A two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0,50 mmol/L Tris-HCl and satisfactory results were obtained using a 0~0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.

Objective To investigate the evolution of surgical treatment and it's influence on prognosis in reptured liver cystic echinococcosis.Method Clinical data of 110 surgically treated cases of reptured liver cystic echinococcosis from January 1990 to December 2008 were retrospectively analysed,and these cases were divided into three groups by different surgical approach;Group A(22 cases):removal of internal capsule,with formaldehyde or hydrogen peroxide regional anthelminthic,with residual cavity side edge of lock stitching or greater omentum filling or drainage after residual cavity closure;Group B (65 cases):Removal of intemal capsule,with hypertonic saline regional anthelminthic,with external capsule extrahepatic partial resection,with residual cavity bile leakage sutured,and with residual cavity open drainage,Group C(23 cases):the total or subtotal external capsule stripping operation,with hypertonic saline regional anthelminthic and drainage.Result The rate of overall postoperative complications of residual cavity was 40.9%、16.9%、0.0%(P<0.05)respectively and the recurrence rate was 18.2%、4.6%、0.0%(P<0.05)respectively;drainage time needed was the longest in group A,and shortest in group C,whereas the operation time was the longest in group C and it costed higher blood loss.Intraabdominal implantation of the hydatid significantly decreased with hypertonic saline treatment.Conclusion Total or near total removal of the external capsule of the hydatic cyst and hypertonic saline treatment with postoperative external drainage is the therapy of choice for reptured liver cystic echinococcosis.

Objective To establish a mouse model of lymph node metastasis of breast cancer cells by luciferase imaging assay,to monitor early process of lymph node metastasis,and to evaluate the effect of X-ray radiation therapy on tumor.Methods The mouse mammary cancer cell line 4T1-Luc expressing luciferase was inoculated subcutaneously into the paw pad of nude mice to establish a model of subcutaneous lymph node metastasis.The lymph node metastasis in nude mice was continuously observed by in vivo fluorescence imaging system,and the nude mice with early lymph node metastasis of breast cancer cells were divided into control group and treatment group randomly.The radiotherapy effect was observed by in vivo fluorescence imaging system and evaluated by the pathological changes of HE staining of tumor tissue.Results A mouse lymph node metastasis model of breast cancer cells was successfully established,and the volume of primary tumor in paw correlated with the fluorescence photon number positively (r =0.958,P ＜ 0.001).On the twenty-fourth day after inoculation,the fluorescence photon number in pad tumor and popliteal fossa tumor of treatment group were significantly decreased in comparison with the control group (t =32.58,P ＜ 0.05),and the inhibition ratio of radiotherapy on tumor growth approached to 85 ％.HE staining showed that the apoptosis and necrosis in irradiated tumor was obviously higher than that in control group.Conclusions Bioluminescence imaging technique can be used to evaluate the effect of X-ray on breast cancer suppression and lymph node metastasis in mice.

OBJECTIVETo detect the expression of TRAIL receptors in fibroblasts of hypertrophic scar in the proliferative stage and explore its significance.

METHODS30 samples of hypertrophic scar were taken from 30 burn cases in the proliferative stage. 30 samples of normal skin were taken as the control. The expressions of TRAIL receptors in the fibroblasts of hypertrophic burn scar and the normal skin were assayed by semiquantitative RT-PCR and flowcytometry.

RESULTSThe expression level of DR5 in the fibroblasts of hypertrophic burn scar is much lower than the control (P < 0.05); the expression level of DcR1 in the fibroblasts of hypertrophic burn scar is much higher than the control (P < 0.05).

CONCLUSIONSThe down-regulated DR5 expression and elevated DcR1 expressions in the fibroblasts of hypertrophic burn scar may attribute to the apoptosis change induced by TRAIL and explain the apoptosis differences between the fibroblasts of hypertrophic scar and normal skin to a certain extent.

Adolescent ; Adult ; Apoptosis ; Burns ; complications ; metabolism ; pathology ; Child ; Child, Preschool ; Cicatrix, Hypertrophic ; etiology ; metabolism ; pathology ; Female ; Fibroblasts ; metabolism ; Humans ; Male ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Young Adult

OBJECTIVETo identify factors associated with YMDD mutation in patients with chronic hepatitis B before and after lamivudine treatment in Zunyi region.

METHODS53 patients with chronic hepatitis B were enrolled in this study, HBV DNA,HBV markers, ALT, AST, TBil, albumin in the serum were examined at 0, 3, 6, 12, 18 and 24 months after lamivudine treatment. HBV genotype and YMDD mutation were determined by sequencing before lamivudine treatment. YMDD mutation was checked again if serum HBV DNA rebound to more than 1 x 10(4) copies/ml after the initial decrease.

