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.

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.

Objective To observe the effect of Succinate injection combined with Bifico on the myocardial enzymes changes of the children with rotavirus enteritis.Methods A total of 146 children with rotavirus enteritis were enrolled and included in this study. Children were randomly divided into the control group (n=73) and the observation group (n=73). The control group was received Bifico on the basis of routine treatment, and observation group was received Succinate injection combined with Bifico. The IL-17, IL-6, TNF-?, LDH, CK, and CKMB was detected by ELISA. The rates of clinical effects was compared.Results After treatment, the IL-17 (22.35 ± 4.21 ng/mlvs. 30.24 ± 6.07 ng/ml,t=2.395), IL-6(31.26 ± 6.14 ng/mlvs. 43.72 ± 8.22 ng/ml,t=2.347), TNF-? (35.62 ± 6.24 ng/mlvs. 49.18 ± 8.72 ng/ml,t=2.421), LDH (135.16 ± 31.25 U/Lvs. 174.08 ± 40.22 U/L,t=2.373), CK (37.82 ± 7.39 U/Lvs. 50.21 ± 11.16 U/L,t=2.385), and CKMB (90.14 ± 11.63 U/Lvs. 113.22 ± 18.35 U/L,t=2.392) in the observation group were lower than those in the control group (P<0.05). The rates of clinical effects was 94.5% (69/73) in the observation group and 83.6% (61/73) in the control group. There was significant difference between the two groups (χ2=2.352,P=0.047).Conclusions The Succinate injection combined with Bifico could reduce the inflammatory indices and alleviate the myocardial injury in the RVE patients.

Objective Serum abnormal prothrombin (PIVKA-Ⅱ) level was detected in pancreatic cancer patients and its clinical value in diagnosing and treating pancreatic cancer was explored.Methods A total of 112 pancreatic cancer patients,28 benign pancreatic diseases patients and 79 healthy controls were collected from Cancer Hospital of Hubei Province.Serum PIVKA-Ⅱ level was determined by chemiluminescent immunoassay (CLIA).CEA and CA19-9 level was detected by electrochemiluminescence immunoassay (ECLIA).Receiver operation characteristic (ROC) curve was drawn and the cut-off value was set.The sensitivity,specificity and accuracy were calculated.Results PIVKA-Ⅱ level in 112 pancreatic cancer patients was 39.0 (22.61,137.67)U/L[median (quartile range)],which was significantly higher than that in 30 patients with benign pancreatic disease [23.0 (17.32,29.67) U/L] and 79 healthy controls [21.21 (17.9,25.12) U/L].The differences were statistically significant (P＜0.01).However,there was no significant difference between benign pancreatic disease patients and healthy controls.ROC analysis showed that area under curve(AUC) for PIVKA-Ⅱ,CEA and CA19-9 was 0.793,0.707 and 0.849.The cut-off value for discriminating pancreatic cancer was established at 32.4,4.7 and 27.0 U/L for PIVKA-Ⅱ,CEA and CA19-9.The sensitivity was 61.6％,38.0％ and 66.1％,and the specificity was 86.3％,96.3％ and 89.9％,and the accuracy was 71.7％,62.3％ and 75.0％,respectively.The sensitivity,specificity and accuracy of PIVKA-Ⅱ combined with CEA and CA19-9 was 90.2％,76.0％ and 84.3％.The sensitivity was obviously higher,while the specificity was lower.Serum PIVKA-Ⅱ level was associated with advanced tumor stage.There were 20 patients who were followed up.PIVKA-Ⅱ level in 15 cases were decreased after surgery or interventional therapy (50.6 U/L vs 110.5 U/L,P ＜0.001),which was increased in 4 of the five cases who received palliative care to a various degree.Conclusions PIVKA-Ⅱ detection could benefit the diagnosis and prognosis monitoring of pancreatic cancer.

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 explore the value of MRI sequence and MRA for diagnosis of moyamoya disease(MMD).Methods Retrospective analysed of 1 8 cases of moyamoya disease clinical datas,1 8 cases were used routine head MRI scan,including T1 WI, T2WI,DWI,transverse and sagittal T2WI sequence FLAIR;MRA three dimensional time of flight(3D-TOF).Results 18 cases of patients with MRA showed bilateral internal carotid artery,anterior cerebral artery distal and proximal middle cerebral artery steno-sis or occlusion of signs.Among them,1 1 cases were found with secondary cerebral infarction,3 cases of cerebral hemorrhage,7 ca-ses of centrum semiovale,corona radiata and periventricular cerebral ischemia,4 cases of cerebral atrophy,8 cases of cerebral soften-ing and gliosis.Conclusion MRI and MRA examination have important value in the diagnosis of moyamoya disease,treatment.

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.