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 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.

AIM:To evaluate the effectiveness and safety between trabeculectomy with collagen matrix versus trabeculectomy with mitomycin C(MMC) for patients with primary open-angle glaucoma(POAG).METHODS:In this prospective randomized comparative study from January 2015 to December 2016.Thirty-two eyes presented with POAG were included in this study, 14 eyes treated by trabeculectomy with subconjunctival implant of collagen matrix (study group) and the other 18 eyes treated by trabeculectomy with mitomycin C.Postoperative IOP, the success rate of operation, number of postoperative glaucoma medications and postoperative complications were recorded.Each patient was followed up at least 6mo.RESULTS:The mean postoperative IOP was statistically different between the study group and the control group after 1d (P<0.05), while not statistically different at 1 and 1mo follow-up (P>0.05), and the mean postoperative IOP was statistically different between the two groups (P<0.05) at 3 and 6mo after surgery.There was no significant difference in the success rate of operation between the two groups at 6mo after operation (P>0.05).The IOP decreased at 1d after openations compared with before, kept stable at 1wk to 6mo.IOP of study group was lowen than control.IOP was controlled by glaucoma medications in the study group by 28% compared to control group by 33% at 6mo after operation, but there was no significant difference.There was no significant difference between the study group and the control group in complications (P<0.05).CONCLUSION:Trabeculectomy with collagen matrix implant is comparable to the use of MMC with a similar success rate in open-angle glaucoma and the range in reducing intraocular pressure was significantly higher than that of MMC and it can significantly avoid the occurrence of low IOP postoperatively, transient anterior chamber, conjunctival wound leakage complications has no advantages compared with the use of MMC.

Objective To discuss which drainage method is more efficient in treatment of choledocholithiasis with obstructive jaundice. Methods Compared the difference of serum total bilirubin,direct bilirubin, the variation of serum total bilirubin and direct bilirubin before and after drainage and daily biliary drainage by endoscopic nasobil-iary drainage (ENBD) and endoscopic nasobiliary drainage with continuous negative-pressure suction after three days. Results The daily biliary drainage about the patients by ENBD with continuous negative-pressure suction is much more effective than the patients by ENBD. The serum total bilirubin and direct bilirubin about the patients by ENBD with continuous negative-pressure suction is less than the patients by ENBD after three days. The variation of serum total bilirubin and direct bilirubin before and after drainage about the patients by ENBD with continuous neg-ative-pressure suction is more than the patients by ENBD. Conclusion The ENBD with continuous negative-pres-sure suction is more effective for choledocholithiasis with obstructive jaundice.

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 reveal the trend of change incidence of hepatitis A(HAV)in Shandong province and explore dynamic analysis methods.Method Dynamic analysis was made on data of hepatitis A in Shandong province from 1990 to 2006.Results The reported incidence of hepatitis A patients dropped from 53.97/100,000 to 1.14/100,000 in Shandong province from 1990 to 2006,which showed a marked downward trend;the groups under the age of 10 dropped from 55.60% to 5.12%,the average age of incidence moved from 14.40 years old to 40.19 years old.Conclusion The incidence of hepatitis A has been changed from prevalent to sporadic in Shandong province,and there was a further downward trend;the age of the high-risk population has been shifted to older age group;the sex ratio of incidence between males and females in the population above 10 years of age had an expansion trend,the age distribution curve differences between males and females was also expanded.