RESULTSHBV genotype in Zunyi region is constitute of B, C and B+C genotype. YMDD mutation occurred in 18 cases after lamivudine treatment, the rate of YMDD mutation was 15.1%, and 34.0% after 1 year and 2 years treatment. There are four types of mutation: rtL180M/M204V, rtL180M/M204I, rtM204I, rtL180M. rtM204V mutation in C gene was always accompanied by rtL180M mutation (100%). The rate of rtL180M/M204V mutation in genotype C group was significantly higher than that in genotype B group (77.8% to 25.0%), the same was true for the rtL180M/ M204I mutation (22.2% to 12.5%). There was no point mutation in genotype C group. The point mutation of rtM204I and rtL180M appeared only in genotype B group. Gender, nation, family history of hepatitis B and HBeAg were not associated with YMDD mutation (P more than 0.05), while the mutation rate was associated with the disease course and severity of disease. YMDD mutation did not occur in patients with low HBV DNA level (less than 10(5) copies/ml).

CONCLUSIONYMDD mutation after lamivudine therapy is associated with HBV genotype and P gene mutation type, and prolonged treatment increases the the mutation rate. In order to reduce the incidence of YMDD mutation, patients with shorter disease course, lower HBV DNA level, more serious liver damage should be treated with lamivudine.

Adult ; Alanine Transaminase ; blood ; Antiviral Agents ; pharmacology ; therapeutic use ; Aspartate Aminotransferases ; blood ; China ; epidemiology ; DNA Mutational Analysis ; DNA Primers ; DNA, Viral ; blood ; genetics ; Drug Resistance, Viral ; Female ; Genotype ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Mutation ; Polymerase Chain Reaction

OBJECTIVETo investigate the effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its receptor on apoptosis in thymus during early post-burn stage in rat with severe burns.

METHODSFifty Wistar rats were randomly divided into sham scald group (SS, n = 10) and burn group (n = 40). The apoptosis in thymus in rats was detected with annexin V/FITC-PI double staining at 4, 12, 24, 48 post-burn hours (PBH). The expression of TRAIL death receptor DR5, DR4 and its decoy receptor DcR1, DcR2 in thymus were detected by RT-PCR and Western blot at above time-points.

RESULTSCompared with that in SS group (6.7 +/- 0.8)%, the apoptosis in the thymus in burn group started to increase at 4 PBH [(17.1 +/- 0.4)%], peaked at 12 PBH [(25.2 +/- 1.1)%], and it was still evidently higher than that in SS group at 48 PBH (P < 0.05). There was no obvious difference in the apoptosis rate in rats in burn group among all the time-points. The expression of DR5 in burn group at each time-points was significantly higher than those in SS group, while that of DcR2 shown an opposite tendency (P < 0.05). The expression of DR4, DcR1 was similar in both groups.

CONCLUSIONThe marked increase in apoptosis rate in rat thymus at early post-burn stage, and the significant change in the expression of DR5 and DcR2 show that TRAIL pathway may participate in apoptosis.

Animals ; Apoptosis ; Burns ; metabolism ; Disease Models, Animal ; Female ; Male ; Rats ; Rats, Wistar ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Thymus Gland ; metabolism

Objective To analyse the relationship between Fourier transform infrared （FTIR） spectrum ofrat's spleen tissue and postmortem interval （PMI） for PMI estimation using FTIR spectroscopy combinedwith data mining method. Methods Rats were sacrificed by cervical dislocation, and the cadavers were placed at 20 ℃. The FTIR spectrum data of rats' spleen tissues were taken and measured at different time points. After pretreatment, the data was analysed by data mining method. Results The absorption peak intensity of rat's spleen tissue spectrum changed with the PMI, while the absorption peak position was unchanged. The results of principal component analysis （PCA） showed that the cumulative contribution rate of the first three principal components was 96%. There was an obvious clustering tendency for the spectrum sample at each time point. The methods of partial least squares discriminant analysis （PLS- DA） and support vector machine classification （SVMC） effectively divided the spectrum samples with different PMI into four categories （0-24 h, 48-72 h, 96-120 h and 144-168 h）. The determination coefficient （R2） of the PMI estimation model established by PLS regression analysis was 0.96, and the root mean square error of calibration （RMSEC） and root mean square error of cross validation （RMSECV） were 9.90 h and 11.39 h respectively. In prediction set, the R2 was 0.97, and the root mean square error of prediction （RMSEP） was 10.49 h. Conclusion The FTIR spectrum of the rat's spleen tissue can be effectively analyzed qualitatively and quantitatively by the combination of FTIR spectroscopy and data mining method, and the classification and PLS regression models can be established for PMI estimation.

