The partial segment of Marek′s disease virus (MDV) glycoprotein B (gB) gene was amplified by PCR. The segment was cloned into pET-28a vector to obtain the recombinant pET-gB plasmid. The recombinant plasmid was transformed into E.coli BL21,and expressed in very high level as inclusion body after induced with 1.0mmol/L IPTG. The inclusion body was solubilized in urea (8mol/L) . The purified protein was obtained by use of His?Bind affinity chromatography. Mice were immunized i.p. by the purified protein to make the polyclonal antibody. The titer of the antibody by indirect ELISA was 1?10~ -5 . Moreover, the analysis by western blot proved that antibody was specific to the recombinant protein. These works lay a favorable foundation for the study of the immune response by MDV gB.

OBJECTIVETo investigate whether aristolochic acid can be transported into human kidney proximal tubular cell (HKC) and its potential mechanism.

METHODSIntracellular aristolochic acid was measured by liquid chromatography-tandem mass spectrometry. The release of lactate dehydrogenase (LDH) induced by aristolochic acid in the presence of organic anion transporter inhibitor (probenecid) or organic cation transporter inhibitor (tetraethylammonium) was evaluated. The effects of probenecid on aristolochic acid induced connective tissue growth factor (CTGF) mRNA and protein expression were also examined by real time polymerase chain reaction and Western blot, respectively.

RESULTSAristolochic acid was detected in the suspension of the denatured HKC after incubation with aristolochic acid sodium salt. The release of LDH from HKC, which was induced by 60 mg/L aristolochic acid sodium salt, was significantly inhibited by 1 mmol/L probenecid (P < 0.01), but not by 1 mmol/L tetraethylammonium. The increased CTGF mRNA and protein expression in HKC stimulated by 40 mg/L aristolochic acid sodium salt was significantly down-regulated by 1 mmol/L probenecid (P < 0.05), with an inhibition rate of 16% and 21%, respectively.

CONCLUSIONAristolochic acid can be transported into HKC by organic anion transport system, and then exerts its biological effects.

Aristolochic Acids ; metabolism ; Connective Tissue Growth Factor ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Kidney ; physiology ; Organic Anion Transporters ; metabolism

Field avian infectious bronchitis virus (IBV) designated as JS/9 5/03, which was isolated from Jiangsu province of china, was cultivated in chicken emb ryo. It's single strain RNA was extracted from purified virus and worked as temp late of reverse transcription polymerase chain reaction (RT-PCR), a pair of pri mer designed according to megalign results of published IBV sequences in Genbank was used to amplify the neucleocapsid gene, the RT-PCR product was sequenced d irectly. Sequence analysis revealed that the sequence of JS/95/03 is most homolo gized with that of M41 strain.

Objective To investigate the roles and significances of MMP-2 and MMP-9 in diffuse proliferative lupus nephritis by repeated renal biopsy.Methods Seventeen patients diagnosed by renal biopsy as WHO typeⅣlupus nephritis were analyzed by immunohistochemistry staining for MMP-2 and MMP-9. Double staining for MMP-2 and MT1-MMP,MMP-9 and CD68 were also performed.Patients had repeated renal biopsy after followed up for 2.5 years.The relationship between expressions of gelatinases and pathological activity index and clinical data were studied.Results MMP-2 immunoreactivity was detected in normal controls and was increased in diffuse proliferative lupus nephritis.MMP-9 staining,which was almost negative in normal giomeruli,was increased much more significantly in diffuse proliferative lupus nephritis. The immunoreactivity of MMP-2 and MMP-9 was positive in MT1-MMP staining and CD68-positive macrophages, respectively.The expression of MMP-2 and MMP-9 was reduced by 70% and 62% in 10 patients whose clinical condition was partially alleviated,while the expressions in 7 patients whose clinical condition was not alleviated,were only reduced by 27% and 32%.The staining for MMP-2 and MMP-9 were correlated with activity index of lupus nephritis and proteinuria.Conclusion Up-regulation of gelatinases expression in diffuse proliferate lupus nephritis is correlated to activity index of the disease.

