Objective To construct plvx-cyclooxygenase-2(COX-2)-DsRed and establish breast cancer cell line MCF7 which overexpressed COX-2, to explore the effect of COX-2 on breast cancer cell.Methods The full-length COX-2 PCR product was obtained by total COX-2 PCR primers and COX-2 cDNA vector.After the PCR product and lentiviral vector plvx-DsRed-Monomer-N1 were cut simultaneously by restriction enzyme BamH1 and Xholl, they were connected and sequenced, to get lentiviral vector plvx-COX-2-DsRed.After selected by puromycin, overexpressed COX-2 breast cancer cell line MCF7-plvx-COX-2-DsRed was obtained.The stable cell line was verified by real time PCR and Western blot.The differences of proliferation ability between stable cell line and normal one were compared by colony formation test and Western blot.Results The lentiviral vector plvx-COX-2-DsRed and stable cell line MCF7-plvx-COX-2-DsRed after selecting were obtained.COX-2 expression level of the stable cell line was 75.29 times as high as that of MCF7, and 64.91 times as high as that of cell line MCF7-plvx-DsRed-Monomer-N1 by PCR assay (P ＜ 0.05), which was consistent with the results of Western blot and microscope photo.MTT results showed that cell line MCF7-plvx-COX-2-DsRed had grown faster than cell line MCF7 and MCF7-plvx-DsRed-Monomer-N1 from the 2nd day (P ＜ 0.05), which was accordant with colony formation assay.MCF7-plvx-COX-2-DsRed cell line had higher c-myc expression and lower β-catenin expression than MCF7 cell and cell line MCF7-plvx-DsRed-Monomer-N1 detected by Western blot relative quantification (P ＜ 0.05).Conclusion The plvx-COX-2-DsRed lentiviral vector and cell line MCF7-plvx-COX-2-DsRed are successfully constructed.COX-2 can increase proliferation of MCF7 cells through up-regulating the expression of c-myc.

Objective To observe the curative effect in patients with systemic lupus erythematosus(SLE) complicated by moderate or severe thrombocytopenia with a blood platelet count(BPC) of under 50 × 109/L and analyze its related factors. Methods We retrospectively analyzed the clinical data on 109 SLE patients with mod-erate or severe thrombocytopenia. Results Of the 109 patients,82(75.2%)had complete response(CR),15 (13.8%)had partial response(PR),and 12(11.0%)had no response(NR),respectively. As compared with the CR+PR group,the NR group had a higher incidence rate of decreased bone marrow megakaryocyte(P < 0.05). However,there were no significant differences between the two groups in SLEDAI scores,rates of positive PAIg and ACA,and levels of C3 and C4(P>0.05 for all comparisons). The total effectiveness rate did not differ signifi-cantly between MP pulse therapy and high-dose corticosteroid therapy. Conclusions A decrease in bone marrow megakaryocytes can be an adverse factorfor affecting the efficacy in patients with SLE complicated by moderate or severe thrombocytopenia.

Objective:To investigate the expression of human epidermal growth factor receptor 2 (HER2) in pancreatic ductal adenocarcinoma(PDAC) and its relationship with the prognosis of patients with PDAC.Methods:From January 2001 to December 2012, 109 paraffin embedded PDAC tissue samples and 27 normal pancreatic tissue samples were collected from the Department of Pathology, Huadong Hospital Affiliated to Fudan University. The expression of HER2 protein in pancreatic tissue was detected by immunohistochemical Envision two-step method. HER2 expression was evaluated according to Hercept test, and its relationship with clinicopathological features and survival time was analyzed.Results:The expression of HER2 protein was negative (-) in 29.4% of PDAC tissues, weakly positive (+ ) in 35.8%, positive (+ + ) in 25.7% and strongly positive (+ + + ) in 9.2%, respectively, and the overexpression rate (+ + , + + + ) was 34.9%; the negative (-) and weakly positive (+ ) expression of HER2 protein in normal pancreatic tissues accounted for 88.9% and 11.1% respectively. There was no expression with positive (+ + ) or strongly positive (+ + + ), therefore, the overexpression rate was 0. The overexpression rate of HER2 protein in PDAC and normal pancreatic tissues was significantly different ( P=0.000). The expression of HER2 protein was significantly correlated with age, and the expression of HER2 protein in patients with PDAC over 65 years old was significantly higher than that in patients with PDAC under 65 years old ( P=0.043), but not with gender, tumor location, tumor grade, T stage, N stage and nerve invasion (all P>0.05). Univariate Cox proportional hazards analysis showed that HER2 expression was associated with postoperative survival time of patients with PDAC ( P=0.032). Multivariate Cox proportional hazards analysis showed that HER2 expression was an independent prognostic factor for survival of patients with PDAC ( P=0.040). The median survival period of patients with HER2 expression + + + was significantly longer than that of patients with HER2 expression -~+ + (128.4 months vs 21.5 months), and the difference was statistically significant ( P=0.038). Conclusions:The overexpression of HER2 in PDAC tissue was related to the age of patients. The survival time of patients with HER2 strongly positive PDAC was significantly longer. HER2 can be considered as an index to evaluate the biological behavior and prognosis of PDAC.

