Preparation of human monoclonal anti-C cell line from peripheral blood B lymphocytes of D--donor / 中国输血杂志

【Objective】 To establish human hybridoma cell lines, secreting monoclonal antibody against antigens of Rh blood system, from a donor with rare D--phenotype. 【Methods】 Peripheral blood B lymphocytes of an O type female donor, lacking C/c/E/e antigens on her erythrocyte, were transformed with Epstein-Barr virus (EBVs). EBVs were harvested from the cultural supernatant of B95-8 cells. The transformed lymphoblastoid cell line (LCL) secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique. Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine, Aminopterin, Thymidine, 2-Deoxycytide, and Ouabain)culture medium, microplate antibody screening and limited dilution subcloning. The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry (FCM). 【Results】 From the cultural supernatant of D--peripheral blood transformed B lymphocytes, 3A6-C6, which agglutinated with R1R1(DCe/DCe)O-type RBCs but not with R2R2(DcE/DcE)O-type RBCs, was screened and preliminarily identified as anti-C. We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells. 【Conclusion】 The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

β-Lactam antibiotics promoting aging and clearance of RBCs would deteriorate the DIIHA / 中国输血杂志

【Objective】 To analyze the influence of β-lactam antibiotics on RBC aging and clearance by detecting various indicators of aging and clearance on RBCs, as well as the differences in phagocytosis for erythrocytes before and after drugs treated in vitro. 【Methods】 RBCs were treated by β-lactam antibiotics, including Penicillin, Cefepime, Cefoperazone and Ceftazidime, and the changing of phosphatidylserine (PS) and clearance related CD markers, including CD35, CD47, CD55 and CD59 on the surface of the RBCs, were detected by flow cytometry at 0h and 24h after drugs treatment. The proportion of acanthocytes by microscope also at 0h and 24h after drugs treatment was calculated. The phagocytosis of drug-treated RBC was detected by monocyte monolayer assay (MMA). Untreated RBCs were incubated in PBS by the same condition as a negative control.The influence of β-lactam antibiotics on RBC aging and clearance by all the results above was studied. 【Results】 Compare to the untreated RBCs, the drug treated RBCs showed a higher PS level on the cell surface. The results showed by percentage as following(0 h vs 24 h): Penicillin 9.42% vs 93.30%, Cefepime 3.88% vs 57.27%, Cefoperazone 4.71% vs 75.75% and Ceftazidime 3.05% vs 43.19%. The acanthocytes ratio was as following(0 h vs 24 h): Penicillin 7.33% vs 86%, Cefepime 2.67% vs 52.67%, Cefoperazone 3.33% vs 67.67% and Ceftazidime 3.33% vs 90.67%. On the opposite, the clearance related CD markers, showed an obviously lower level after drugs treated(0 h vs 24 h): CD35: Penicillin 7.36% vs 11.87%, Cefepime 0.14% vs 28.51%, Cefoperazone 11.85% vs 21.55% and Ceftazidime 7.63% vs 8.73%; CD47: Penicillin 1.22% vs 9.13%, Cefepime 1.80% vs 0.86%, Cefoperazone 0.08% vs 6.85% and Ceftazidime 1.54% vs 5.50%; CD55: Penicillin 14.46% vs 44.31%, Cefepime 17.27% vs 38.41%, Cefoperazone 19.28% vs 33.28% and Ceftazidime 14.62% vs 34.13%; CD59: Penicillin 4.71% vs 20.56%, Cefepime 4.03% vs 7.60%, Cefoperazone 5.91% vs 22.38% and Ceftazidime 5.93% vs 30.89%. Drug-treated RBCs attached more to monocytes than untreated RBCs. 【Conclusion】 The β-lactam antibiotics could induce the changing of PS and the clearance of related CD markers on surface of RBCs. They also could lead acanthocytes and make the RBCs more susceptible to phagocytosis by monocytes. The β-lactam antibiotics could promote the RBCs aging and clearance, which might deteriorate the DIIHA.

