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The feasibility of bone marrow stromal cells autologous transplantation for rabbit model of dilated cardiomyopathy induced by adriamycin was studied. Twenty rabbits received 2 mg/kg of adriamycin intravenously once a week for 8 weeks (total dose, 16 mg/kg) to induce the cardiomyopathy model with the monitoring of cardiac function by transthoracic echocardiography. Marrow stromal cells were isolated from cell-transplanted group rabbits and were culture-expanded on the 8th week. On the 10th week, cells were labeled with 4,6-diamidino-2-phenylindole (DAPI), and then injected into the myocardium of the same rabbits. The results showed that viable cells labeled with DAPI could be identified in myocardium at 2nd week after transplantation. Histological findings showed the injury of the myocardium around the injection site was relieved with less apoptosis and more expression of bcl-2. The echocardiography found the improvement of local tissue movement from (2.12±0.51) cm/s to (3.81±0.47) cm/s (P＜0.05) around the inject site, but no improvement of heart function as whole. It was concluded bone marrow stromal cells transplantation for dilated cardiomyopathy was feasibe. The management of cells in vitro, the quantity and the pattern of the cells transplantation and the action mechanism still need further research.
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To investigate the effects of metallothionein (MT) on isolated rat heart, 16 Wistar rats were randomly divided into 2 groups. In control group (group C), distilled water was injected intraperitoneally and 24 h later isolated hearts were perfused with Langendorff and stored at 4 degrees C for 3 h with histidine-tryptophan-ketoglutarate (HTK) solutions, and then isolated hearts were perfused for 2 h by Langendorff. In experimental group (group E), 3.6% ZnSO(4) was injected intraperitoneally, 24 h later isolated hearts were perfused by Langendorff and stored at 4 degrees C for 3 h with HTK solutions, and then the isolated hearts were perfused for 2 h with Langendorff. MT content, the recovery of hemodynamics, myocardial water content (MWC), lactate dehydrogenase (LDH) and creatine kinase (CK) leakage, adenosine triphosphate (ATP) and malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, myocardial cell Ca(2+) content, Ca(2+)-ATPase activity of mitochondria ([Ca(2+)-ATPase](m)) and its Ca(2+) content ([Ca(2+)](m)), synthesizing ATP activity of mitochondria ([ATP](m)), and the ultrastructure of cells were examined. There were a significant increase in group E in hemodynamic recovery, ATP content, SOD activity, [Ca(2+)-ATPase](m) activity, [ATP](m) activity, and substantial reduction in MWC, LDH and CK leakage, MDA content, myocardial cell Ca(2+) content, [Ca(2+)](m) content, and the ultrastructural injury were obviously milder than that of group C. This study demonstrated that MT has protective effects on isolated rat heart.
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Cardiotónicos/farmacología , Creatina Quinasa/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Metalotioneína/biosíntesis , Metalotioneína/farmacología , Miocardio/metabolismo , Miocardio/ultraestructura , Distribución Aleatoria , Ratas Wistar , Superóxido Dismutasa/metabolismo , Sulfato de Zinc/farmacologíaRESUMEN
The feasibility of bone marrow stromal cells autologous transplantation for rabbit model of dilated cardiomyopathy induced by adriamycin was studied. Twenty rabbits received 2 mg/kg of adriamycin intravenously once a week for 8 weeks (total dose, 16 mg/kg) to induce the cardiomyopathy model with the monitoring of cardiac function by transthoracic echocardiography. Marrow stromal cells were isolated from cell-transplanted group rabbits and were culture-expanded on the 8th week. On the 10th week, cells were labeled with 4,6-diamidino-2-phenylindole (DAPI), and then injected into the myocardium of the same rabbits. The results showed that viable cells labeled with DAPI could be identified in myocardium at 2nd week after transplantation. Histological findings showed the injury of the myocardium around the injection site was relieved with less apoptosis and more expression of bcl-2. The echocardiography found the improvement of local tissue movement from (2.12+/-0.51) cm/s to (3.81+/-0.47) cm/s (P<0.05) around the inject site, but no improvement of heart function as whole. It was concluded bone marrow stromal cells transplantation for dilated cardiomyopathy was feasibe. The management of cells in vitro, the quantity and the pattern of the cells transplantation and the action mechanism still need further research.
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To investigate the effects of metallothionein (MT) on isolated rat heart, 16 Wistar rats were randomly divided into 2 groups. In control group (group C), distilled water was injected intraperitoneally and 24 h later isolated hearts were perfused with Langendorff and stored at 4℃ for 3 h with histidine-tryptophan-ketoglutarate (HTK) solutions, and then isolated hearts were perfused for 2 h by Langendorff. In experimental group (group E), 3.6% ZnSO4 was injected intraperitoneally, 24 h later isolated hearts were perfused by Langendorff and stored at 4℃ for 3 h with HTK solutions, and then the isolated herts were perfused for 2 h with Langendorff. MT content, the recovery of hemodynamics, myocardial water content (MWC), lactate dehydrogenase (LDH) and creatine kinase (CK) leakage, adenosine triphosphate (ATP) and malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, myocardial cell Ca2+ content, Ca2+-ATPase activity of mitochondria ([Ca2+-ATPase]m) and its Ca2+ content ([Ca2+]m), synthesizing ATP activity of mitochondria ([ATP]m), and the ultrastructure of cells were examined. There were a significant increase in group E in hemodynamic recovery, ATP content, SOD activity, [Ca2+-ATPase]m activity, [ATP]m activity, and substantial reduction in MWC, LDH and CK leakage, MDA content, myocardial cell Ca2+ content, [Ca2+]m content,and the ultrastructural injury were obviously milder than that of group C. This study demonstrated that MT has protective effects on isolated rat heart.
