Objective To investigate the stimulating effects of activin A(ACT-A) and follistatin(FS) on the secretion of type I collagen(Col I) and the expression of Col I mRNA of cultured rat renal interstitial fibroblasts in vitro.Methods Rat renal interstitial fibroblasts isolated from normal SD rat renal medulla were cultured in vitro.The cells were divided into three groups:different concentrations of ACT-A(group A),different concentrations of FS(group F),and different concentrations of FS plus 30ng/ml ACT-A(group A+F).The expression of Col I mRNA in cultured rat renal interstitial fibroblasts was detected by RT-PCR,and the expression of Col I protein in cultured rat renal interstitial fibroblasts was examined by immunocytochemistry.Results Renal interstitial fibroblasts were successfully cultured in vitro.In present study,the expression of Col I mRNA increased significantly in cultured rat renal interstitial fibroblasts after stimulated by ACT-A in dose-dependent manner(P0.05).FS could inhibit the effects of ACT-A(30ng/ml) on Col I mRNA and Col I protein of the cultured renal interstitial fibroblasts in dose-dependent manner in A+F group(P

Objective To elucidate the MRI appearance of prostatic abscess,the DWI and enhanced MRI features.Methods 12 cases of prostatic abscesses were retrospectively analyzed,the clinical symptom mainly manifested as lower urinary tract symptoms and fever.All of the patients were given routine MR examination including DWI sequence,6 patients received further enhanced MR examination.Results In the 12 cases,there were 4 cases behaved as single type,8 cases as multifocal type.The abscess showed iso-or slightly hypo-signal intensity on T1 WI,hyper-signal intensity on T2 WI,markedly high signal intensity on DWI and correspond-ing markedly low signal intensity on ADC.Complete abscess walls showed iso-or slightly hyper signal on T1 WI,hypo-signal inten-sity on T2 WI.The mature abscess walls were thin and smooth,which showed homogeneously ring enhanced in 4 cases.The imma-ture abscess walls showed uneven thickness and moderately enhanced in 2 cases.Septum in the abscess could be found in 4 cases, which showed similar enhancement to the abscess walls,while the abscess cavity showed non-enhanced.Abscesses involved the sur-rounding structures in 2 cases,the involved area showed obvious hyper-signal on T2 WI fat-suppression sequence.Conclusion DWI is the best sequence in the diagnosis of prostatic abscess,the markedly high signal intensity on DWI is the characteristic sign.The enhanced MRI showed the walls and septa clearly,the extent and involvement of adjacent structures.The multi-parametric MRI is a prominent procedure in the diagnosis of prostatic abcess.

Objective To investigate the effect of PTEN protein on the proliferation of rat renal fibroblasts resulted from the stimulation of TGF-?_1. Methods In vitro, the cultured rat renal fibroblasts were transfected with the adenovirus containing PTEN. Invert fluorescent microscope was used to detect the GFP expression, meanwhile PTEN mRNA was determined by RT-PCR method. 48h after the transfection, TGF-?_1 was added into the culture medium in a concentration of 10ng/ml. After another 24h, the proliferation rate of rat renal fibroblasts was determined by means of MTT method. Immunocytochemistry was also used to detect the expression of PCNA. Results As GFP positive fibroblasts were detected,the expression of PTEN mRNA was significantly increased in the rat renal fibroblasts which were transfected with the adenovirus containing PTEN. Accompanied with the expression of PTEN, the proliferation of cultured rat renal fibroblasts resulted from the stimulation of TGF-?_1 was obviously inhibited, and the expression of PCNA was significantly decreased. Conclusion PTEN could suppress the proliferation of rat renal fibroblasts caused by stimulation of TGF-?_1, and it might be useful to inhibit renal fibrosis and delay the rate of renal impairment.

Objective To clone the human gene of Hepatitis B virus e antigen binding protein 1 (HBEBP1), which was screened with yeast two-hybrid system and bioinformatics techniques, to construct prokaryotic expression vector of pET-32a(+)-HBEBP1, and to induce the expression of recombinant protein in E. coli BL21. Methods The DNA fragment of HBEBP1 of about 372bp was amplified by reverse transcription PCR (RT-PCR), in which the mRNA was taken from HepG2 cells as the template, and cloned into pGEM-T vector. After restriction enzyme digestion identification and sequencing, the correct target DNA fragment was inserted into inducible prokaryotic expression vector pET-32a(+) and then transformed into competent E. coli BL21. By restriction enzyme digestion and PCR, the positive transformed clones were identified and induced with IPTG to obtain fusion protein. The HBEBP1 fusion protein was analyzed by Western blotting hybridization. Results The 372bp DNA fragment of HBEBP1 was amplified by RT-PCR. The recombinant expression vector pET-32a(+)-HBEBP1 was constructed successfully. After transformation with pET-32a(+)-HBEBP1 and induction with IPTG, the recombinant target protein of about 33kD was obtained, which was consistent with our anticipation. Western blotting assay showed that the protein had good specificity. Conclusions The recombinant prokaryotic expression vector pET-32a(+)-HBEBP1 is constructed, and the HBEBP1 gene is cloned successfully. The HBEBP1 fusion protein could be expressed in prokaryotic expression system of E. coli. These results lays a foundative for studying the immunogenicity and the biological characteristics of the HBEBP1 protein.