Effects of different doses of dexmedetomidine on cardiac conduction system / 临床麻醉学杂志

Objective To investigate the effect of dexmedetomidine on cardiac conduction system at different loading doses.Methods Eighty male patients with orthopedic surgery,aged 20-65 years,falling into ASA physical atatus Ⅰ or Ⅰ,were randomly divided into groups D1,D2,D3 and C with 20 in each.Groups D1,D2 and D3 were infused with dexmedetomidine 0.3,0.5 and 0.8μg/ kg using a micro-pump for 10 min,group C infused 0.9％ NaCl solution in the same manner.MAP,HR,SpO2 were recorded and ECG was traced before injection (T1),5 min (T2),10 min (T3) after injection and 10 min after the end of pumping (T4).P wave duration,P-R interval,QRS time,and QTc value were calculated.Results There was no significant difference in SpO2,P wave duration,P R interval and QRS time among the four groups.There was no significant difference in MAP,HR and QTc value in group C and group D1.Compared with that in group C,MAP was significantly decreased,HR was significantly slowed down and QTc value was significantly shortened in group D2 and D3 from T2 to T4 (P ＜ 0.05).Conclusion Dexmedetomidine does not affect the cardiac conduction system.0.5 μg/kg and 0.8μg/kg dexmedetomidine can effectively shorten the QT interval.To avoid severe bradycardia,patients with low heart rate should use no more than 0.5 μg/kg dexmedetomidine.

Surgical and micro-invasive treatment of pancreatic duct stones: an analysis of 42 patients / 中华肝胆外科杂志

Pancreatic duct stone is a sequel of chronic pancreatitis and may be found in the main ducts,side branches or parenchyma.These stones obstruct the pancreatic ducts and produce ductal hypertension,which leads to pain,the cardinal feature of CP.Surgical operation has been the preferred treatment of pancreatic duct stones in many domestic and external pancreatic medical centers.Lithotomy by longitudinal pancreatic duct incision and Roux-en-Y anastomosis of pancreatic duct to jejunum is the main and effective surgical procedure,while micro-surgery was also rational for the treatment of pancreatic duct stones.However,further studies with a larger sample size and longer follow-up duration are needed to improve the surgical technique and verify our initial results.

Lung protective effect of Shenfu injection on the legs' ischemia-reperfusion injury / 临床麻醉学杂志

Objective To discuss the lung protective effect of Shenfu injection on the legs' ischemia-reperfusion injury.Methods Sixty patients (6 males, 24 females, ASA grade Ⅰ or Ⅱ)with unilateral lower limb surgery, were randomly divided into Shenfu injection group (group SF, n=32) and control group (group C, n=28).All patients were treated by combined spinal epidural anesthesia, with the same dose of local anesthetic (0.75% bupivacaine 1.5 ml and the same pressure (300mm Hg) of tourniquet.Patients in group SF were given intravenous infusion of Shenfu injection with the dose of 1 ml/kg (added in normal saline 100 ml) 30 min before apply tourniquet and 1 ml/kg (added in nomal saline 50 ml) 5 min before tourniquet deflation.Patients in group C were injected with equal dose of compound sodium lactate at the same time.Recorded the hemodynamic changes before apply tourniquet (T0) and 5 min (T1), 15 min (T2) and 30 min (T3) after tourniquet deflation.Took venous blood to determine concentrations of plasma thromboxane B2 (TXB2) and malondialdehyde (MDA).Results Compared with T0, MAP in the two groups were significcantly lower at T1-T3 (P＜0.05).MAP in group SF at T2 and T3 were higher than those in group C (P＜0.05).There was no significant difference in HR between the two groups.TXB2 and MDA in group SF at T2 and T3 were significantly lower than those in group C (P＜0.05 or P＜0.01).Conclusion Shenfu injection with antioxidation has a protective effect on lower limb ischemia-reperfusion lung injury induced by tourniquet.

Solid-phase screening of antibodies in blood and its effect on clinical blood transfusion / 中国输血杂志

Objective Detecting antibodies against RBCs,platelets,lymphocytes,and neutrophils by a solid-phase antibodies screening system.Methods mono-layer blood cell immobilized in the bottom of U-microplate respectively to prepare the solid-phase antibody screening system.Then detecting the antibodies exist or not in 2 150 random blood donor and 440transfusion patients' samples.Analyze the influence of antibodies against blood cells to the transfusion effect.Results 53RBC antibodies,22 platelet antibodies,13 lymphocyte antibodies,55 neutrophil antibodies were detected in random blood donor samples;31 RBC antibodies,38 platelet antibodies,24 lymphocyte antibodies,43 neutrophil antibodies and 115 mixed (two or more antibodies against RBC,platelets,lymphocytes or neutrophils exist at the same time) were detected in transfusion patients' samples after detection.233 suspected adverse reaction happened after transfusion,which antibodies were detected in 133 samples.Conclusion The antibodies against blood cells solid-phase screening system can applied in pre-and post-transfusion detection.The solid-phase screening method is more sensitivity than serologic tube test and flow cytometry.Existing of antibodies influence the transfusion effect.

