Objective:To construct tetracycline regulating expression system of HBV-S gene,and study its regulating expression of HBVAg by tetracycline in HepG 2 cells. Methods:Insert HBs gene into the multiple cloning site of pcDNA4,which contain tetracycline operator in the promoter,and the constructed vector with pcDNA6 was co-transferred into HepG2 by means of lipofectamine,and use 400?g/ml Zeocin and 10?g/ml Blasticidin to kill the untransfected host cell line. After 20 days’select,the cell line which express HBsAg stablely after induced by tetracycline was generated. Then the HBsAg expressing was induced by varying concentration of tetracycline(0.5?g/ml,1.0?g/ml,2.0?g/ml,3.0?g/ml).Amount of HBsAg was detected by means of MEIA (Microparticle Enzyme Immunoassay) to determine the appropriate concentration of tetracycline. Result:HBsAg had been expressed at low quantity without tetracycline in transfected HepG2. However the S/N of HBsAg were high but not different after induced by varying concentration of tetracycline. Conclusion:Tetracycline regulating expression system of HBV-S gene was constructed successfully and HBsAg was highly expressed in the vector induced by tetracycline.

A hybridoma cell strain 6H_3(a kind of IgG_1)which could secrete the monoclonal antibody to human H blood group substance was yielded through PEG—fusion of the SP2/0 cells and BALB/C mouse spleen cells immunized by H blood group substance. The agglutination titers of cell culture supernatant and the ascites fluid were 512 and 16384,respectively. Based on hemagglutination specificity test,absorption and eluate test,and hemagglutination inhibition assay,it was showed to be specific only for the human H blood group substance. The ability of hybridoma cells to secrete the monoclonal antibody wasn't be changed when it has been stored in the liquid nitrogen for a year. The agglutination activity of monoclonal antibody has remained stable when it has been kept at 56℃ for 30 min,and repeatedly subjected to freezing and thawing tests at —20℃ and 37℃,respectively. It can be expected to replace the traditional anti-H reagents and to be used for clinical blood typing.

Objective To develop a duplex digital PCR(dPCR) for evaluation of the stability of luciferase(Luc) gene in Luc2P reporter cell lines(CHO-K1,Hacat,HEK293 and UT7).Methods Genomic DNAs of Luc2P reporter cell lines were extracted,a duplex dPCR was developed to determine the copies of reference gene RPP30 and the target gene Luc,and the relative copy number of Luc(copies Luc/copy RPP30) was employed as the indicator for the stability evaluation of Luc gene;The developed method was verified for the specificity,precision,linearity,accuracy and durability,and analyzed for the applicability,according to the related requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).Results All the original cells without reporter gene transfection were negative,while all the four reporter cell lines were positive,and the negative and positive regions in dPCR results were clearly distinguished;The relative standard deviation(RSD) of the eight repeated detections of the same genomic DNA sample and the six independent extractions of genomic DNA sample of the same cell were all less than 10%,and the linear fitting R~2 values were more than 0.99 for both Luc and RPP30.The spike recoveries of five groups of samples detected by the developed method were between 50% and 100%,and the results of chip-type dPCR and droplet-type dPCR were consistent.This method distinguished the relative copy number of Luc in different cell clones,and the results of detecting the relative copy number of Luc in three passages(P8,P12 and P31) were highly consistent.Conclusion The developed duplex dPCR method has good specificity,precision,linearity,accuracy,durability and applicability,and might be used to evaluate the stability of Luc gene in Luc2P reporter cell lines.

