Objective To investigate the applieation of restriction display (RD) technique in the preparation of HCV probes of clinical genotyping microarray. Methods Restriction enzyme Sau3A Ⅰwas chosen to digest the full-length HCV cDNAs of three distinct subtypes, i.e.1a, 1b and 2a. The resultant restrictive fragments were then ligated with universal adapters. PCR primers were designed to match the universal adapters but with one "nesting" base overhanging at the 3′- end. The PCR reactions were performed by ten pairs of different primer combinations. The differential genes were separated through polyacrylamide gel electrophoresis and silver staining. The second-round PCR was performed using the isolated bands as PCR templates. The purified PCR products were then cloned into T-vectors. The recombinant plasmids were extracted from positive recombinant clones and the target gene fragments were sequenced. Results The target HCV gene fragments ranging from 200 to 900bp were isolated and sequenced, which were correlated precisely with the RFLP (Restriction Fragment Length Polymorphism) prediction. A total of 66 different fragments were obtained, averaging about 22 for each subtypes. These fragments could be further used as probes in HCV microarray preparations. Conclusion RD technique is of great value in obtaining a large number of equal sized gene probes, which provide a swift protocol in generating DNA probes for the preparation of microarrays.

To study cardiac function in an ovine model of chronic heart failure in relation to cardiac collagen and its phenotype, 11 sheep were developed into chronic heart failure by sequential microembolisation. Cardiac collagen contents and phenotypes were determined after hemodynamic evaluation at 6 months. The results showed that collagen contents and type Ⅰ /type Ⅲ collagen ratio in the left ventricle were increased significantly in heart failure group as compared with those in the normal group. Collagen concentration in left ventricle correlated significantly to left ventricular ejection fraction, left ventricular end diastolic pressure, dP/dt min , left ventricular diastolic diameter, and diastolic interventricular septum thickness.Our conclusion is increase in collagen content and type Ⅰ/Ⅲ collagen ratio may be partly responsible for the reduction of cardiac function seen in this model of heart failure.

Objective To establish a method for determination of imatinib mesylate liposome and related substances. Methods The liquid chromatography was carried out on a Kromasil C18 column. The mobile phase A consisted of methanol-octane sulfonate solution(42:58). The mobile phase B consisted of methanol-octane sulfonate solution(4:96). The flow rate of gradient elution was 1. 2 mL·min-1 . The detection wavelength was 268 nm. The column temperature was room temperature. Results The intermediates and degraded substances could be seperated under the selected chromatographic conditions. Imatinib mesylate showed a good linear relationship within 1-100μg·mL-1,r=0. 999 1(n=5). Conclusion The method is specific, accurate,sensitive,and simple,and can be used for quality control of imatinib mesylate liposome.

Objective To investigate the prevalence of healthcare-associated infection(HAI)in medical institutions in Pudong New Area.Methods Ten medical institutions in Pudong New Area were selected as monitored hospitals, cross-sectional survey on HAI in each hospital at a given day of November 2013 and November 2014 were conduc-ted.Prevalence of HAI in different levels of hospitals were compared.Results The prevalence rates of HAI in 2013 and 2014 were 4.04% and 3.75% respectively,there was no significant difference between two years(χ2 =0.709,P=0.400).The prevalence rates of HAI in primary,secondary,and tertiary hospitals in 2013 were 0.66%,3.32%, and 4.60% respectively,in 2014 were 0,3.52%,and 4.01 % respectively,prevalence rates of HAI in different lev-els of hospitals of each year were significantly different (all P <0.05).Prevalence rates of HAI were high in depart-ments of neurosurgery,hematology,intensive care units(ICUs),and gerontology.The prevalence rate of infection in ICUs increased obviously,from 10.09% to 18.78% (χ2 =3.921 ,P =0.048),departments of gerontology de-creased obviously,from 10.07% to 5.02%(χ2 =5.698,P =0.017).The main HAI sites were lower respiratory tract (36.72%),upper respiratory tract (9.96%),and urinary tract (12.89%).172 pathogenic isolates were de-tected in 2013,and 177 were detected in 2014,gram-negative bacteria,gram-positive bacteria,and fungi accounted for 60.74%,26.37%,and 12.89% respectively.Constituent ratios of pathogens causing HAI between two surveys were not significantly different (χ2 =5.819,P =0.830).Conclusion Among different levels of hospitals in Pudong New Area,tertiary hospitals have the highest prevalence rate,HAI in ICU increases obviously,the main HAI site is lower respiratory tract,the main pathogens are gram-negative bacteria.

