Objective:To investigate whether the overexpression of Numb gene can effectively intervene the progression of cholestatic liver fibrosis (CLF) in adult liver.Methods:Twenty-four SD rats were randomly divided into sham operation (Sham, n=6), common bile duct ligation (BDL, n=6), empty vector plasmid (Numb-EV, n=6) and numb gene overexpression group (Numb-OE, n=6). The CLF model was prepared by common bile duct ligation. Simultaneously, the model was established, and the adeno-associated virus (AAV) carrying the cloned numb gene was injected into the rats' spleens. Samples were collected at the end of four weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb), serum total bilirubin (TBil), serum total bile acid (TBA), liver histopathology, liver tissue hydroxyproline (Hyp) content, and alpha smooth muscle actin (α-SMA), cytokeratin (CK) 7, and CK19 expression conditions were determined in liver tissue. An analysis of variance was used to compare the means of multiple groups. Results:Compared with the Sham group, the Numb mRNA level in the rat liver tissue was significantly decreased in the BDL group (0.872±0.237 vs. 0.452±0.147, P=0.003). Compared with the Numb-EV group, the Numb mRNA level in the liver tissue was significantly increased in the Numb-OE group (0.487±0.122 vs. 1.094±0.345, P＜0.01). Compared with the Sham group, the Hyp content (μg/L) (288.46±49.49 vs. 901.98±271.85, P＜0.01) and the α-SMA mRNA level (0.858±0.234 vs. 8.976±1.398, P＜0.01) were significantly higher in the BDL group. Compared with the Numb-EV group, the Hyp content (864.32±113.54 vs. 580.44±171.77, P=0.039), the α-SMA mRNA level (6.138±1.443 vs. 1.322±0.859, P＜0.01) and the protein levels were significantly reduced in the Numb-OE group. Compared with the Sham group, the serum ALT, AST, TBil, and TBA levels were significantly increased in the BDL group ( P＜0.01), and the ALB content was significantly decreased ( P＜0.01). Compared with the Numb-EV group, AST and TBil levels were significantly reduced in the Numb-OE group ( P＜0.01), as were the ALT and TBA levels ( P＜0.05); however, the ALB content was significantly increased ( P＜0.01), and the differences were statistically significant. Compared with the Sham group, the mRNA expression levels of CK7 and CK19 were significantly increased in the BDL group (1.40±0.42 vs. 43.78±7.56; 1.11±0.51 vs. 363.81±134.84, P＜0.01). The mRNA expression levels of CK7 and CK19 were significantly reduced in the OE group (343.19±81.22 vs. 3.22±2.34; 40.53±14.02 vs. 15.68±9.36, P＜0.01). Conclusion:Overexpression of the Numb gene can inhibit CLF progression in the adult liver, which may become a new target for CLF therapy.

Objective:To evaluate the immunological efficacy of a novel DNA vaccine against West Nile virus (WNV) in a mouse model.Methods:A DNA vaccine VRC-prME expressing the precursor membrane (prM) and envelope protein (E) of WNV Xinjiang strain (XJ11129-3) was constructed and its ability to express virus-like particles was verified in vitro. C57BL/6 mice were immunized twice with VRC-prME via intramuscular injection combined with electroporation with an interval of four weeks. Enzyme-linked immunoassay (ELISA) was used to detect serum antibodies after immunization. WNV (NY99 strain) single-round infectious particles were used to detect neutralizing antibodies. Cellular immune responses were analyzed by enzyme-linked immunoblot assay (ELISPOT) and intracellular cytokine staining (ICS). Results:VRC-prME induced a strong Th1-biased antibody response in mice that could cross-neutralize the WNV (NY99 strain) single-round infectious particles two weeks after the boost immunization. Moreover, the vaccine also elicited antigen-specific multifunctional CD8 + T cell responses (IFN-γ, IL-2, TNF-α). Conclusions:The novel DNA vaccine prepared in this study, expressing the prME protein of WNV XJ11129-3 strain, could induce stronger humoral and cellular immune responses in mice, which was worthy of further research and development for the prevention of WNV infection in China.