Objective To investigate the association between the lumbar vertebral fracture damage degree with the fracture classification,injury score,kyphosis deformity and nerve function injury.Methods According to the damage degree of posterior lig ament complex(PLC),the patients were divided into the PLC intact group and PLC injury group.Its relationship with PLC injury was researched by evaluating the fracture classification,injury score and nerve function injury situation in the two groups.Results The LCS score and TLICS score in the PLC injury group were (7.1±0.8) points and (8.2±0.6) points,which were higher than (5.7±0.5) points and (4.6±0.7) points in the PLC intact group.The Denis score in the PLC injury group was more serious.The Cobb angle in the PLC injury group was 29°,and which in the PLC intact group was 19°.The proportion of nerve function insufficiency in the PLC injury group was 89％,while which in the PLC intact group was only 60 ％.Conclusion The thoracolumbar vertebral fracture is closely related with PLC.PLC damage degree is positively correlated with the fracture classification,injury score,kyphosis deformity and nerve function injury degree.

Acute Disease ; Child ; Cytidine Deaminase ; genetics ; Humans ; Leukemia ; genetics ; Polymorphism, Single Nucleotide ; genetics

The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs, which is crucial for the quality control of products. In order to ensure that the disulfide bonds are correctly paired, disulfide bond analysis is an essential part of peptides and protein drug characterization. Mass spectrometry can be used to analyze disulfide bonds. However, insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site. Conventional collision-induced dissociation (CID) and high-energy induced cleavage (HCD) cannot accurately locate the complex disulfide bond. In our study, three methods were used to localize the complex disulfide, including enzyme digestion combined with key peptide fragment in source decay (ISD) fragmentation method, enzyme digestion combined with partial reduction alkylation method, intact protein source ISD and electron transfer dissociation (ETD) cleavage method, The applicability of insulin aspart, insulin lispro and insulin glargine were also investigated. This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues, and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

Objective To investigate the biochemical properties of rat improved perfusion-acellular pancreatic bioscaffold (APB).Methods The fresh pancreas from 10 rats were perfused through portal vein.The histological structure,ECM composition and DNA quantification of APB were evaluated.For the biocompatibility study,a 0.5 cm2 APB construct was surgically placed within a dorsal subcutaneous pocket of mice.Results The pancreatic tissue become translucent and the macroscopic three-dimensional architectures of native pancreas are retained after decellularization.The extracellular matrix (ECM) ultrastructures were well preserved.Immunofluorescence staining showed that collagen Ⅰ,Ⅳ,fibronectin and laminin were maintained in the APB after decellularization.DNA quantification of APB decreased significantly (P ＜ 0.05).The histological analysis of the subcutaneous implantation site showed the presence of immunological response surrounding the partially degraded APB.The histological remold score was 10.4 ± 1.8 at 14 days and 13.8 ± 1.3 at 28 days.The proliferation of AR42J cell line with the expression of amylase as the marker of exocrine acinar cells can be detected on the recellularized APB.Conclusion APB in this study met the stringent requirement to define a successful decellularization.The technique allowed the efficient generation of APB with preserved 3D architecture and represented a biocompatible scaffold capable of integrating within host tissue.

Chicken embryo fibroblasts(CEFs)are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus(AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR(QPCR)analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4(RPL4)and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ)are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene(ACTB)and the ribosomal protein L4(RPL4)gene are the best references.

ObjectiveTo investigate the roles of 11 β-hydroxysteroid dehydrogenase type 1 ( 11 β-HSD1 )on hippocampus of rat with chronic unpredictable mild stress (CUMS).MethodsTwenty-four male SpragueDawley rats were randomly divided into control group and depressive model group. Chronic unpredictable mild stress (CUMS) was used to make up depressive animal model.Behavioral changes were recorded by body weight measuring,sucrose consumption test (SCT) and open field test (OFT),respectively.The mRNA transcription of 11β-HSD1 in hippocampus tissues of the rats were detected by real-time RT-PCR,and the protein expression of 11β-HSD1 were detected by western blot and immunofluorescence.ResultsBcforc starting CUMS protocol,the rats exhibited equivalent weight and sucrose consumption.Twenty-eight days after CUMS protocol,behavior parameters such as body weight,sucrose consumption,nunber of crossing,and number of rearing were significantly decreased in rats exposed to CUMS group compared with control group (P ＜ 0.05,P ＜ 0.01 ).Correspondingly,realtime RT-PCR assays showed the mRNA expression of 11 β-HSD1 in the hippocampus of CUMS group,which was (31 ±9) ％ lower than that of control group.Meanwhile,the protein expression of it in CUMS group was lower than that of control group (P ＜ 0.05 ).Inmunofluorescence revealed that the number of positive 11 3-HSD1 cells was high (223 ± 13) in the control group,while the number was decreased prominently (92 ± 11 ) in the CUMS group (P ＜ 0.01 ).ConclusionDepressive behavior of rats is induced and the expression of 11 β-HSD1 in the hippocampus is decreased prominently by CUMS,the mechanism of which is at least related to the low expression of 11β-HSD1 and disturbance of glucocorticoid metabolism caused by CUMS.