognosis of breast cancer (BC) patients treated with neoadjuvant chemotherapy of TA regimen (taxane and antharcycline drugs).@*Methods@#439 female BC patients were recruited and treated with neoadjuvant chemotherapy of TA regimen. A blood sample (2 ml) of peripheral blood was collected from each patient before chemotherapy. Tagging SNPs (tag-SNPs) were selected. We investigated the association of tag-SNPs with prognosis, by Sequenom Mass ARRAY system platform, characterizing tag-SNPs. The hazard ratio (HR) and 95% confidence interval (CI) for progression or death were calculated by multivariable-adjusted Cox regression model.@*Results@#Seven tag-SNPs (rs11045689, rs200104106, rs3764006, rs3834935, rs4149117, rs7305323 and rs73241801) were selected for study. Compared with individuals carrying the rs11045689 GG genotype, individuals carrying rs11045689 AA genotype performed worse PFS and OS, with the HR (95% CI) for progression being 1.39 (1.11~1.75) and the HR (95% CI) for death being 1.38 (1.04~1.83). Compared with individuals carrying the rs73241801 CC genotype, individuals carrying rs73241801 TT genotype performed better OS (P=0.041), with the HR (95% CI) for death being 0.65 (0.44~0.94). The number of risk allele was significantly associated with PFS (P=0.012) and OS (P=0.017) of BC patients by accumulation analysis. Compared with individuals carrying one or less than one risk allele, individuals carrying four risk alleles performed worse PFS and OS, with the HR (95% CI) for progression being 1.37 (1.09~1.72) and the HR (95% CI) for death being 1.36 (1.02~1.81).@*Conclusion@#The variations of rs11045689 and rs73241801 in SLCO1B3 gene were significantly associated with prognosis of BC patients treated with neoadjuvant chemotherapy of TA regimen, which might serve as biomarkers for predicting prognosis of BC patients treated with neoadjuvant chemotherapy.
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Objective To study the mechanism of carboxypeptidase E ( CPE ) in promoting the migration of lymphocytes and their subsets through vascular endothelial cells. Methods CRISPR/Cas9 technology was used to prepare cpe gene-knockout MS1 (Cpe-/-MS1) cells. Adhesion ability of lymphocytes to MS1 and Cpe-/-MS1 cells was analyzed with adhesion assay. Expression of adhesion molecules on these cells were detected by RT-PCR and flow cytometry. Transwell model was used to compare the difference in the transmigration of lymphocytes and their subsets through MS1 and Cpe-/-MS1 cells. Results Cpe-/-MS1 cells were successfully obtained. Under the stimulation of TNF-α, the adhesion ability of lymphocytes to MS1 cells was much better than that of Cpe-/-MS1 cells. Moreover, adhesion molecules expressed on MS1 cells were significantly more than those on Cpe-/-MS1 cells. The percentages of lymphocytes and their sub-sets that transmigrated through MS1 cells were significantly higher than those through Cpe-/-MS1 cells. Con-clusion CPE involved in the adhesion of lymphocytes to vascular endothelial cells and the transmigration of them through vascular endothelial cells, which was of great significance for understanding the migration of lymphocytes across vascular endothelial cells to peripheral lymph nodes.
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Objective To study the function of gene 0955 in Streptococcus suis type 2 98HAH33. Methods Growth condition of wild-type, mutant and complemented strains of Streptococcus suis type 2 98HAH33 was compared at different stages. Differences in adhesion ability to host cells and anti-phagocyto-sis among these strains were compared by using bacterial adhesion test and analyzing their survival rates in blood. Mouse and piglet models were used to evaluate their virulence. Results The growth of the mutant and the complemented strains was slightly slower than that of the wild type strains in logarithmic growth phase, but no significant difference was found in plateau phase. Bacterial adhesion test showed that gene 0955 might encode a new adhesion factor of Streptococcus suis type 2 98HAH33. Blood bactericidal test sug-gested that gene 0955 was not associated with anti-phygocytosis. Animal experiments showed that gene 0955 might be a novel virulence gene of Streptococcus suis type 2 98HAH33. Conclusion Gene 0955 might en-code a novel adhesion factor and virulence factor of Streptococcus suis type 2 98HAH33.
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Objective To evaluate the efficacy and safety of peroral direct cholangioscopy (PDCS) in the treatment of refractory common bile duct stones after retrograde cholangiopancreatography (ERCP).Methods Data of 13 patients who underwent peroral direct cholangioscopy for refractory common bile duct stones after retrograde cholangiopancreatography at our institution from April 2012 to August 2014 were retrospectively analyzed.Completion rate,side-effects and complications were summarized.Results Peroral direct cholangioscopy was successfully performed with electrohydraulic lithotripsy and stones were removed in 12 of the 13 patients.Lithotripsy and stone removal failed in one patient,though the ultraslim endoscope accessed distal common bile duct near the stone.Stones were removed directly with extraction basket under PDCS in 7 patients,and duodenoscopy was performed in 5 patients due to too many stone fragments.Stone removal was successfully completed at one time in 9 cases.Stones removal failed in 3 cases at one time because of too many stone fragments,but was successful with duodenoscopy a week later after a temporary biliary tract stenting.Andoxygen saturation decreased in one patient due to vomit during the operation.Sputum in oropharyngeal area was immediately sucked out and the gastric juice at the bottom of the stomach was also sucked by endoscopy.Oxygen saturation returned to normal levels and the subsequent operation was not affected.No aspiration pneumonia occurred after the operation.Hyperamylasemia occurred in two patients,and postoperative biliary tract infection occurred in one,but the situation was controlled after appropriate treatment.Conclusion PDOS using an ultraslim endoscopy is an effective and feasible endoscopic procedure for the refractory common bile duct stones.This study provides a new method for the treatment of refractory common bile duct stones.
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Objective To investigate the diagnostic and therapeutic value of peroral cholangioscopy for residual stones after retrograde chalangiopancreatography (ERCP).Methods The soft-tipped guidewire (0.021in) was linked to 3-0 silk thread on the front of extraction balloon catheter outside as a guide device.After retrograde chalangiopancreato-graphy (ERCP) for common bile duct stones,extraction balloon with the guide device was sent to intrahepatic bile duct by duodenoscopy,and residual stones were observed and removed with a stone basket directly under ultrathin upper endoscope if the residual stones were small ; if the stones were large,they were crushed with electrohydraulic lithotripsy before being removed.Results The ultrathin upper endoscope were successfully inserted into hilar bile duct in 42 cases of 46 patients,and failed in 4 cases,and the success rate of insertion was 91.3％.The mean time was 11.3 min from the mouth into hilar bile duct.Stones were found larger than 4 mm in diameter in 6 of 42 patients,and stone residual rate was 14.3％.The biggest stone was 10 mm× 12 mm in diameter.Stones were found in in 2 of 27 patients after ERCP,and the stone residual rate was 7.4％.Stones were found in 4 of 15 patients after ERCP basket lithotripsy,and the stone residual rate was 26.7％.Stones were removed directly in 5 in 6 cases with extraction basket,they were crushed in 1 case by electrohydraulic lithotripsy and then removed with basket.No serious complications were observed.Conclusion The application of peroral cholangioscopy using an ultrathin upper endoscope is feasible.The method is a useful endoscopic procedure for extraction of residual stones,which helps to avoid repeated treatment.