Objective To analyze the abnormalities of local chemokines in patients with vitiligo and to explore the effect of tacrolimus on the secretion of chemokines in keratinocytes.Methods Blister fluids of 50 patients with vitiligo were collected,including lesion areas and normal areas.Luminex was used to analyze the concentration of local chemokines in patients with vitiligo to determine whether the chemokines were closely related to vitiligo.The effect of tacrolimus on chemokine secretion of was analyzed by Western blot in HaCaT cells.Results By Luminex analysis of blister fluid,it was found that CXCL9 and CXCL10 were significantly higher in the leukoplakia of vitiligo,and there was a significant difference,compared with the blister fluid in the normal site (P＜0.01).IFN-γ significantly stimulated the keratinocyte cell line HaCat to express CXCL9 and CXCL10.After pretreatment of HaCaT cells with 20 mg tacrolimus,the expression of CXCL9 and CXCL10 was significantly decreased,compared with the blank control (P＜0.01).Conclusions The leukoplakia chemokines CXCL9 and CXCL10 are highly expressed in vitiligo patients.The tacrolimus can significantly reduce the expression of CXCL9 and CXCL10 in keratinocytes under stress,and it therefore plays a therapeutic role in vitiligo.

To confirm the etiology of a dead case for a 6 year-old female Ailurus fulgens,one strain of the predominant bacteria from pathologic tissues(heart,liver,spleen,lung and other samples) of the dead Ailurus fulgens were examined and isolated.The isolate was named R1 and no other bacteria were isolated.The bacterial etiological examination(morphological characteristics,biochemical characteristics and 16S rDNA gene detection)of R1 showed that it was identifed as K.peneumoniae.Artificial infection to mice about R1 was also conducted in this study.R1 had strong pathogenicity to mice and the LD50 is 6.5 × 104 CFU/mL.Moreover,the clinical and pathological features of the dead mice were consistent with that of the Ailurus fulgens.To find effective therapeutic drugs of curing other Ailurus fulgens,antibiotic sensitivity test of R1 was conducted,and the results revealed that R1 was highly sensitive to cefotaxime et al,moderately sensitive to amikacin and resistant to penicillin.These data showed that K.peneumoniae was bacterial pathogen leading to death of the Ailurus fulgens and it had strong resistance to penicillins,macrolides and virginiamycin and it had broad drug resistance spectrum.However,R1 is sensitive to cephalosporins and aminoglycoside antibiotics.

Objective:To investigate whether endogenous hydrogen sulfide (H2 S)was involved in the pathogenesis of osteoarthritis (OA)and its underlying mechanism,to detect H2 S and its synthases ex-pression in knee cartilage in patients diagnosed with different severity of OA,and to explore the transcrip-tion and expression of gene MMP-13 in chondrocytes treated with IL-1βor H2S.Methods:Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2 S content using methylene blue assay.Articular cartilages of the patients who underwent knee arthroplasty were collected for the cell culture of relatively normal chondrocytes.The chondrocytes were cultured to the P3 generation and H2 S molecular probes were used for detection of endogenous H2 S generation in the chondrocytes.Immunocytochemistry was used to detect the localization of H2 S synthases including cystathionine β-synthase (CBS),cystathionine-γ-lyase (CSE),and mercap-topyruvate sulfurtransferase (MPST)in OA chondrocytes.Western blot was used to quantify the protein expressions of CSE,MPST,and CBS in cartilage tissues of the patients who were diagnosed with OA and underwent knee arthroplasty.The relatively normal human chondrocytes were cultured to passage 3 and then divided into 4 groups for different treatments:(1 )the normal control group,no reagent was added;(2)the IL-1βgroup,5 μg/L of IL-1βwas added;(3)the IL-1β+H2S group,200 μmol/L of NaHS was added 30 min before adding 5 μg/L of IL-1β;(4)the H2 S group,200 μmol/L of NaHS was added. The transcription and expression of gene MMP-13 in chondrocytes of each group were determined with Real-time PCR and Western blot,respectively.And the total NF-κB p65 and phosphorylated NF-κB p65 in chondrocytes were detected with Western blot.Results:The content of H2 S in the synovial fluid of degenerative knee was (14.3 ±3.3)μmol/L.Expressions of endogenous H2 S and its synthases including CBS,CSE and MPST were present in the cytoplasm of chondrocytes.CSE protein expression in Grade 3 (defined by outerbridge grading)cartilage tissues was significantly increased as compared with that of Grade 1 cartilage tissues (1.67 ±0.09 vs.1.26 ±0.11,P<0.05).However,no significant difference of CBS or MPST expression among the different groups was observed.The expression of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (1 .87 ±0.67 vs.0.22 ± 0.10,P<0.05 ),and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (0.55 ±0.11 vs.1.87 ±0.67,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The transcription of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (31.40 ±0.31 vs.1.00 ±0.00,P<0.05), and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (24.41 ± 1.28 vs.31.40 ±0.31,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The total NF-κB p65 in the IL-1βgroup was significantly higher than that in the normal chondrocytes (2.13 ±0.08 vs.0.73 ±0.08,P<0.05),and that in the IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (1 .24 ±0.13 vs.2.13 ±0.08,P<0.05 ),and that in the H2 S group had no significant difference compared with that in the normal control group.The phosphorylated NF-κB p65 in IL-1βgroup was significantly higher than that in the normal chondrocytes (1.30 ±0.13 vs.0.19 ±0.04,P<0.05),and that in IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (0.92 ±0.26 vs.1.30 ±0.13,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.Conclusion:H2 S affected the cartilage degeneration by partly inhibiting the degradation of extracellular matrix.

