Early Diabetes Impairs Bone Marrow Derived Endothelial Progenitor Cells Function Which Not Related To Oxidative Stress Mediated Mechanism

1019 words - 5 pages

2. MATERIALS AND METHODS
2.1. Animals and type 1 diabetic mouse model
All the experimental protocols described in this study were approved by the Animal Care Committees of Juntendo University. Eight weeks old C57BL/6J mice (20 – 25 g) were used for the experiments. Diabetes (DM) were induced by injection intra-peritoneally of Streptozotocine (STZ, 50 mg/kg, Sigma, St. Louis, MO) dissolved in citrate buffer (pH 4.5) for 5 consecutive days, as previously described [41]. Seven days after the initial injection, mice with blood glucose level equal or higher than 300mg/dL were decided as diabetic mice (DM), while those whose blood glucose level < 300 mg/dL will receive additional STZ injection (50mg/kg) for 3 consecutive days. Diabetic mice were considered if their blood glucose levels were 300 mg/dL or higher which maintained at least 4 weeks.
2.2. Bone marrow (BM)-derived KSL cells Isolation.
BM cells were harvested from femur, tibia, pelvis and humerus bones of DM and control mice as previously described [10]. BM mononuclear cells were washed with PBS-EDTA and removed the erythrocytes by ammonium chloride. The cells were then labeled by biotin-conjugated antibody cocktail (CD3e, CD45/B220, Ly-6G and Ly-6C, CD-11b, TER-119) for 20 min at 4°C (all antibodies obtained from eBioscience, San Diego, CA) and followed by anti-biotin microbeads labeled (Miltenyi, Auburn, CA). The microbeads labeled cells were separated on a sorting system (AutoMACS, Militenyi) to obtain the negative fraction of lineage negative (Lin-) cells. The Lin- cells were counted and stained with APC labeled Sca-1 and PE-labeled c-Kit antibodies (eBioscience, San Diego, CA) for 20 min at 4°C, washed with FACS buffer once and suspended in 20% FBS in Iscove modified Dulbecco medium (Gibco,Carlsbad, CA). The cells were then sorted for c-Kit+, Sca-1+ and Lin- (KSL) cells by FACS Aria (BD, San Jose, CA). Sorted KSL cells were incubated at 37 °C for 1 hour after sorting before using for the following experiments.
2.3. Detection of protein carbonyls as oxidative stress markers.
The levels of 2,4-dinitrophenyl hydrazine (DNPH) for protein carbonylation was measure as oxidative stress markers by ELISA. The proteins were extracted from the cells in RIPA buffer (Thermo Scientific, Rockford, IL) with Protease inhibitor cocktail (Roche, Penzberg, Germany). The protein amounts were measured using BCA protein assay kit (Thermo Scientific). Briefly the equal amount of proteins were precipitated with 20% Trichloroacetic acid (TCA) and re-solved with 2N HCl. Then the proteins were labeled with DNPH, precipitated with ethyl acetate-ethanol (1:1) and wash the precipitates with the same solution for removing excess amount of DNPH. The precipitates were solved in 8M urea and measured protein amount using BCA kit. The extracts were diluted with coating buffer (15 mM Na2CO3-35 mM NaHCO3-0.02% NaN3 in H20) to adjust the protein amounts and then a 96-well ELISA plate were coated each wells at 4°C...

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