Diagnostic value of double contrast-enhanced ultrasound in obstructive jaundice / 中华超声影像学杂志

Objective To evaluate the preoperative value of dual contrast-enhanced ultrasound (DCEUS) on obstructive jaundice.Methods Seventy-nine patients with obstructive jaundice were included.DCEUS (percutaneous transhepatic contrast-enhanced cholangio-ultrasonography combined with intravenous contrast-enhanced ultrasound) was performed preoperatively.The biliary obstruction plane,degree and cause were observed.After surgery,the diagnostic accuracy of DCEUS was compared with final pathologic results respectively.Results The overall accuracy of DCEUS in determining the flat,degree and cause of biliary obstruction was 98.7％,98.7％ and 93.7％,respectively.The DCEUS and golden standard were both almost perfect for assessing biliary obstruction with Kappa values of 0.979,0.837 and 0.975(P =0.000).The overall diagnostic accuracy of obstruction combined with obstruction plane,the extent and cause was 92.4％.Conclusions DCEUS could be considered a feasible,reliable,and exhaustive method for preoperative evaluation of obstructive jaundice.

The expression differences of miRNA in lung tissue and mast cells of asthmatic mice / 中华实用儿科临床杂志

Objective To study miRNA expression differences in ovalbumin(OVA)- induced murine asthma models of mice and mast cells stimulated by inflammatory cytokines stimulation,and to better understand asthma deve-lopment so as to provide potential target for its prevention and treatment. Methods OVA - induced murine asthma models were validated by detecting cells in bronchoalveolar lavage fluid(BALF)and histopathology. And miRNA ex-pression differences in the lung tissues between the model group and the normal control group were detected by real -time polymerose chain reaction PCR . After tumor necrosis factor - α(TNF - α),interleukin 12(IL - 12)stimulation, miRNA expression differences in murine mast cells P815 were detected. Results The number of total cells and eosino-phil cells both increased in BALF of the model group[(12. 8 ± 2. 2)x 107 / L vs(5. 6 ± 2. 5)x 107 / L,t = 4. 760,P ﹤0. 05;(6. 6 ± 1. 9)x 107 / L vs(0. 8 ± 0. 8)x 107 / L,t = 8. 068,P ﹤ 0. 05]. In addition,histopathology showed more inflammatory cell infiltration in the model group than that in the normal control group,indicating that the models were validated. The expression of miRNA - 155 was up - regulated approximately 5. 0 - fold in the lung tissues of the model group(P ﹤ 0. 05),while miRNA - 192 showed no differences compared with the controls. After TNF - α and IL - 12 stimulated P815 mast cells,miRNA - 192 expressions in P815 were expression in P815 was up - regulated approximate-ly 1. 9 - fold and 1. 7 - fold after TNF - α and IL - 12 stimulation,respectively(P ﹤ 0. 05). Conclusions It is conclu-ded that miRNAs are differentially expressed in the presence of OVA - induced murint asthma models and mast cells stimulated by inflammatory cytokines. These differentially expressed miRNAs may regulate the function of mast cells and involved in the pathogenesis of asthma.

Analysis of the factors associated with scientific research management at the First Affiliated Hospital of Zhejiang University Medical School / 中华医学科研管理杂志

In recent years,scientific research of our hospital has achieved remarkable progress with the implementation of discipline-centered ideology,personnel training strategy and adequate incentive policy.The paper briefly introduces the achievements in scientific research and the measures adopted,and analyzed the key factors associated with science research management.

Expression,Purification,Preparation of Polyclonal Antibody and Subcelluar Localization of the NS2 Protein of Periplaneta fuliginosa Densovirus / 微生物学通报

NS2 is a nonstructural protein of Periplaneta fuliginosa densovirus(PfDNV) with a molecular mass of 30 kD,whose function is not yet clearly understood. In order to study the expression,subcellular distribution and the function of NS2 protein,the coding region of NS2 was amplified from the hindgut tissue of cockroaches infected with PfDNV by RT-PCR and then the recombinant prokaryotic expression vector pET28a-NS2 was constructed. The recombinant plasmid was transformed into E. coli BL21(DE3) to express the 6?His fusion protein in the bacteria. After purification,the fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. The specificity of the anti-NS2 antibody was successfullyproved by western blotting on the eukaryotic expressed products of NS2 protein.Meanwhile,the full sequence of ns2 gene was also cloned into the eukaryotic expression vector pAC. The recombinant plasmid pAC-NS2 was then transfected into Schneider line 2(S2) cells to express NS2 protein in the insect cells. The subcellular localization of NS2 in the insect cells was then investigated by indirect immunofluorescence technique using the anti-NS2 polyclonal antiserum. The confocal laser scanning microscope observation showed that NS2 protein was located primarily in the cytoplasm with some punctate nuclear staining.