Objective To investigate androgen receptor (AR) expression in triple negative breast cancer(TNBC) patients and its predictive value for clinical outcomes.Methods Five hundred and sixty-eight primary breast cancer patients who received surgery treatment from January 2008 to January 2010 were included in this study.Tissue sections of surgical resection specimens were underwent estrogen receptor (ER),progesterone receptor (PR),and human epidermal growth factor receptor 2 (HER2) immunohisto chemical staining,according to the judgment for triple negative breast cancer and non triple negative breast cancer.Data on age,family history,menstruation,tumor size,tumor stage,tumor grade,pathological type and lymph node metastasis were collected for analysis.Immunohistochemical analysis of AR was done in tissue sections of triple negative breast cancer patients,Kaplan-Meier method was used to compare survival between AR(+) and AR(-) groups,and Log-rank method was used to determine the association between AR-positive expression and 5-year overall survival as well as disease free survival.Results Among 568 breast cancer patients,174 (30.6％)TNBC patients were identified,including 84 patiens with AR (-) expression and 90 patients with AR (+) expression.During 5 years' follow-up,reccurrence rate was 35.7％ (30/84) in AR (-) group and 20.0％ (18/90) in AR(+) group;5-year overall survival rate was 73.8％ in AR(-) group and 81.1％ in AR(+) group.There was no significant association between AR(+) expression and 5-year death or recurrence rate,while after adjusting for age,menstruation,tumor size and lymph node metastasis,AR (+) expression was significantly associated with decreased 5-year death and recurrence rate,adjusted hazard ratio (HR) was 0.794,95CI (0.430-1.021) and 0.722,95CI (0.451-0.965) respectively.Conclusion AR (-) expression is associated with increased risk of death and recurrence and is a promising predictive marker for poor clinical outcomes.

As an emergent attack,dyspnea can be caused by a variety of diseases,therefore,it is difficult and important as to the differential diagnosis in clinical practice.In the past,Physicians identified the cause of dyspnea primarily based on the clinical symptoms,signs and laboratory tests.However,dyspnea often misdiagnosed because the similarity and overlapping of clinical manifestations among many different diseases and lack of specificity for those conventional lab tests.Until recently,B-type natriuretic peptide (BNP) has emerged as a highly sensitive and specific index to identify heart failure,and has made it reality that rapid and accurate differential diagnosis of dyspnea in the lab.In the last couple of years,the differential diagnosis of dyspnea has been remarkably improved because of the increasing new test reagents,advanced devices and better assay techniques.The present article will review the laboratory differential diagnosis of dyspnea between cardiovascular and pulmonary diseases.

BACKGROUND:Membrane guided bone regeneration technology has become an important method in repairing bone defects. With the deepening of the research, related concept and the mechanism of membrane guided bone regeneration have been gradual y confirmed, but there are stil some unresolved issues.
OBJECTIVE: To review the classification of membrane tubes, performance, disadvantages and advantages in membrane guided bone regeneration, as wel as some unresolved issues in application and research.
METHODS:The first author searched PubMed and CNKI databases to retrieve articles about the discovery of membrane guided bone regeneration and the concepts, classification of membrane tubes, performance, disadvantages and advantages during bone defect treatment, which were published from 1963 to 2013. The key words were“guided bone regeneration, guided tissue regeneration, bone defect treatment”in English and Chinese, respectively.
RESULTS AND CONCLUSION:Membrane guided bone regeneration technique is a most promising treatment for bone defects, but for the treatment of long tubular bone defects, it is stil in the experimental stage. Currently, there is no membrane tube for long-segment bone defects. According to the material sources, the membrane tubes can be divided into:non-biological material, such as polytetrafluoroethylene, polylactic acid, silica gel, titanium film;biological materials, such as col agen membrane, chitin membrane, polyhydroxybutyrate. The membrane tubes can also be classified into nondegradable materials and biodegradable materials. Biodegradable materials have good histocompatibility and no cytotoxicity, which can degrade in a certain period after implantation;part of the membrane can also al ow free exchange of tissue fluid and nutritional substances. But there are stil some shortcomings that the degradation time is difficult to control and the volume is difficultly maintained under the membrane tube. New bone formation in non-biodegradable materials is complete. In the process of osteogenesis, the membrane tube cannot be absorbed and has to be removed secondarily, though it has good histocompatibility and better therapeutic outcomes. In the future, we should further improve membrane performance, so that the membrane tube can play a dual role, fixation and guided bone regeneration;a series of animal studies should be conducted to study the effect of stress on the membrane tube and osseointegration within the membrane tube, to master the law of osseointegration of membrane tubes, thereby providing evidence for repair of long tubular bone defects.

