ABSTRACT
IL-17 Receptor D (IL-17 RD) is a cytokine receptor that mediates IL-17 signaling and plays an important role in responding to the invasion of extracellular pathogens and many inflammatory and autoimmune diseases such as rheumatoid arthritis. In this study we report the generation of a mouse monoclonal antibody against human IL-17 RD. The recombinant human IL-17RD extracellular domain (hIL-17RD-ECD) was produced in the baculovirus expression system and purified from culture medium of sf9 insect cells. The purified protein was used as a T-dependent antigen to immune Balb/C mice. B cells from the spleen of immunized mice were fused with murine cell SP2/0. Hybridoma cell lines were screened for the production of the monoclonal antibody against hIL-17-RD-ECD using ELISA. A hybridoma cell line 1F8 was found to have a high production of the antibody, which was further confirmed for the specificity by both western blot and ELISA analyses. The monoclonal antibody obtained from hybridoma 1F8 was characterized to be IgG1+Kappa subclass. This study provided a base for the further therapeutic application of the antibody on the autoimmune disease including rheumatoid arthritis.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Baculoviridae , Insecta , Genetics , Metabolism , Mice, Inbred BALB C , Receptors, Interleukin-17 , Genetics , Allergy and Immunology , Recombinant Proteins , Genetics , Allergy and ImmunologyABSTRACT
Human tumor suppressor gene beclin 1 regulates cell growth through autophagy. The mRNA expression of beclin 1 was reported to be down-regulated in breast cancer with high frequency of loss of heterozygosity (LOH). However, there was no report about the expression levels or the regulatory mechanisms of beclin 1 in gastric and colorectal cancer. Both the mRNA and protein expression levels of beclin 1 was detected in the tissues of gastric and coiorectai cancers, as well as the aberrant DNA methylation and LOH related to the expression of beclin 1. By comparing with normal tissues adjacent to the tissue of these tumors, it was found that beclin 1 mRNA expression levels were significantly decreased in gastric tumor tissue. Furtherly by explorating the 5' region of beclin 1 gene sequence, a large and dense CpG island was discovered and meanwhile methylations in the promoter and the intron 2 regions of beclin 1 were found in both gastric and colorectal tumors. And LOH was found in gastric tumors. These findings suggested that aberrant DNA methylation, as well as LOH, were involved in the regulation of beclin 1 expression in gastric and colorectal cancer.
ABSTRACT
Objective To study the association between the plasma homocysteine level and coronary artery disease(CAD), and the methylenetetrahydrofolate reductase (MTHFR) A1298C polymorphism, the methionine synthase (MS) A2756G polymorphism and their associations to the plasma homocysteine level and CAD in the elderly . Methods One hundred and twenty-nine elderly patients with CAD documented by coronary angiogram and 48 elderly patients with normal coronary angiographic results were included in this study. Plasma homocysteine level were measured by fluorescence polarization immunoassay (FPIA) method and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyse the MTHFR A1298C and MS A2756G genotypes. Results The plasma homocysteine level was significantly higher in CAD group than that in the control group〔(16.2?8.6) ?mol/L vs (12.7?5.0) ?mol/L,P0.05);the prevalence of MTHFR 1298CC homozygous in the CAD patients was significantly less than that in the control group (3.1% vs 14.6%, P
ABSTRACT
Wild-type p53 has been shown to be involved in several aspects of cell growth control. In order to study the role of wild-type p53 in the growth of lung adenocarcinoma cell line GLC-82, recombinant p53 adenovirus vector and the control LacZ gene were constructed. Infection conditions were detected by biochemistry staining for LacZ s expression product ?-gal, immunohistochemical analysis for p53 protein expression, and PCR technique for the infected cells. The cell growth rate and cell cycle were analysed with flow cytometry. The results showed that the constructed recombinant adenovirus vector could infect cells with high level expression of (?-gal and p53 protein and p53 cDNA could be found in the infected cells. The wild-type p53 could inhibit GLC-82 cell proliferation and cause cellular death. These results indicated that wild-type p53 gene mediated by adenovirus vector might be used in the treatment of lung adenocarcinoma.
ABSTRACT
In order to study the bladder carcinoma cell growth suppression by introduction of foreign retinoblastoma (Rb) gene and explore a gene therapy approach for bladder cancer, a replication-deficient adenovirus vector encoding a wild-type Rb, AdCMVRb, was constructed and transfected into the cultured human bladder carcinoma cell line EJ. The efficiency of gene transfection and expression was detected by immunochemical staining, Western blotting and polymerase chain reaction. The role of Rb in suppressing EJ growth was observed by cell-counting, [3H]thymidine incorporation and flow cytometry. The results showed that wild-type Rb gene could be transfected effectively into cultured EJ with Ad-CMVRb and could arrest the cells at GO/Gl phases of the cell cycle, leading to inhibition of DNA synthesis. The results demonstrated the potential of adenovirus-mediated Rb gene therapy for bladder cancer.