ABSTRACT
<p><b>BACKGROUND</b>Tubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-β1 (TGF-β)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT.</p><p><b>METHODS</b>HK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-β1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-β1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-β1 receptor antagonist (TGF-β1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-β1.</p><p><b>RESULTS</b>HK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-β1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-β1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-β1RA (P < 0.05).</p><p><b>CONCLUSION</b>C3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-β1/CTGF signaling pathway in vitro.</p>
Subject(s)
Humans , Blotting, Western , Cadherins , Genetics , Cell Line , Cell Transdifferentiation , Complement C3a , Pharmacology , Complement C5a , Pharmacology , Epithelial Cells , Cell Biology , Immunohistochemistry , Microscopy, Electron, Scanning , Myofibroblasts , Cell Biology , Real-Time Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To prepare and characterize the monoclonal antibody (mAb) against human SOCS3.</p><p><b>METHODS</b>BALB/c mice were immunized with recombinant GST-SOCS3 protein, from which the spleen cells were isolated and fused with Sp2/0 cells. After several rounds of screening and cloning, the hybridoma cell strain secreting anti-SOCS3 mAb was obtained, whose specificity was evaluated using ELISA and Western blotting, and the titer, immunoglobulin subtype and affinity of the mAb were also measured.</p><p><b>RESULTS</b>The hybridoma cell strain secreting anti-SOCS3 mAb was identified to belong to IgG2a subtype. The mAb titers in cultural supernatant and acetic fluid were 1:640 and 1:25600, respectively, as determined by ELISA with affinity reaching 4.84x10(6) L/mol.</p><p><b>CONCLUSION</b>The success in anti-SOCS3 mAb preparation provides the basis for further study of the negative regulation of cytokine signal transduction and the immunoregulation in microorganism infections.</p>
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Hybridomas , Bodily Secretions , Mice, Inbred BALB C , Recombinant Fusion Proteins , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of gastrin in human gastric cancer cell line SGC-7901 and the effects of gastrin-17 and anti-gastrin mAb on its growth.</p><p><b>METHODS</b>The expression of gastrin was determined by immunohistochemistry with anti-gastrin mAb prepared by our group. In a series of experiments, the growth of SGC-7901 cells was evaluated by MTT assay on cells grown in serum-free medium and treated with gastrin-17 and/or anti-gastrin mAb.</p><p><b>RESULTS</b>Immunohistochemical examination of SGC-7901 cells revealed a specific gastrin immunoreactivity. Gastrin-17 significantly stimulated cell growth at the concentrations of 1 x 10(-9) mol/L approximately 1 x 10(-5) mol/L in a dose-dependent manner. The growth of SGC-7901 cells treated with anti-gastrin mAb, either alone or in combination with gastrin-17 (1 x 10(-7) mol/L), was significantly inhibited.</p><p><b>CONCLUSION</b>Growth of human gastric cancer cells SGC-7901 can be stimulated in an autocrine fashion. The gastrin-stimulated growth of gastric cancer cells can be blocked by anti-gastrin mAb bound specifically with gastrin. Further study on the significance of anti-gastrin mAb in designing immunotherapy targeting to gastrin or gastrin receptor is warranted.</p>