Objective Objective To explore the reasons of lung hypervolemia after liver transplantation and the corresponding treatment strategies.Method 291 patients received liver transplantation,in which 35 cases underwent pulmonary edema at early stage (pulmonary hypervolemia group),and the rest without pulmonary hypervolemia served as control group.Average central venous pressure (CVP) was recorded pre-,intra-and post-operatively.Total intake,total discharge and fluid balance were also recorded intraoperatively and 3 days post-operatively.Result In pulmonary hypervolemia group,CVP was (12.33 ± 5.08),(14.33 ± 3.03) and (16.50 ± 4.57) mmHg pre-,intra-and post-operatively,significantly higher than that in control group (P＜0.05 for all).Total intake,total discharge and fluid balance in pulmonary hypervolemia group were significantly higher than those in control group intraoperatively and 3 days post-operatively (P＜0.05 for all).After diuretic therapy and hemodialysis,30 cases in pulmonary hypervolemia group recurred,and 5 cases died of infection.Conclusion Pulmonary hypervolemia at early stage after liver transplantation is related to fluid balance.The reasonable control of total intake,total discharge and fluid balance is necessary.

Objective:To make an assessment on the genotoxicity caused by black carbon ( BC ) and ozonized black carbon (O3-BC).Methods: In this study, 74 healthy male ICR mice [weighed (28 ± 1.5) g] were randomly divided into 7 groups, including one phosphate buffer solution ( PBS) control group and six particles exposed groups by intratracheal instillation with either BC or O 3-BC at the doses of 50, 100, 200 μg/mouse, respectively.There were 12 mice in the groups of 200μg/mouse and 10 mice in others.The mice were sacrificed 24 h after four intratrachealinstillations .The activities of catalase ( CAT) in serum and the levels of malondialdehyde ( MDA) in lung tissue homogenate were measured . As the DNA damage mark , 8-hydroxyguanosine ( 8-OHdG ) in urine and serum were quantified with ELISA method.Micronucleus test was used for potential genotoxicity of BC and O 3-BC.Hematoxylin and eosin staining was used to stain lung paraffin section .Results:The mice were in good condition during instillation , and the liver coefficient of the test groups was significantly lower than that of the control group (P<0.05).The activities of CAT in serum significantly increased in the 100 μg/mouse and 200μg/mouse groups after being exposed to these two kinds of particles .The micronucleus rate in allthe BC and O3-BC exposed groups increased ( P <0.05), but there was no statistically significant difference among the groups in the levels of 8-OHdG in serum and urine and MDA in lung tissue homogenate .In-flammatory response was found in the lung tissue under the microscope after exposure to BC and O 3-BC. Conclusion:Intratracheal instillation of BC and O 3-BC induced increasing of oxidative stress and genetic damage in mice .But there was no significant difference between these two particles in toxicity .Whether the genotoxicity of O 3-BC is higher than that of BC or not is uncertain .Further research is needed .

OBJECTIVETo investigate the relationship between level of Th17/CD4+T cell ratio in peripheral blood and postoperative complications in patients with hepatitis B virus (HBV)-related cirrhosis after orthotopic liver transplantation (OLT).