OBJECTIVE: To find out the prevalence of lower urinary tract symptoms (LUTS) and analyze the characteristics of the urodynamics diagnosis in female outpatients. To study the natural history of detrusor underactive (DU) by the followed up. METHODS: A retrospective study of the female LUTS outpatients in Peking University People's Hospital from Jan. 2005 to Dec. 2015 was performed. The basic information and the urodynamic results of a total of 656 female outpatients were collected. The distribution of storage symptoms, voiding symptoms, complications and urodynamic diagnosis were analyzed. The symptoms and urodynamic results were compared among the groups, which were divided according to age, urodynamic diagnosis and diabetes mellitus. A follow-up of 163 female DU outpatients was performed, including the treatments and the American Urological Association symptoms scores (AUAss). RESULTS: Frequency (25.03%) is the most common symptom in female outpatients, followed by stress urinary incontinence (20.04%), urgency (19.97%), and difficulty of voiding (17.32%). Stress urinary incontinence (SUI) accounted for the first (36.04%) of the whole outpatients, followed by the DU (24.08%), and bladder outlet obstruction (17.58%). The patients aged 51-60 years occupied the peak of almost all the diagnosis. There was a higher proportion of the young female patients than that of the middle and old patients diagnosed with no abnormal after the urodynamic study. The first, strong, urge and maximum bladder capacity were significantly larger in DU patients with diabetes than without diabetes. Follow-up results of the DU patients showed there was no significantly difference of the AUAss scores in both the two groups before and after the follow-up, but the quality of life decreased significantly. CONCLUSION Female LUTS outpatients showed a main complaint of storage symptoms. SUI ranked the first in female patients with LUTS. With the increase of age, bladder sensation and detrusor function decrease. In elderly patients, DU became the first ranked disease instead of SUI. Diabetes can affect the sensory function of bladder in patients with DU, and then increase the difficulty of voiding. The patients with DU, absent from treatment, experienced a lower quality of life.
Aged ; Female ; Follow-Up Studies ; Humans ; Lower Urinary Tract Symptoms ; Middle Aged ; Outpatients ; Quality of Life ; Retrospective Studies ; Urodynamics

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.
Animals ; Apoptosis ; Autophagy ; Hypoxia ; Mice ; Myocytes, Cardiac ; Signal Transduction

BACKGROUNDEarly goal-directed therapy (EGDT) has become an important therapeutic management in early salvage stage of septic shock. However, splenic organs possibly remained hypoperfused and hypoxic despite fluid resuscitation. This study aimed to evaluate the effect of EGDT on hepatic perfusion in septic shock patients.

METHODSA prospective observational study was carried out in early septic shock patients who were admitted to Intensive Care Unit within 24 h after onset and who met all four elements of the EGDT criteria after treatment with the standard EGDT procedure within 6 h between December 1, 2012 and November 30, 2013. The hemodynamic data were recorded, and oxygen metabolism and hepatic functions were monitored. An indocyanine green clearance test was applied to detect the hepatic perfusion. The patients' characteristics were compared before treatment (T0), immediately after EGDT (T1), and 24 h after EGDT (T2). This study is registered at ClinicalTrials.org, NCT02060773.

RESULTSTwenty-one patients were included in the study; however, the hepatic perfusion data were not included in the analysis for two patients; therefore, 19 patients were eligible for the study. Hemodynamics data, as monitored by pulse-indicator continuous cardiac output, were obtained from 16 patients. There were no significant differences in indocyanine green plasma disappearance rate (ICG-PDR) and 15-min retention rate (R15) at T0 (11.9 ± 5.0%/min and 20.0 ± 13.2%), T1 (11.4 ± 5.1%/min and 23.6 ± 14.9%), and T2 (11.0 ± 4.5%/min and 23.7 ± 15.3%) (all P > 0.05). Both of the alterations of ICG-PDR and R15 showed no differences at T0, T1, and T2 in the patients of different subgroups that achieved different resuscitation goal numbers when elected (P > 0.05).

CONCLUSIONThere were no hepatic perfusion improvements after EGDT in the early phase of patients with septic shock.

TRIAL REGISTRATIONClinicaltrials.gov NCT02060773 (https://clinicaltrials.gov/ct2/show/NCT02060773).

Aged ; Aged, 80 and over ; Cardiac Output ; physiology ; Disease Management ; Female ; Fluid Therapy ; Hemodynamics ; physiology ; Humans ; Intensive Care Units ; statistics & numerical data ; Male ; Middle Aged ; Prospective Studies ; Shock, Septic ; therapy