Objective To detect the influence of the perfusion quantity and distribution of bone cement by percutaneous ky-phoplasty(PKP) on the early treatment result of thoracolumbar osteoporotic compression fractures(OVCF) .Methods From May 2011 to May 2013 ,62 cases of osteoporotic fractures of thoracic or lumber vertebra were treated by PKP .CT scans were performed postoperatively to analysis the distribution of the bone cement in the vertebra .According to the bone cement distribution on the transverse plane CT film ,the results were classified into four degrees :excellence ,good ,fair and poor .The cases were followed-up regularly .Preoperative and postoperative visual analogue scale(VAS) ,oswestry dysfunction index(ODI) ,height of the operated ver-tebra ,cobb angle ,the incidences of complications during and after the surgery were compared between groups of different degrees of bone cement distribution and different amount of bone cement injection .Results Among the 62 cases ,the follow-up time ranged from 3 to 36 months[average(10 .5 ± 5 .3)months] .In all of the cases ,there was statistically significant difference between the pre-operative and postoperative VAS scoring(P< 0 .05) .3 months after suergery ,there were no statistically significant influence on the results of VAS scoring ,the ODI scoring ,the height lost of the operated vertebra and the improvement of the Cobb angle(P> 0 .05) . In cases of bone cement injection more than 5 mL ,adjacent vertebra fractures happened in 3 cases 6 months postoperatively and 6 cases 12 months postoperatively .In cases of bone cement injection less than 4 mL ,there were only 2 cases of adjacent vertebra frac-tures happened 12 months posoperatively .The degree of vertebra height lost between the bone cement excellent group and poor group was statistically significant in 6 months and 12 months postoperatively .In cases when the distribution of bone cement was ex-cellent ,the improvement of pain and function was significantly different(P< 0 .05) .Conclusion OVCF is treated by PKP .Through conventional operation ,the ultra-early(within 3 months)efficacy is excellent ,in cases of different amount of bone cement injection and different degree of bone cement distribution .However ,with appropriate amount of bone cement ,the more eventfully and sym-metrically the distribution of the bone cement is ,the better of the early clinical results ,probably .

Objective To explore the feasibility of establishment of NOD/SCID-mouse model with multiple myeloma by using plasma cells from myeloma patients.Methods The femurs and tibias were removed from the New Zealand white rabbits;the muscles,periosteum and cartilage tissues were cleared.Then each bone was cut into two pieces gently along its middle.The NOD/SCID mice weighing 25 - 30 g (4 - 6 weeks)were anesthetized by intraperitoneal injection;rabbit bone was inserted into the right side of the mouse back and engraftment of the bones was allowed to take place after 4 weeks.The 5000 000 purified plasma cells which expressed CD38 +/CD45 - were immunofluorescence labeled and then injected slowly into the implanted rabbit bone through the distal end.The mice were observed weekly;the plasma cells growth in mice was screened by the living-imaging system and the tumor from the mice was determined by biopsy.Results The implanted rabbit bone survived after 4 weeks.The tumor in mice was observed 2 weeks after the purified myeloma cells were injected into the rabbit bone,and it reached 100 mm3 after 8 weeks.Results of the living-imaging system showed that the myeloma cells had uptake in the rabbit bone after 2 weeks of injection and this phenomenon was more pronounced after 8 weeks of injection (2.4×10 4 vs .1.5× 10 5 ,P < 0.05 ).The tumor infiltrated with numerous plasma cells and osteoclasts increased upon the biopsy. Conclusion Rabbit bone marrow implanted into NOD/SCID mice can effectively support local injection of plasma cells of multiple myeloma patients,and the NOD/SCID-mouse model of myeloma has been established.This model can be used to study in vivo experiments related to myeloma and clinical therapeutic approaches for this disease.