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The expression and changes of local cytokines network were detected in heart transplantation in rats, so as to determine the role of cytokines in the acute rejection of rats of heart transplantation. Allografts were divided into 4 groups (n=12 in each group): group A (control), group B (IL-2 monoclonal antibody-treated), group C (CsA-treated) and group D (IL-2 monoclonal antibody+CsA-treated). Hearts from DA rats were transplanted into a cervical location in Wistar recipients. The local expression of IL-1β, IL-2, CD25, IL-4, IL-5, IL-6, IL-10, TNFα and INFγ was detected at day 1, 3, 5, 7, 9, 11 and 14 by reverse transcription polymerase chain reaction. The results showed that the survival time of allografts was 8.3±1.7, 29.2±7.1 (P＜0.05), 26.4±5.7 (P＜0.05) and 55.0±10.6 (P＜0.01) days respectively in groups A, B, C and D. The expression of IL-1β, IL-4, IL-10and IFNγ was up-regulated, and that of IL-2, CD25, IL-5, IL-6 and TNFα was significantly inhibited in group A; The expression of IL-1β, IL-5, IL-6, IL-10 and IFNγ was up-regulated, and that of IL-2,IL-4 and TNFα was significantly down-regulated in group B; The expression of IL-1β, IL-2, CD25,IL-5, TNFα and IFNγ was up-regulated, and that of IL-4, IL-6 and IL-10 was significantly down-regulated in group C; The expression of IL-14, Il-5, IL-6 and Il-10 was up-regulated, and that of IL-1β, IL-2, CD25, TNFα and IFNγ was significantly down-regulated in group D. In conclusion,cytokines play an important role in the development of acute transplantation rejection. Different cytokines play different roles in different local environments.
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The expression and changes of local cytokines network were detected in heart transplantation in rats, so as to determine the role of cytokines in the acute rejection of rats of heart transplantation. Allografts were divided into 4 groups (n=12 in each group): group A (control), group B (IL-2 monoclonal antibody-treated), group C (CsA-treated) and group D (IL-2 monoclonal antibody+CsA-treated). Hearts from DA rats were transplanted into a cervical location in Wistar recipients. The local expression of IL-1beta, IL-2, CD25, IL-4, IL-5, IL-6, 1L-10, TNFalpha and INFgamma was detected at day 1, 3, 5, 7, 9, 11 and 14 by reverse transcription polymerase chain reaction. The results showed that the survival time of allografts was 8.3+/-1.7, 29.2+/-7.1 (P<0.05), 26.4+/-5.7 (P<0.05) and 55.0+/-10.6 (P<0.01) days respectively in groups A, B, C and D. The expression of IL-1beta, IL-4, IL-10 and IFNgamma was up-regulated, and that of IL-2, CD25, IL-5, IL-6 and TNFalpha was significantly inhibited in group A; The expression of IL-10, IL-5, IL-6, IL-10 and IFNgamma was up-regulated, and that of IL-2, L-4 and TNFalpha was significantly down-regulated in group B; The expression of IL-1beta, IL-2, CD25, IL-5, TNFalpha and IFNgamma was up-regulated, and that of IL-4, IL-6 and IL-10 was significantly down-regulated in group C; The expression of IL-14, 11-5, IL-6 and 11-10 was up-regulated, and that of IL-10, IL-2, CD25, TNFalpha and IFNgamma was significantly down-regulated in group D. In conclusion, cytokines play an important role in the development of acute transplantation rejection. Different cytokines play different roles in different local environments.
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Summary: Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow cytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90 %. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs.
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Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow cytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90%. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs.
