Cytokine Antibody Array of Rattus Norvegicus Bone Marrow-Derived Mesenchymal Stem Cells

Background and Aim: Growth factors and cytokines secreted from mesenchymal stem cells (MSCs) as well as other types of cells relate tocell fates toward survival versus death and interaction versus protection,etc. It is now well documented that the main stem cell properties,including self-renewal, multi-lineage differentiation, and tissueengraftment, are principally influenced by the local microenvironmentand soluble molecular signals. Therefore, the identification of thesefactors is very important for further consideration. The aim of this studyis the identification of secreted cytokines from rat bone marrow-derivedMSCs.Methods: In this study, 6- to 8-week-old male Rattus rats were euthanized,bone marrow from tibia and femur was obtained by flushing and MSCswas isolated by the Ficoll-Paque method. MSCs was cultured until reachconfluency. For antibody array, Rat Cytokine Antibody Array–Membrane(ab133992, Abcam) consisting of a total 34 different cytokine antibodiesspotted in duplicate onto two membranes were used. For this purpose,supernatants from cells of the passage 3-6 were collected at culture days7. Array membranes, each in separate wells of provided 8-well plate, wereincubated for 30 minutes in 2 mL of blocking buffer, further incubatedfor 2 hours in a shaker at room temperature with 2 mL of fresh culturemedium. After the membranes were thoroughly washed with wash bufferI and II, 2 μL of biotin-conjugated was added to each membrane, andthe mixture was incubated on a shaker 4oC overnight. Following thewash, the membranes were incubated with HRP-conjugated streptavidinfor 2 hours at room temperature. Proteins were detected by detectionbuffer C and D provided in kit and signals were captured by CCDcamera. The exposure time was 5 and completed within 20 minutesas chemiluminescence signals will fade over time. Arrays images wereprocessed with Image J software.Results: The cytokine antibody array membrane incubated for 2 hourswith a fresh culture medium as suggested in the manufacturer’s protocolyielded a number of spots whose intensities were significantly strongerthan the background level, indicating that culture medium of bonemarrow-derived MSCs featured a single predominant hybridizationsignal for TIMP-1 (tissue inhibitor of metalloproteinases-1).Conclusion: The significant expression of TIMP-1 suggests that thiscytokine as well other secreted cytokines could be involved in thecell interactions. The identity of another molecule involved in theanti-proliferative effect of bone marrow-derived MSCs requires furtherinvestigation.

کلیدواژه ها:

Cytokine secretion profile ، Rat bone marrow-derived MSCs ، TIMP-1