OBJECTIVETo compare the efficacy and adverse events of adjusted BACOD (bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone) regimen (continuous intravenous infusion) and conventional BACOD regimen (conventional intravenous drip) in the treatment of relapsed and refractory diffuse large B cell lymphoma (DLBCL).

METHODSRetrospective analysis of 63 cases of relapsed or refractory DLBCL patients was performed, 32 patients received conventional BACOD regimen and 31 patients received adjusted BACOD regimen.

RESULTSThe response rates for adjusted group and conventional group were 87.1%(27/31)and 62.5%(20/32), respectively, during a median follow-up of 14(7-84) months. The difference was statistically significant between the two groups (P=0.025). The main adverse events were myelosuppression, gastrointestinal adverse reactions were rarely serious, and there were no serious liver and kidney toxicity. The median overall survival (OS) was 33 months for adjusted group and 12 months for conventional group, there was statistical differences (P=0.019). The median progression free survival (PFS) was 11 months and 8 months for two groups, the difference was not statistically significant (P=0.095). 1-year survival rates were 68.8% for adjusted group and 44.3% for conventional group, there were no statistical differences (P=0.055). The expected 3- and 5-year survival rates of adjusted group were significantly higher than that of conventional group (47.1% vs 12.8%, P=0.002; 37.7% vs 8.5%, P=0.006, respectively).

CONCLUSIONCompared with the conventional BACOD regimen, the adjusted BCOAD regimen is effective and well tolerated in patients with relapsed or refractory DLBCL, the overall response rate and OS increased.

Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; Bleomycin ; Cyclophosphamide ; Dexamethasone ; Doxorubicin ; Female ; Humans ; Lymphoma, Large B-Cell, Diffuse ; drug therapy ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Vincristine

Objective To investigate the dynamic change and the clinical curative effect evaluation of plasma (1-3)-beta glucan D (BG) in the patients with pulmonary disease complicating fungal infection .Methods The MB-80 miroorganism dynamic rapid de-tection system and fungi BG detection kits were adopted to detect plasma BG content before and after treatment in 87 cases of pul-monary disease complicating fungal infection and the controls .The sputum culture in the patients was performed before and after treatment .Results Plasma BG levels before antifungal therapy ,at 1 ,2 weeks after treatment in 87 patients were (162 .81 ± 70 .03) , (15 .89 ± 30 .88) and (4 .58 ± 7 .87)pg/mL ,which in the control group was (5 .62 ± 1 .83)pg/mL ,plasma BG level had statistical differences between before treatment and at 1 ,2 weeks after treatment in the patients with the control group (P<0 .05);Plasma BG levels between at 1 week after treatment with at 2 weeks after treatment and the control group had statistically significant differ-ences (P<0 .05) .Among 87 patients ,66 cases were positive sputum culture at 1 week after antifungal drug treatment and 9 cases were positive sputum culture at 2 weeks after treatment .Conclusion Continuously monitoring the patient′s plasma BG level com-bined with the sputum fungal culture results ,clinical symptoms and lung shadow in X-ray has certain clinical value to judge the anti-fungal effect .

A label-free electrochemical immunosensor using hollow structure nanomaterials based on its ordered porous and big surface area was designed. Au nanocage, with good conductivity, catalysis, and biocompatibility, was prepared and modified on the surface of glassy carbon electrode with graphene to immobilize antibody of microcystin directly. In the absence of microcystin, biosensor can obtain high current response signal of electrochemical probe ( [ Fe( CN) 6 ] 3-/4-. When microcystin was combined with its antibody specifically, the charge density and mass transfer resistance on the surface of electrode increased, resulting in a decrease of the corresponding peak current of [ Fe ( CN ) 6 ] 3-/4-. This change was in proportion to the concentration of microcystin indirectly. Experiment conditions such as cultivation time of antigen and concentration of antibody were optimized. The results showed wide linear range of 0. 05 μg/L-1. 0 mg/L and the detection limit of 0. 017 ng/mL. This sensor has good stability and simple production procedure. This sensor provides a new and simple means for the ultrasensitive determination of microcystins in real water samples.