ABSTRACT
<p><b>OBJECTIVE</b>To explore the role of Bid protein in the mitochondria and endoplasmic reticulum (ER) associated apoptotic pathway.</p><p><b>METHODS</b>Apoptosis of MUTZ-1 cells induced by homoharringtonine (HHT) was measured by FACS. Mitochondria and ER associated apoptotic pathway was detected by RT-PCR and Western blotting. And the translocation of Bid protein was measured by laser scanning confocal microscope (LSCM).</p><p><b>RESULTS</b>After exposure of MUTZ-1 to HHT at 0.05 microg/ml for 24 h, typical ER-stress phenomenon induced apoptotic cells and release of Ca2+ from the cytosolic Ca2+ storage and the loss of mitochondrial membrane potential were observed. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor were markedly increased at 4 h after 0.05 microg/ml HHT treatment and peaked at 12 h, then decreased steady. Activation of caspase protein was also observed at 8 h. The translocation of Bid protein from ER to mitochondria was observed at 12 h after HHT treatment.</p><p><b>CONCLUSION</b>HHT can induce MUTZ-1 cells apoptosis. The cell death may be likely mediated by the ER stress pathway as well as mitochondrial pathway and Bid protein may be the cross talk of the two apoptotic pathways.</p>
Subject(s)
Humans , Apoptosis , Physiology , BH3 Interacting Domain Death Agonist Protein , Metabolism , Physiology , Calcium , Metabolism , Caspase 3 , Metabolism , Caspases, Initiator , Metabolism , Cell Line , DNA-Binding Proteins , Metabolism , Endoplasmic Reticulum , Metabolism , Physiology , Harringtonines , Pharmacology , Mitochondria , Metabolism , Physiology , Regulatory Factor X Transcription Factors , Transcription Factors , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore whether transplantation of autologous bone marrow stem cells might augment angiogenesis and collateral vessel formation in a canine model of hindlimb ischemia.</p><p><b>METHODS</b>CD34(+) stem cells were centrifugation through Ficoll and an immune magnetic cell sorting system from bone marrow (20 ml) of canine (n = 5) and induced into endothelial cells with VEGF in vitro, and expression of von Willebrand factor. Bilateral hindlimb ischemia was surgically induced in canines and Dil fluorescence labeled autologous stem cells were transplanted into the ischemic tissues.</p><p><b>RESULTS</b>Four weeks after transplantation, fluorescence microscopy revealed that transplanted cells were incorporated into the capillary network among preserved skeletal myocytes. The stem cells transplanted group had more angiographically detectable collateral vessels, a higher capillary density (12.0 +/- 2.8 vs. 5.0 +/- 1.6 per field; t = 4.17 P < 0.05) and a higher ABI (0.58 +/- 0.14 vs. 0.32 +/- 0.11; t = 2.95, P < 0.05).</p><p><b>CONCLUSIONS</b>Direct local transplantation of autologous bone marrow CD34(+) stem cells seems to be a useful strategy for therapeutic neovascularization in ischemic tissues in adults, consistent with "therapeutic vasculogenesis."</p>
Subject(s)
Animals , Dogs , Female , Male , Antigens, CD34 , Cell Differentiation , Disease Models, Animal , Endothelial Cells , Cell Biology , Hematopoietic Stem Cell Transplantation , Methods , Hematopoietic Stem Cells , Chemistry , Cell Biology , Physiology , Hindlimb , Ischemia , Therapeutics , Neovascularization, PhysiologicABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of tumor necrosis factor alpha (TNFalpha) on the expression of phospholamban (PLB) and sarco (endo) plasmic reticulum Ca(2+)-ATPase (SERCA2a) and concentration of intracellular free calcium in myocardiocytes.</p><p><b>METHODS</b>The neonatal rat myocardiocytes were randomly divided into 6 groups: treatment with different concentrations of TNFalpha (1,10,30,50,and 70 microg/L, respectively) and without TNFalpha (control). The mRNA and protein expression of PLB and SERCA2a were detected with one-step reverse transcription-polymerase chain reaction and Western blotting. The changes of intracellular free calcium concentration ([Ca2+]i) in cultured single neonatal rat cardiomyocyte were determined with Fluo-3/AM loading by laser scanning confocal microscopy. RESULTS TNFalpha significantly increased the expression of PLB mRNA and protein in a dose-dependent fashion. The ratio of PLB/beta-actin mRNA in myocardiocytes incubated with 10,30,50, and 70 microg/L TNFalpha significantly increased by 66%, 106%, 141%, and 189% compared with control (P < 0.05), and protein levels significantly increased by 30%, 48%, 73%, and 114% respectively compared with control (P < 0.001), but there was no significant difference in PLB mRNA expression between the group treated with 1 microg/L TNFalpha and control group. TNFalpha had no effect on the expression of mRNA and protein of SERCA2a. TNFalpha (50 microg/L) incubated with cell for 24 hours diminished delta[Ca2+]i of single neonatal rat cardiomyocyte about 33% stimulated by isoproterenol (P < 0.01), but had no effect on delta [Ca2+]i of cardiomyocyte without isoproterenol stimulation.</p><p><b>CONCLUSION</b>TNFalpha can increase the expression of PLB and decrease delta[Ca2+]i in cardiomyocytes, which may be related with its negative inotropic effects on cardiomyocytes.</p>