Sirolimus inhibits the differentiation, proliferation and migration of endothelial progenitor cells in vitro / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effect of sirolimus on differentiation, proliferation, adhesion and migration of endothelial progenitor cells (EPC) in vitro.</p><p><b>METHODS</b>(1) Mononuclear cells (MNC) were isolated from rat bone marrow by Ficoll density gradient centrifugation and cultured on fibronectin-coated culture dishes with or without sirolimus (0.01 - 100 ng/ml) for 12 days. (2) After 8 days cultured, attached cells were treated with sirolimus (0.1 - 200 ng/ml) or vehicle for various time points (12 h, 24 h, 48 h and 96 h). EPC were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under laser confocal immunofluence microscope. EPC proliferation, migration were assayed with MTT assay and modified Boyden chamber assay respectively.</p><p><b>RESULTS</b>EPC number differentiated from MNC at 12 days was significantly lower in sirolimus treated cells in a dose-dependent manner than that of vehicle-treated cells. Sirolimus also significantly inhibited the proliferative, migratory and adhesive capacity of EPC in a time and dose dependent manner.</p><p><b>CONCLUSION</b>Present results suggested that sirolimus could inhibit EPC differentiation from MNC and reduce the proliferation, migration and adhesion capacities of EPC.</p>

Relationship between hypoxia-induced apoptosis and caspases-3 activation, intracellular calcium overload in cardiomyocytes / 中国应用生理学杂志

<p><b>AIM</b>To explore the effects of hypoxia on Caspases activation in cardiomyocyte and role of intracellular calcium in this event in cardiomyocytes.</p><p><b>METHODS</b>After hypoxia 0 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, apoptotic cell percentage was determined with Hoechst 33342 straining. Expressions of Caspases-3 mRNA and release of mitochondrial cytochrome c in primary culture of cardiomyocytes were determined by using RT-PCR and Western blotting respectively.</p><p><b>RESULTS</b>Elevation of Cyt c in cytosol was in accordance with the decline in mitochondrial Cyt c content. Significant increase in Cyt c in cytosol appeared at 12 h post hypoxia and peaked at 24 h while Cyt c in mitochondria could not be detected at 24 h post hypoxia. Hypoxia up-regulated Caspases-3 mRNA expressions beginning at 3 h post hypoxia. Intracellular calcium overload occurred earlier than release of mitochondrial Cyt c and the activation of Caspase-3 during the hypoxic insult. Inhibition of Caspase-3 activation and pretreatment with calcium chelator BAPTA/AM offered a marked protective effect on hypoxia induced cardiomyocyte apoptosis.</p><p><b>CONCLUSION</b>Hypoxia can induce mitochondrion-dependent Caspase-3 activation in cardiomyocytes and therefore leads to cell apoptosis. Increase of intracellular Ca2+ plays an important role in the activation of Caspase-3 and the induction of apoptosis in cardiomyocytes.</p>

Mechanisms of morphine-evoked changes of intracellular calcium in primarily cultured hippocampal neurons / 中国应用生理学杂志

<p><b>AIM</b>In order to explore the neurobiological mechanism of morphine addiction and treatment methods, the acute and chronic effects of morphine on the intracellular free calcium concentration ([Ca2+]i) in cultured hippocampal neurons were investigated.</p><p><b>METHODS</b>Changes of [Ca2+]i induced by morphine in primarily cultured hippocampal neurons were measured by confocal laser scanning microscopy using Ca(2+) -sensitive dye fluo-4 as the calcium fluorescent probe.</p><p><b>RESULTS</b>Morphine actually induced the increase in [Ca2+]i of hippocampal neurons. This process could be blocked by naltrindole (delta opioid receptor antagonist) pretreatment, but not by CTOP (micro opioid receptor antagonist) pretreatment. Pretreatment of the cells with thapsigargin almost completely blocked morphine-evoked response; while pretreatment of the cells with verapamil partially inhibited this response. After exposure to 100 micromol/L morphine for 24 h, intracellular [Ca2+]i increased and the increase could be intensified after adding 10 micromol/L naloxone to the medium.</p><p><b>CONCLUSION</b>Morphine induced the release of Ca2+ is mainly from inositol 1, 4, 5-trisphosphate (IP3) sensitive stores in hippocampal neuron of rats through activation of delta2 subtype opioid receptor.</p>