Surgical management of pancreatic duct stones / 中华普通外科杂志

Objective To evaluate surgical management of pancreatic duct stones.Methods From 1997 to 2007, 24 cases of pancreatic duct stones underwent surgical treatment, the clinical data were retrospectively analyzed. Results In this study, 17 cases underwent lithotomy by longitudinal pancreatic duct incision, Roux-en-Y anastomosis(side-to-side) of pancreatic duct to jejunum, extra drainageof the main pancreatic duct was done in two cases, hepaticojejunostomy in three cases, pancreaticcystojejunostomy in one case. One case suffered from postoperative bleeding at pancreatic ojejunostomy, one from stress ulcer, and both were cured by conservative treatment. Three cases underwent pancreaticeduodenectomy, anastomosis bleeding occurred in one patient, and was cured by conservative method. One case underwent duodenum-preserving resection of the head of the pancreas, 2 cases underwent distal pancreatectomy, one case underwent lithotomy by pancreatic duct incision and primary closure, no postoperative complications occurred among those patients. 21 cases were followed up, results were excellentin 17 patients. Conclusions Lithotomy by longitudinal pancreatic duct incision, Roux-en-Y anastomosisof pancreatic duct to jejunum is the main and effective surgical procedure, while duodenum preserving pancreatic head resection and lithotomy by pancreatic duct incision and primary closure are also rational for the treatment of pancreatic duct stones.

Different KEL gene mRNA transcripts in reticulocyte and non-reticulocyte cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the difference of the transcripts between reticulocyte and non-reticulocyte cells in human blood.</p><p><b>METHODS</b>Genomic DNA, reticulocyte RNA and total RNA of K-, K+ and Kell-null(K0) were extracted, then PCR, reverse transcription-PCR(RT-PCR) and nested PCR followed by sequencing or cloning-sequencing were used to analyze the KEL gene mRNA exons 1-19 and exons 2-8. Four kinds of monoclonal antibodies were labeled to detect the expression of Kell glycoprotein on red cells or leukocytes with flow cytometry.</p><p><b>RESULTS</b>In reticulocyte, only one normal KEL transcript faithful to the genomic structure was found in all tested samples except K0 which had 4 different transcripts. Sequence analysis of exons 2-8 of total RNA confirmed the alternative KEL transcripts existed in different samples, mostly caused by abnormal splicing, among them, skipping of exon 3 and a 16 bp insertion of intron 6 at the beginning of exon 7 were the most frequent. Although only one band was observed after amplifying the exons 1-19 from total RNA, the sequencing result showed it was a mixture of different sequences. There was strong expression of Kell glycoprotein on red cells except K0, but no or low expression on leucocytes by flow cytometry.</p><p><b>CONCLUSION</b>Alternative transcripts of KEL gene exist in different cells, which would be responsible for different Kell glycoprotein expression patterns on different cells. This study suggested that reticulocyte RNA was more suitable than total RNA for molecular study of KEL gene transcription.</p>

Candida in Nosocomial Infection:A Clinical Study and Drug Sensitivity Analysis / 中华医院感染学杂志

