Objective:To construct and express anti CD3/anti CD20 Diabody and identify its biological activity.Methods:PCR and overlap PCR were used to construct anti CD3/anti CD20 Diabody.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by both the detection of Western blot and size exclusion chromatography;its antigen binding activity was examined by FACS and rosetting assay.Results:The data of DNA sequence showed that the anti CD3/anti CD20 Diabody was corrected.The Diabody was recovered in high yield(up to 1 mg/ml) after E taq purification and predominantly(90%) as a dimer.The Diabody binded Jurkat cells(CD3 +) and Daudi cells (CD20 +),respectively.Furthermore,the Diabody was capable of simultaneous binding to Jurkat cells and Daudi cells as shown by cellular rosetting.Conclusion:The anti CD3/anti CD20 BsF(ab') 2 was first recast into the Diabody format and succeeded to obtain high level expression.The results of some biological activity experiments indicated that the Diabody could bind to Jurkat cells and Daudi cells.

Aim Toexamineliverdamagebyrifampi-cin and hepatic gene expression related to bile acid me-tabolisminmice.Methods Adultmalemicewere given rifampicin(180 mg·kg-1 ,po)daily for 30 days and(90 mg·kg-1 ,po)daily for 90 days,blood bio-chemistry,histopathology,and gene expression were examined.Results Rifampicinincreasedanimalliver index and serum enzyme activities. Histopathology showed steatosis and spotted feathery-like degenera-tion.Rifampicin increased the expression of CYP7A1 after 30 and 90 days of administration,along with in-creased FXR and SHP.Rifampicin reduced the expres-sion of BSEP after 30 days of high dose administration. Conclusion Repeatedadministrationofrifampicin may cause liver injury and intrahepatic cholestasis in mice,and these effects are associated with the altera-tion of gene expression related to bile acid metabolism.

ObjectiveTo investigate the clinical application of tacrulimus (TAC, FK506) in children with primary nephrotic syndrome (NS). MethodsSixty-five primary NS children received routine or decreased-dosage glucocorticosteroid according to clinical NS types after hospitalization. At the same time, TAC was given orally with the dosage of 0.1 to 0.15 mg/kg, once every 12 hours, for 6 to 24 months. And the serum concentration of TAC was monitored during the course. ResultsAfter the treatment of TAC for 1 to 2 months, 65 patients were recovered with gradually reduced urinary protein, rapidly increased serum albumin, and improvement of cholesterol and triglycerides. Total remission rate was 83.1% and onset time was 7 to 54 days. Twelve cases experienced recurrence. Increased CD4, as well as 3/3 or 3/1 TAC genotype, indicated higher remission rate. Various pathological types had different remission rates or ratio, which were as follows: minimal change nephropathy (96.4%), mesangial proliferative glomendonephritis (90.0%), membranous nephropathy (2/3), membranous proliferative glomerulonephritis (3/5), focal segmental glomerulosclerosis (4/9). The patients would recover in the course of treatment under the conditions of TAC initial dose as 0.1 to 0.15 mg /kg per 12 hours and controlled serum concentration as 5 to 10 g/L. During the treatment, 12 cases appeared gastrointestinal symptoms, mainly as anorexia, nausea and vomiting, 1 abdominal pain, 2 headache, 1 tremor, 1 paresthesia, 3 insomnia, 4 transient increased Scr, 8 slightly increased NAG, 6 increased C3 and α-2 macroglobulin. The symptoms disappeared within one week or after stopping TAC. ConclusionsTAC is effective in primary NS children, even with abnormal liver function or tuberculosis infection. TAC can also be a substitute to cyclosporine A.

Objective Mizoribine ( MZR) is a new immunosuppressant , however , little domestic research has been done on MZR for treatment of nephrotic syndrome in children .This study was to investigate curative effect and adverse reaction of MZR in the treatment of children with frequently relapsing nephrotic syndrome , using prospective controlled trials . Methods A total of 59 pa-tients with frequency relapsing nephrotic syndrome were randomly divided into two groups .29 patients of treatment group were treated with MZR +glucocorticoid , while 30 patients of control group were given Tripterygium wilfordii ( TW)+glucocorticoid treatment , and the course of treatment lasted for 12 months.24-hour urine protein, urinary N-acetyl β-glucosidase (NAG), serum albumin, serum cholesterol, serum creatinine, recurrence frequency, and average prednisone dosage were observed . Results At the end of treat-ment, Serum albumin in treatment group was higher than that in control group [(40.95 ±6.12)g/L vs (30.25 ±9.02)g/L], and Se-rum cholesterol ([5.45 ±0.82]mmol/L vs [7.53 ±2.74]mmol/L), urinary protein ([0.89 ±0.52]g/24 h vs [1.63 ±2.02]g/24 h), urinary NAG enzyme ([21.43 ±14.16]U/g· Cr vs [41.67 ±12.35]U/g· Cr) levels were lower compared with control group . There was significant difference between the two groups .In terms of mean recurrence times , no significant difference was found at 6th months of follow-up between the two groups, however, treatment group had lower recurrence rate than control group at 3rd month, 9th month, 12th month of follow-up, which was of significant difference .The average amounts of hormone of treatment group were lower than those of control group ([0.56 ±0.16] mg/kg· d vs [0.72 ± 0.34]mg/kg· d)、([0.64 ±0.35]mg/kg· d vs [0.67 ±0.52]mg/kg· d)、([0.53 ±0.41] mg/kg· d vs [0.83 ±0.37] mg/kg· d)、([0.34 ±0.15] mg/kg· d vs [0.54 ±0.26] mg/kg· d) at 3rd month, 6th month, 9th month, 12th month of follow-up, which was of significant difference . Conclusion Compared to Tripterygium wil-fordii combined with hormone therapy , MZR combined with prednisone therapy in children with recurrent NS frequency can reduce the relapse rate and dosage of corticosteroid to improve the clinical remission rate .