Objective To evaluate the value of renal parenchymal volume and thickness by non-contrast spiral CT in evaluating the differential glomerular filtration rate (GFR) for chronic obstructed kidneys, and to compare the correlations between the two morphologic indices of renal parenchyma and the GFR for chronic obstructed kidneys. Methods Seventy-one patients who had a diagnosis of unilateral chronic upper urinary tract obstruction were included in this analysis. (1) The renal parenchymal volume was mea-sured by non-contrast spiral CT. Both kidneys were scanned by non-contrast spiral CT. The renal parenchymal area of each section was marked manually. Renal parenchymal volume was calculated as the sum of renal parenchymal area multiplied by the width of each section. The volume percentage of obstructed kidney (%CTvol) was also calculated. (2) Renal parenchymal thickness was measured on the first and last non-contrast CT image levels from the anterior, posterior and lateral locations of the kidney that clearly contained the collecting system. The mean of these measurements was defined as the renal parenchymal thickness. The differential renal parenchymal thickness of the obstructed kidney (%CTt) was defined as the percentage of the obstructed renal parenchymal thickness to the total renal parenchymal thickness for both kidneys. GFR was determined with 99Tcm-DTPA dynamic imaging system by Gates method. The differential GFR for obstructed kidney (%GFR) was the GFR percentage of obstructed kidney to the total GFR for both kidneys. The Pearson relation test was carried out between the %CTvol, %CTt and the %GFR respectively. Results %CTvol and %CTt correlated well with %GFR in chronic obstructed kidneys among the 71 test group patients. Pearson correlation coefficient r was 0.80 (t=11.20, P＜0.05) and 0.66 (t=7.24, P＜0.05), respectively. The linear correlation equation respectively was %GFR=0.05+0.80×%CTvol (F=125.48, P＜0.05) and %GFR=0.12+0.66×%CTt (F=52.36, P＜0.05). Conclusions Renal parenchymal volume and thickness by non-contrast spiral CT might be used as clinical practical parameters to evaluate the differential GFR for chronic obstructed kidneys. Renal parenchymal volume is more accurate than renal parenchymal thickness.

Objective To assess the clinical value of measuring the concentration of serum osteoprotegerin (OPG) in detecting the bone metastases in patients with prostate cancer. Methods The concentration of serum OPG in 40 patients was determined by ELISA. The data of ECT bone scan and Gleason score was collected simultaneously. The correlations between serum OPG and bone metastases, Gleason score were tested. Results The concentration of serum OPG in patients with bone metastases by ECT scan was( 16 237. 19 ±5144. 26) ng/L,which was significantly higher than the concentration in patients without bone metastases , which was (12 123.32 ±4136. 50)ng/L. There was no significant correlation between serum OPG and Gleason score. Conclusions The serum OPG has an important clinical value in prediction of prostate cancer with bone metastases. There is no significant correlation between serum OPG and the Gleason score.

Background and purpose: Uridine diphosphoglucu-ronosyl transferase 1A1 (UGT1A1) is an important enzyme for metabolism of irinotecan. The activity of UGT1A1 enzyme was significantly affected by the gene polymorphism. This study aimed to investigate the correlation of UGT1A1*28 and *6 gene polymorphisms with irinotecan-based chemotherapy in colorectal cancer(CRC). Methods: Analysis of UGT1A1*28 and *6 gene polymorphisms was performed in 160 gastrointestinal cancer patients admitted to Zhongshan Hospital Fudan University from Apr. 2013 to Dec. 2013 by amplifying the gene fragments using PCR, STR and Sanger sequencing. Eighty-two cases with CRC treated with irinotecan were chosen to observe the adverse events during chemotherapy. The incidence of different genotypes was compared. Results:The distribution of the genotypes in 160 gastrointestinal cancer patients was as followed:UGT1A1*28 wild-type genotype TA6/6 (124, 77.5%), heterozygous genotype TA6/7 (33, 20.5%), and homozygous genotype TA7/7 (3, 2.0%);UGT1A1*6 wild-type genotype GG (105, 65.6%), heterozygous genotype GA (48, 30.0%), and homozygous genotype AA (7, 4.4%). In the 82 CRC cases, the incidences of grade 3 and 4 neutropenia in the patients carrying UGT1A1*28 (TA6/7+TA7/7 ) were higher than those in the WT genotype (TA6/6) (58.3%vs 0.0, P<0.001), and increased the total incidence of adverse events (76.0%vs 45.6%, P<0.001). There was no signiifcant relevance between UGT1A1*6 genotype, age, gender chemotherapy and adverse events. Conclusion:In the CRC cases with irinotecan-based chemotherapy, the UGT1A1*28 (TA6/7+TA7/7) genotype signiifcantly increased the risk of grade 3 and 4 neutropenia. Detecting UGT1A1 gene polymorphisms may guide individualized treatment and predict adverse events.

The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

Objective To observe the shenqifuzheng injection (SFI) combined with PG therapy (gemcitabine and cisplatin) in patients with non-small cell lung cancer (NSCLC). Method 76 patients with NSCLC were selected from August 2011 to August 2013 and randomly divided into observation group and control group. Each group had 38 cases. The control group received gemcitabine and cisplatin, observation group were gave SFI on the basis of control group. The total efficiency, disease control rate, quality of life and incidence of adverse reactions were observed after treatment in each group. Results The total effective rate was 47.37%in observation group , 42.11%in control group, there was no significant difference between two groups. The disease control rate in observation group was 81.58%higher than 57.89%in control group, the difference was statistically significant (χ2=3.990, P<0.05). The improvement rate in quality of life was 60.53%in observation group better than 31.58%in the control group , the difference was statistically significant (χ2=5.296, P<0.05).The decrease incidence of white blood cell (χ2=4.491, P<0.05) and platelets (χ2=4.491, P<0.05) in observation group were significantly lower compared with the control group, while, there were no difference of liver damage and gastrointestinal reactions between two groups. Conclusion SFI is helpful to improve the efficacy of PG therapy in patients with NSCLC and relieve the side effects of chemotherapy.