METHODSFifty-one patients with HBV-related cirrhosis who received OLT were enrolled in this study. Flow cytometry analysis was performed to measure the proportion of Th17 cells to CD4+T cells at the following time points:pre-OLT, and post-OLT days 7, 14 and 21. The relevant hepatic biochemistry indexes, serum concentration of FK506 and level of procalcitonin (PCT) were detected for all patients after OLT.

RESULTSThe transplant recipients were divided into four groups according to the postoperative complication, which included acute rejection (AR, n=12), postoperative infection (POI, n=10), transient intrahepatic cholestasis (TIHC, n=12) and no complications (n=17). The Th17/CD4+T cell frequencies were notably higher in the AR patients after OLT (vs. before OLT, P less than 0.01) and this increase was positively correlated with rejection activity index (RAI; r=0.759, P=0.004). Up to post-OLT day 14, the frequencies of Th1 7/CD4+T cells in the POI group were similar to those of the AR group but they decreased to near-baseline level at post-OLT day 21.Furthermore, the percentage of Th17/CD4+T cells in the POI group was positively correlated with PCT (r=0.768, P=0.010). The frequencies of Th17/CD4+T cells in the TIHC and no complications groups showed a slowly decreasing trend after OLT and became markedly lower than the before OLT levels (P<0.01). The concentration of FK506 in the AR group was significantly lower than that in the other groups at post-OLT day 14 (P=0.000).

CONCLUSIONSTh17/CD4+T cell level in peripheral blood might be a useful marker for risk assessment and monitoring of OLT postoperative complications, such as acute rejection and postoperative infection, in the early stage, and might help to improve patient prognosis by allowing for timely application of anti-rejection and antibacterial agents.

Calcitonin ; Calcitonin Gene-Related Peptide ; Hepatitis B ; Hepatitis B virus ; Humans ; Liver Cirrhosis ; Liver Transplantation ; Postoperative Complications ; Protein Precursors ; Th17 Cells

Objective To characterize the whole-sequence of plasmid pB557-NDM isolate from Enterbacter cloacae and elaborate its antibiotic-resistant mechanisms .Methods Antibiotic resistance genes were determined by PCR , followed by amplicon sequencing .The activity of class A/B/D carbapenemases was determined by modified Carba NP test .Conjugation experiments were performed to verify the transferability of plasmid pB 557-NDM.The minimum inhibitory concentration (MIC) values of bacterial strains were tested using VITEK 2.The genetic structure, mobile elements and antibiotic-resistant mechanisms of transferable plasmid pB 557-NDM were determined by a whole genome sequencing method .Results The modified CarbaNP test showed that B557 and B557-EC600 had class B carbapenemase activity , and that the blaNDM was carried by plasmid pB557-NDM.This plasmid could be transferred into E.coli through conjugation experiments and therefore could confer corresponding antibiotic resistances to the transconjugant B 557-EC600.Plasmid pB557-NDM was an IncA/C2 plasmid, whose total length was 141.65 kb, and the GenBank accession number was KX786648.It had two inserted regions.One was the blaCMY-6 region where the blaCMY gene was carried by a transposition unit IS Ecp1-blaCMY , the other was the blaNDM-1 region which consisted of a ΔTn1696-In46-rmtC-ISKpn14-ΔTn125 complex structure.Conclusion The production of plasmid pB557-NDM in strain B557 contributes most to its high resistance to many antibiotics .The blaNDM-1 gene is carried in a trancated transposition ΔTn125.

Objective To investigate the risk factors of developing cardiac arrest in postreperfusion syndrome ( PRS) of orthotopic liver transplantation ( OLT ).Methods Clinical data of 192 patients who underwent OLT in the Third Hospital of Hebei Medical University from 2003 to 2013 were retrospectively analyzed.Among them, 38 patients developed PRS.According to the occurrence of cardiac arrest or not , the patients were divided into 2 groups, including 7 cases in cardiac arrest group (5 males and 2 females) and 31 cases in non-cardiac arrest group (23 males and 8 females).The probable influence factors of cardiac arrest were selected, including gender, age, preoperative cardiac indexes ( electrocardiogram or color doppler ultrasound of heart ) , preoperative albumin , borderline pH value during opening circulation , borderline temperature, borderline blood potassium level , blood calcium level after opening , degree of donor fatty liver , time of occluding inferior vena cava , and cold ischemia time of donor liver.Comparison of data between two groups was used t-test or Fisher exact probability test.Rick factors with significance differences between two groups were analyzed by unconditional Logistic regression analysis.Results Probable risk factors of developing cardiac arrest in PRS included borderline pH value <7.35 during opening circulation , borderline temperature<36 ℃during opening circulation , borderline blood potassium level >4 mmol/L during opening circulation and moderate fatty liver ( all in P<0.05 ).Results of unconditional Logistic regression analysis showed that moderate fatty liver was an independent risk factor of cardiac arrest in PRS.Conclusions Moderate fatty liver is an independent risk factor of cardiac arrest in PRS.The rational application of liver donors and paying more attention to perioperative treatment of recipients have important significance for reducing the incidence of cardiac arrest.

