ABSTRACT
Objective To prepare the human bone sialoprotein (BSP) monoclonal antibodies (mAb)with high titer and specificity and identify its characterization,which is based on further studying BSP as clinical biomarker for breast cancer metastasizing to bone. Methods BALB/c mice were immunized with purified recombinant BSP protein.Cell fusion was performed between mouse splenic cells and myeloma cells (Sp2/0), and then the hybridoma cell lines secreting mAb against BSP antigen were screened and cloned. The ascites were prepared and purified with Protein G affinity chromatography.The titer and subtypes of mAb against BSP were identified and measured by ELISA and Western blotting analysis. ResultsNine hybridoma cell lines that stably secreted mAb against BSP were successfully obtained.Two of them,D001 and D002,were further identified, which belonged to the subtypes of IgG1 and κ light chain. The two antibodies titers in culture supernatant were 1∶5120 and 1∶10 240, respectively, and those in the ascites fluid were 1∶25 600 and 1∶51 200,respectively.Results of Western blotting analysis and immunohistochemistry showed that the two antibodies could specifically bind with BSP derived from human breast cancer cells.ConclusionNine mAb against BSP have been successfully prepared which can be used for further studying the biological properties of BSP and reveal its relationship with data from clinic patients.
ABSTRACT
The purpose of this study was to employ microarray analysis to evaluate differential gene expression in synovial tissue samples obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) to study the expression profile of apoptosis-associated genes in these tissues. Four samples were obtained from RA-affected patients and three from osteoarthritis patients. After total RNA was extracted from synovial tissue, the RNA was processed using two-cycle target labeling, followed by hybridization and scanning procedure. The GeneChip Human Genome U133 Plus 2.0 containing 900471 gene loci was used and eight genes associated with apoptosis were identified with a selected p value<0.05 and a twofold change in expression in rheumatoid samples compared to osteoarthritis tissues. Anti-apoptotic genes were generally upregulated whereas apoptotic genes were downregulated suggesting that these genes may play a role in the pathogenesis of RA. Furthermore, these genes may serve as novel therapeutic targets for the treatment of RA.
Subject(s)
Apoptosis , Arthritis, Rheumatoid/metabolism , Gene Expression Profiling , Osteoarthritis/metabolism , Actinin/genetics , Adaptor Proteins, Signal Transducing/genetics , Adolescent , Adult , Aged , Arthritis, Rheumatoid/etiology , Arthritis, Rheumatoid/pathology , Granzymes/genetics , Humans , Middle Aged , Osteoarthritis/etiology , Osteoarthritis/pathology , Receptor, IGF Type 1/geneticsABSTRACT
Objective To study effect of Tongbiling (TBL) on VEGF mRNA expression in RA synovlcytes cultured in vitro and explore its mechanism. Method Synovial cells from the knee joint in patient with RA were digested, divided and separately cultured after arthroscopy. Blank controls:DMEM culture containing 10% FBS in saline;Low-dose admission group of TBL:DMEM culture containing 10% FBS in 50 mg/L TBL;High-dose admission of TBL:DMEM culture containing 10% FBS in 200 mg/L TBL. RNA were distilled. Expression of VEGF mRNA were detected with RT-PCR. Result Compared with control group, expression of VEGF mRNA in low and high-dose admission groups of TBL both decreased (P