Objective To investigate the risk factors of post-pancreaticoduodenectomy complications.Methods The clinical data of 207 patients with pancreatic carcinoma or peri-ampullary carcinoma who underwent pancreaticoduodenectomy at the Anhui Provincial Hospital from Dec.2007 to Dec.2012 were studied.Seventeen clinicopathologic factors to correlate with the postoperative mortality and methods were selected for univariate analysis and multivariate analysis using logistic regression.Results Univariate analysis showed the major risk factors of postoperative mortality and morbidity were a history of previous operation,pre-operative drainage,total serum bilirubin level,alanine aminotransferase level,serum albumin level,serum pre-albumin level,parenchyma texture and pancreatic duct diameter (P＜0.05).Multivariate analysis showed alanine aminotransferase level,parenchyma texture and pancreatic duct diameter were independent risk factors of postoperative complications.Pancreatic duct diameter was the independent risk factor of pancreatic fistula.Pancreatic fistula was the independent risk factor of hemorrhage.Conclusion The postoperative complications of pancreaticoduodenectomy was closely related to alanine aminotransferase level,parenchyma texture and pancreatic duct diameter.

Objective:To observe whether the in-flow anesthetic gas absorber can reduce the recovery time after stopping inhalation of enflurane or isoflurane. Methods: In fixed tidal volume, minute volume and fresh gas flow, the recovery time of isoflurane or enflurane anesthesia with or without in-flow anesthetic gas absorber were compared after closing the vaporizer.Results: With in-flow anesthetic gas absorber, the time for anesthetic gas concentration in circle to reduce the MAC to 0.3was 3.3±0.5 min for isoflurane, which was significntly shorter than that without absorber (20±0.3min) ( P＜0.01 ) and the time for enflurane was 3.5±0.5min and 25±0.1min respectively. Conclusion:Anesthetic gas absorber can reduce the recovery time of either enflurane or isoflurane inhalational anesthesia significantly.

Objective To investigate the mechanism of anireflux procedures in treating sliding hiatal hernia and the effectiveness of the method of cardia position by clock to evaluate the outcome of antireflux procedures. Methods From 1992 to 2008, 136 patients with sliding hiatal hernia underwent transabdominal antireflux surgery: 27 patients received Nissen procedure and 109 recieved floppy Nissen operation. Results There is no postoperative death. Within one month after operation, in the Nissen group, 2 patients developed severe dysphagia and 1 had regurgitation; in the floppy Nissen group, 9 patients had regurgitation, and 3 had dysphagia. During the over-2-year follow-up period, 2 patients developed severe dysphagia,1 had nausea and 2 had significant reflux in the Nissen group. While 3 patients recturred, 2 had dysphagia and 7 had significant reflux in the floppy Nissen group. The effective rates of the Nissen group and the floppy Nissen group were 81.5% and 88.1%, respectively. There was no significant difference in postoperative tests between two groups ( P ＞ 0.05 ). Tests of cardia position by o'clock showed that cardia positions of 80. 1% patients before surgery were located at sites between 10 and 11 o'clock and those of 91.7% after surgery being at 9 o' clock or less. Conclusions The Niseen and the floppy Nissen fundoplication can reach good long-term results in treatment of sliding hiatus hernia. Mechanism of these antireflux procedures include, by fundoplication, increasing esophageal sphincter pressure, prolonging the sphincter length, deepening His angle and prolonging abdominal esophagus, etc. Among them, the last one, descending the cardia position to 9 o'clock or less is vital to antireflux.The test of cardia position by o'clock is practically important to reach the successful result.

At present, there are 8 sources of neural stem calls (NSCs), such as the early embryo, umbilical cord blood, brain tissue of adults and bone marrow, which are used for experiments. Four methods are also provided to isolate NSCs, for example, the serial passage and the gene transduction. Most of the hotspots at home and abroad are on the modulation of NSC transplantation in vivo, to make it proliferate and differentiate in hand. Most findings are achieved through experiments on rodents, no matter in vitro or vivo, which indicate that the proliferation and differentiation of NSCs can be affected by a variety of biological factors and drugs. Even more, the effects are multiple according to the factors and drugs of different kinds and the same kind of different concentrations, even the same factor would have different roles in different process of the culture of NSCs. The survival of NSCs during transplantation can also be affected by local microenvironment. Neither the NSC endogenous nor exogenous treatment is efficient. Furthermore, the body also rejects the exogenous NSCs. Taken together, it is tough for us to conclude that a factor does have some effects on the NSCs. Moreover, there are still a series of key problems unveiled clinically, such as how to perfect transplantation and whether to choose exogenous or endogenous NSCa for transplantation.