Objective To explore a novel and highly specific small-molecule TNFβinhibitor by using computer-aid?ed virtual screening and cell-based assays in vitro. Methods Computer-aided drug design and virtual screening were used to design and identify chemical compounds that targeted TNFβbased on the crystal structure of the TNFβ-TNFR1 com?plex. The effect of the small-molecule compound against TNFβ-induced cytotoxicity of L929 cells was detected by MTT as?say, and the efficacy of the compound to inhibit TNFβ-induced apoptosis of L929 cells was determined by flow cytometry as?say. The impact of the compound on L929 cell cycle was examined by Propidium Iodide (PI) staining and flow cytometry, and the influence of the compound on TNFβ-triggered signal pathway was analyzed by Western blot assay and Ultra VIEW VOX 3D Live Cell Imaging System. Results No.35 compound (named as C35 thereafter) could effectively inhibit TNFβ-induced cell death in a dose dependent manner, and the half-maximum inhibition concentration (IC50) was 8.19μmol/L. Furthermore, C35 had lower cytotoxicity and minimal effect on L929 proliferation. Here we further revealed that C35 could affect TNFβ-induced apoptotic pathway by blocking the activation of Caspase 3, and markedly reduce L929 cell apoptosis induced by TNFβ. Conclusion A novel TNFβsmall-molecule inhibitor was identified by combining computer-aided virtual screening with functional assays, and which could block TNFβ-triggered apoptotic pathway and efficiently inhibit the cell death in?duced by TNFβ.

Objective Gitelman Syndrome is a disease caused by the mutation of Na-Cl cotransporter gene(SLC12A3).The article studied the significance of diagnosis and identification by genetic mutation. Methods We collected the clinical data, then we sequenced the SLC12A3 gene by the first sequencing technology and MLPA. Results SLC12A3 complicated heterozygotic mutation was observed.One of them showed c.1964G>A, p.(Arg655His) and exon 8 deletion mutation, the other showed c.2543A>T, p.(Asp848Val) and c.976delG, p.(Val326fs) mutation of SLC12A3 gene in children. Conclusion The final diagnosis depended on gene diagnosis. Pediatrician must recognize the manifestations to advoid misdiagnosis.

Aim To construct and express anti-CD20Fab-LDP,generate anti-CD20Fab-LDM and identify its biological activity.Methods PCR and overlapping PCR were used to construct anti-CD20Fab-LDP.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS.Specific killing activity in vitro of anti-CD20Fab-LDM was analyzed by MTT.Results The data of DNA sequence showed that anti-CD20Fab-LDP was correct.The fusion protein was recovered in high yield(up to 4 mg·L~(-1))after proteinG purification.The fusion protein could bind to Raji cells(CD20+),and similar affinity data were obtained with anti-CD20Fab.Anti-CD20Fab-LDP showed potent cytotoxicity to Raji cells with IC_(50) values of 0.9×10~(-10) mol·L~(-1).Conclusions Anti-CD20Fab-LDP with high level expression was successfully obtained and could bind to Raji cells cells.Anti-CD20Fab-LDM showed specific killing activity to Raji cells in vitro.

Aim To determine whether membrane cytokeratin 8(CK8 )and BCRP expression cooperatively contributed to multidrug resistance(MDR)in MCF-7/MX cells.Methods MCF-7/MX cells were transfected with specific anti CK8-siRNAs and anti BCRP-siRNAs via LipofectAMINE2000.The expression of CK8 and BCRP was determined using Western blot,and membrane staining was observed by laser confocal microscopy.Sensitivity to chemical drugs was examined by Sulforhodamine B method.Results The expression levels of cell surface CK8 and BCRP were obviously reduced by siRNAs,and inhibition of CK8 and BCRP expression could effectively restore the sensitivity to drugs and reverse MDR phenotype of MCF-7/MX cells.Conclusions CK8 together with BCRP may play significant roles in conferring the multifactorial MDR phenotype of MCF-7/MX cells,but may act independently via potentially different mechanisms.Combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.

Aim To investigate the potential suppressing effect of calmodulin antagonist EBB on metastasis-associated properties of human metastatic ovarian clear cell adenocarcinoma cell ES-2.Methods MTT assay was used to assess the growth inhibition of EBB on ES-2 cells.The invasive capacity and motility potential were determined by Transwell chamber assay and Wound assay.[Ca2+]i was observed under the laser scanning confocal microscopy.Results EBB inhibited the proliferation of ES-2 cells in vitro.The IC50 on ES-2 cells was(13.67?1.56)?mol?L-1.The invasive ability and motility potential of ES-2 cells were decreased after exposure to 3,7 and 14 ?mol?L-1 EBB respectively(P

To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Solubility ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor Receptor-1 ; genetics ; isolation & purification ; metabolism