OBJECTIVE To investigate the relevant factors and drug sensitivity of Candida in nosocomial infection from a class A comprehensive hospital in Hangzhou in order to prevent and control nosocomial infection.METHODS We collected and identified clinic samples from inpatients during from Jan 2004 to Dec 2006 and drug resistance test was performed for Candida strains.The CHROMagar candida color medium and the Analytic Products Inc(API)20CAUX identification system were used to isolate and identify Candida strains from inpatients.The Etest was used to study the antifungal sensitivity.The data were analyzed by WHONET-5 software and were determined by the Clinical and Laboratory Standard Institute(CLSI)method.RESULTS Totally 264 strains of Candida were detected in the three years.The detection ratio in each year was 78 strains(29.5%),87 strains(32.9%),and 99 strains(37.5%),respectively.The C.albicans was the most commonly isolated species(65.5%)and then was C.tropicalis(15.1%)in all Candida species.The detection ratio was the highest in sputum samples(108 strains,40.9%)and the patients with respiratory tract infection(58 strains,22%).The results of antifungal drug sensitivity test showed that Candida were the most susceptive to amphotericin B,followed by fluconazole.The results of change showed that Candida remained stable susceptive to amphotericin B,but susceptive ratio to fluconazole was descent in the three years.CONCLUSIONS Candida in nosocomial infection are an increasing tendency and C.albicans is the commonest.Sensitivity of Candida to various antifungal drugs is different and there is a developing change in susceptive ratio to antifungal drugs.Strengthening supervising relevant factors and improving the immunity of organism are the main measures to prevent Candida in nosocomial infection.Antifungal drug should be used reasonably based on drug sensitive test.

Expression of recombinant N-glycosylation interleukin 24 and study of inducing tumor cells apoptosis in vitro / 中国药理学通报

Aim To obtain recombinant human IL-24 secretorily expressed in Pichia pastoris,and study the activity of inducing tumor cells apoptosis of this N-glycosylation protein. Methods By the recombinant plasmid ?/pUC18,the confirmed IL-24 gene was inserted between the sites of BamH Ⅰ and EcoR Ⅰ of expression plasmid pPIC9K. The recombinant plasmid IL-24/pPIC9K was transformed into P. pastoris strain GS115. Yeast transformants were induced for expression of recombinant human IL-24 with methanol. The desired protein was identified with Tricine-SDS-PAGE and Western blot.Amount of IL-24 was assayed with ELISA and the glycosylation was analyzed by PNGase F.The activity of inducing tumor cells apoptosis was confirmed by MTT assay and Hoechst assay in vitro.Results Recombinant expression plasmid IL-24/pPIC9K was successfully constructed. 5 transformants were screened with G418 and PCR. Induced with methanol,the expression level of IL-24 was (81.31?14.46) mg?L-1 at flask fermentation,and 70 % IL-24 generated N-glycosylation.Recombinant IL-24 induced apoptosis in MCF-7 cells,but not in NHLF.Conclusion The secretorily expression of the N-glycosylation IL-24 protein in P. pastoris and the study of inducing tumor cells apoptosis lay the foundation for the further study of molecular mechanism of IL-24 on anti-tumors and the potential application.

Primary common bile duct closure after choledochotomy / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the rationality and feasibility of primary closure of the common bile duct after choledochotomy for common bile duct calculi.</p><p><b>METHODS</b>From January 1990 to June 2001, 386 patients with the evidence of stones in the common bile duct underwent choledochotomy. Among them, 215 received primary closure of the common bile duct (group A) and 171 T-tube drainage (group B). The patients with emergency operations were excluded. Intraoperative choledochoscopy or cholangiography was routinely performed to rule out the possibility of retained stones. The duct was meticulously stitched using 0/3 to 0/5 absorbent sutures for primary closure. A T-tube was placed in the subhepatic space in the patients of both groups.</p><p><b>RESULTS</b>Postoperative bile leakage was seen in 9 patients of group A and in 5 of group B, respectively (P > 0.05), and no reoperations were necessary. After surgery, the average time and volume of transfusion was 4.9 days and 9.1 liters in group A, versus 7.3 days and 12.8 liters in group B (P < 0.01). The patients in group B had a longer postoperative hospital stay than the those in group A (average 17.6:10.0 days, P < 0.01). T-tube removal resulted in bile peritonitis in 5 patients at day 16, 17, 19, 21 and 22 after surgery in group B, and 3 patients required repeated surgery.</p><p><b>CONCLUSIONS</b>Primary closure of the common bile duct after choledochotomy is safe, effective, and inexpensive in selected patients with common bile duct calculi, and should be regarded as an alternative procedure.</p>

Studies on antioxidation of extract from Ruppia rostellata koch / 中国海洋药物

Extract from Ruppia rostellata Koch was divided into three parts: petroleum ether part, CH2Cl2 and water-soluble part. Each part of them was studied upon it's antioxidation. The results indicated that the active compounds were only in the water-soluble part. In soybean oil, the antioxidation of water-soluble part was stronger than that of BHA and V. E. , In lard, the antioxidation of water-solubile part was a little weaker than that of BHA and V.E.