Objective:To analyze the clinical characteristics of novel coronavirus (COVID-19) infection in liver transplant recipients.Methods:Clinical data of 130 liver transplant recipients with COVID-19 infection followed in Hebei Medical University Third Hospital from November 2022 to January 2023 were retrospectively collected, including 103 males and 27 females, aged (53.6±11.4) years. The severity of COVID-19 infection, its clinical symptoms, and negative conversion time for each recipient were analyzed and compared.Results:For the disease severity, 121 (93.1%) were mild, 5 (3.8%) were moderate, 3 (2.3%) were severe, and 1 (0.8%) was critically severe. The most common symptoms in the 126 patients with mild to moderate COVID-19 infection were successively fever, fatigue, cough and myalgia, with a negative conversion time of (10.1±4.5) (3.0-29.0)d. In those who underwent transplantation less than 12 months ( n=28), those who were taking mycophenolate mofetil ( n=53) and those who were taking methylprednisolone ( n=10), the negative conversion time was (11.6±5.1) d, (11.4±5.4) d, and (13.4±6.4) d, respectively, longer compared to those who underwent transplantation more than 12 months (9.7±4.2 d, n=98), and who were not taking mycophenolate mofetil (9.2±3.4 d, n=73) or methylprednisolone (9.8±4.2 d, n=116, all P<0.05). The negative conversion times were longer in recipients with symptoms (eg. fatigue and cough) than those in asymptomatic recipients (11.0±5.1 d vs. 9.0±3.3 d, t=2.64, P=0.009, and 11.4±5.2 d vs. 9.0±3.4 d, t=2.92, P=0.004). Conclusion:The COVID-19 infection in liver transplant recipients was mainly mild and moderate. The most common symptoms are fever, fatigue, cough and myalgia. The short time (less than 12 months) after liver transplantation, oral mycophenolate mofetil and methylprednisolone, with symptoms (fatigue and cough) could be associated with a prolonged negative conversion time.

Objective:To investigate the causes of immune failure in the population with high vaccination rate of measles and rubella vaccine in Beijing by detecting the IgG antibody affinity in suspected cases of measles and rubella.Methods:Serum samples of 276 suspected cases of measles and rubella were tested for IgM and IgG antibodies by enzyme-linked immunosorbent assay (ELISA). The affinity of IgG antibody was detected, and the relative affinity index was calculated.Results:Among the 276 suspected cases, 104 were measles and 108 were rubella. Six measles cases had vaccination history and were caused by primary immunization failure ( n=3) and secondary immunization failure ( n=3). Twelve rubella cases had vaccination history and were due to primary immunization failure ( n=4) and secondary immunization failure ( n=8). Specific high-affinity antibodies were detected in nine measles cases and seven rubella cases without vaccination history, which indicated that these cases were reinfected. In the cases without measles or rubella, other pathogenic infections including mixed infections were detected, which were mainly caused by EB virus. Conclusions:Both primary and secondary immunization failure occurred in the population with immunization history. Reinfection was found in the patients who had not received vaccination against measles or rubella. Other pathogenic infections were existed among the cases without measles or rubella. Thus, misdiagnosis was responsible for the increased proportion of measles and rubella patients with immunization history in suspected cases in recent years. Full-course vaccination was conducive to produce high-affinity antibodies against measles and rubella. A supplementary vaccination campaign should be launched to consolidate the immune barrier against measles and rubella in key population or high-risk population, aiming to block the circulation of measles virus and achieve the goal of eliminating measles.