To obtain non-pathogenic rabies virus glycoprotein(RV-G),we expressed RV-G in Saccaromyces Cerevisiae(S.cerevisiae).In our study,tat-G fusion gene was cloned into the expression vector pYes2.0,which allows expression of a foreign gene in the yeast cells under the control of GAl1 promoter.Transforma-tion was performed by using lithium-treated yeast cells and several Ura+-tranformants were isolated.Ac-cording to the relative mobility in SDS-PAGE,we know probably two forms(designated as yGI and yGⅡ) of RV-G analogues produced in S.cerevisiae,their molecular weights were estimated as 66 kD and 56 kD,respectively.On the other hand,there was a specific band about 56 kD shown in western blot result.Com-bining precursors’ achievements,we will draw a conclusion that trans-membrane domain(TD) and cyto-plasmic domain have a negative regulation on RV-G antigen immunogenicity in S.cerevisiae.

Objective To study the expression of interleukin-1β in aortic dissections and aneurysms. Methods Aortic specimens were obtained from patients with type Ⅰ thoracic aortic dissection (11 cases),ascending thoracic aortic aneurysms (10 cases),and healthy organ donors (7 cases).Expression of interleukin-1β,matrix metalloproteinase-9,and signal transduction factors phospho-p38 and phospho-JNK were detected by real time RT-PCR,Western blot,and immunohistochemistry,respectively.TUNEL staining was performed to detect apoptosis of media cells. Results Apoptosis in the media of thoracic aortic dissection and ascending thoracic aortic aneurysms was dramatically higher than control group.Expression of interleukin-1β gradually increased in an order of control group,thoracic aortic dissection to ascending thoracic aortic aneurysms ( P ＜ 0.01,respectively).Expression of matrix metalloproteinase-9significantly increased in the media of thoracic aortic dissection and ascending thoracic aortic aneurysms compared with control group (P ＜ 0.01,respectively).There were positive correlations between interleukin1 β and matrix metalloproteinase-9,interleukin-1β and phospho-p38 in thoracic aortic dissection ( P ＜ 0.01,respectively),interleukin-1β and apoptosis in ascending thoracic aortic aneurysms (P ＜ 0.01 ).Conclusions Interleukin-1β and interferon-γ might effect the formation of thoracic aortic dissection and ascending thoracic aortic aneurysms possibly through the up-regulation of matrix metalloproteinase-9 and apoptosis of media cells in humans.

Objective:To evaluate the immunogenicity of an experimental SARS-CoV inactivated vaccine.Methods:The virus suspension of F69 strain was inactivated with 0.4% formaldehyde and purified,then used as the immune antigen combined with Freund′s adjuvant.Eight adult New Zealand rabbits were immunized 4 times with this vaccine.12 sets of rabbit serum were sampled from the third day to 74th day after first immunization.Titers of specific IgG antibody and neutralizing antibody were determined by indirect ELISA and micro-cytopathic effect neutralizing test.Results:Rapid and potent humoral immune responses were induced by F69 inactivated vaccine in all eight immunized rabbits.Both specific IgG antibody and neutralizing antibody all peaked just with 2 vaccinations,with the maximum titer of 1∶81 920 and 1∶20 480 respectively about 6 weeks after first immunization.Across neutralizing reaction existed between F69 and Z2-Y3 strains.Conclusion:F69 inactivated vaccine owns strong immunogenicity.Similar antigenic epitopes exist between the F69 strain and Z2-Y3 strain,which ensured the cross neutralizing reaction.

Objective To analyze the prevention and treatment of postoperative hemorrhage after pancreaticoduodenectomy. Methods The clinical data of 142 patients undergoing pancreaticoduedenectomy from Jan 1995 to Dec 2008 were retrospectively analyzed. Results The incidence of postoperative hemorrhage was 14.1% (20/142), the mortality caused by this complication was 35% (7/20). Among these patients, intra-abdominal hemorrhage occurred in seven cases, and gastrointestinal hemorrhage occurred in 14 cases, with one case suffering both. There were three and four cases of early and delayed intra-abdominal hemorrhage respectively. Early and delayed gastrointestinal hemorrhage occurred in three and eleven cases respectively. Univariate analysis showed that operative blood loss, blood transfusion, infection and pancreatic fistula were significantly associated with postoperative hemorrhage. Multivariate analysis using Logistic regression identified two variables as independent factors associated with postoperative hemorrhage, namely, infection(OR=6.918) and pancreatic fistula(OR=3.948). Conclusions The incidence and mortality of hemorrhage after pancreaticoduodenectomy is still high. Skillful operation and prevention of pancreatic fistula, infection and stress ulcer are the key paints for reducing postoperative hemorrhage. Proper treatments should be used according to the site, onset and severity of hemorrhage.

Objective To explore the efficiency of transcervical video-assisted mini-incision thyroidectomy for thyroid tumor. Methods 53 patients with thyroid tumors who underwent the video-assisted mini-incision thyroidectomy from Jan. 2006 to Dec. 2009 in the Department of Thyroid Surgery, Yantai Yuhuangding Hospital,were retrospectively studied. Results All of the patients, except 5 cases converting to open thyroidectomy, underwent the video-assisted mini-incision thyroidectomy successfully. The temporary recurrent laryngeal nerve injury occurred in 1 patients. The subcutaneous fluid accumulation occurred in 3 patients. The skin burns around the incisions occurred in 3 patients. There was no complication of hematoma, hypocalcemia, or superior laryngeal nerve injuries. The follow-up period was from 3 months to 35 months. There was no evidence of recurrence and metastasis by postoperative ultrasonic and CT scan and radioiodine scintigraphy. Conclusions The video-assisted mini-incision thyroidectomy is feasible, safe, and with the advantage of cosmetic results. For thyroid tumor,especially thyroid cancer, longer follow-up is necessary to draw definitive conclusion on recurrence and survival rate.

ObjectiveTo evaluate the safety and effectiveness of fast track surgery (FTS) in l aparoscopy-assisted radical distal gastrectomy (LADG) for gastric cancer.MethodsSixty-one patientswith distal gastric cancer were randomly divided into three groups:FTS + LADG group (n =19) undergoing LADG and FTS treatments,LADG group (n =22) undergoing LADG and traditional perioperative cares,and FTS + ODG ( open distal gastrectomy) group ( n =21 ) undergoing ODG and FTS treatments.FTS treatments included avoidance of mechanical bowel cleansing,restrictive perioperative intravenous infusion,early ambulation,early enteral nutrition.The age,sex,body weight,anastomotic mode,number of lymph node dissected,and tumor stage,serum albumin (ALB),blood urea nitrogen (BUN),C-reaction protein (CRP),flatus time,postoperative hospital stay,medical cost,and postoperative complications were compared between three groups. ResultsThe level of ALB in FTS + LADG group were higher than in LADG group at the 4th and 7th day after surgery ( P ＜ 0.05,P ＜ 0.01 ).Compared to LADG group,the variation of ALB from preoperation to 4th day after surgery in FTS + LADG group and FTS + ODG group was significant( P ＜ 0.01,P ＜ 0.05 ).CRP level between FTS + LADG group and FTS + ODG group were different significantly at 4th and 7th day after surgery ( P ＜ 0.05,P ＜ 0.05).FTS + LADG group has earlier recovery of gastrointestinal peristalsis than other two groups ( P ＜ 0.05,P ＜ 0.05 ).The medical cost in FTS + LADG group was less than in LADG group ( P =0.003 ),but higher than in FTS + ODG group (P ＜0.01 ).ConclusionsThe practice of FTS in LADG was safe,effective,improves nutritional status,eases stress reaction,accelerates gastrointestinal peristalsis and postoperative rehabilitation.