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Integrina beta1/análisis , Receptores de Hialuranos/análisis , Células de la Médula Ósea/citología , Proliferación Celular , Separación Celular , Células Cultivadas , Citometría de Flujo , Células Madre Mesenquimatosas/citologíaRESUMEN
<p><b>OBJECTIVE</b>To investigate the role of anti- interleukin-2 receptor (CD25) monoclonal antibody in the regulation of cytokine mRNA expression of IL-1beta, IL-2, CD25, IL-4, IL-5, IL-6, IL-10, tumour necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) in cardiac allografts to elucidate its immunological mechanism and role in rats that have undergone cardiac transplantation.</p><p><b>METHODS</b>These in vivo studies were conducted using a rat MHC mismatch SD to Wistar heterotopic cardiac transplant model. Simulect, an anti-CD25 antibody, was used to prevent allograft rejection. An increase in the rate of allograft survival was observed. Rats were sacrificed on day 1, 3, 5, 7, 9, 11, 14 post-transplantation and hearts were harvested for further study. Cytokine mRNA expression was determined by semiquantitative RT-PCR.</p><p><b>RESULTS</b>In the control group, cardiac allografts were rejected at 8.3 +/- 1.7 days after transplantation (x +/- s). The rats who received CsA rejected the cardiac allograft at 26.4 +/- 5.7 days post-transplant. Allograft survival of Simulect-treated rats was 29.2 +/- 7.1 days (P < 0.05 vs controls). Rats treated with simulect and CsA had the longest survival of 55.0 +/- 11.6 days (P < 0.001 vs controls). CD25 mRNA expression in the heart tissue samples of treated rats was undetectable or very weak. However, the untreated group, CD25 expression increased, although anti-CD25 decreased this CD25 expression in the heart graft. Furthermore, in untreated allografts, IL-2, TNFalpha and IFN-gamma were strongly expressed, an effect that markedly decreased after simulect treatment. Finally, IL-4, IL-5, IL-6 and IL-10 expression was strong in anti-CD25-treated allografts.</p><p><b>CONCLUSIONS</b>These results suggest that anti-CD25 antibody treatment may not only neutralize CD25 activity but also play a role in altering cytokine mRNA expression and prolong the survival of allografts.</p>
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Animales , Masculino , Ratas , Anticuerpos Monoclonales , Usos Terapéuticos , Citocinas , Genética , Supervivencia de Injerto , Trasplante de Corazón , Alergia e Inmunología , ARN Mensajero , Ratas Sprague-Dawley , Ratas Wistar , Receptores de Interleucina-2 , Fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HomólogoRESUMEN
The cardioprotective effects of melatonin on recovery of rat donor hearts after 12 h of preservation were investigated. Wistar rats weighing 200 to 250 g (n=24) were randomly divided into 3 groups. In the non-storage group (n=8), donor hearts were not stored. In the melatonin group (n=8), donor hearts were stored in 4 degrees C St. Thomas solution with melatonin (0.1 mmol/L). In the control group (n=8), donor hearts were stored in 4 degrees C St. Thomas solution only. The coronary flow (CF), cardiac function, coronary vasodilatory response, creatine kinase (CK) and high energy phosphate levels were measured after the hearts had been preserved for 12 h. Transmission electron microscopy was used to examine the microstructural changes after 12 h of preservation. The recovery of cardiac function and coronary vasodilatory response were significantly improved in the melatonin group (P<0.01). CK release decreased greatly in the melatonin group (P<0.01). High energy phosphate levels were significantly better preserved in the melatonin group (P<0.01). Histological findings were much better in the melatonin group than in the control group. These results suggest that melatonin has cardioprotective effects on the recovery of rat donor hearts after 12 h of preservation.
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Animales , Humanos , Masculino , Ratas , Cardiotónicos , Farmacología , Creatina Quinasa , Metabolismo , Depuradores de Radicales Libres , Farmacología , Trasplante de Corazón , Radical Hidroxilo , Melatonina , Farmacología , Daño por Reperfusión Miocárdica , Miocardio , Metabolismo , Preservación de Órganos , Métodos , Distribución Aleatoria , Ratas Wistar , Factores de TiempoRESUMEN
To study the effects of different pH HEPES-KH reperfusate solution on immature myocardial protection, isolated perfused Langendorff model from immature rabbit hearts were developed formed. Control group (C) was perfused only with pH 7.4 HEPES-KH solution for 90 min. Ischemia/reperfusion group (group I/R) was perfused with pH 7.4 HEPES-KH solution before ischemia or after ischemia. Experimental group (group E), after ischemia, was perfused with pH 6.8, pH 7.1 and pH 7.4 HEPES-KH solutions for 5 min, 5 min, and 20 min, respectively. The left ventricular function recovery, MWC, LDH and CK leakage, MDA, ATP content, and SOD activity were determined. Our results showed that the left ventricular function recovery, ATP content and SOD activity in group E were higher than those of group I/R (P < 0.05). MWC, MDA content, LDH and CK leakage in group E were lower than those of group I/R (P < 0.05). These findings suggested that pH paradox might be one of important mechanisms for immature myocardial ischemia-reperfusion injury, and acidic perfusate, at the beginning of reperfusion, might attenuate pH paradox and ameliorate functional recovery in isolated perfused immature rabbit hearts.
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Animales , Femenino , Masculino , Conejos , Soluciones Cardiopléjicas , Farmacología , Corazón , Paro Cardíaco Inducido , Concentración de Iones de Hidrógeno , Isquemia Miocárdica , Metabolismo , Daño por Reperfusión Miocárdica , Metabolismo , Miocardio , Metabolismo , Distribución Aleatoria , Superóxido Dismutasa , MetabolismoRESUMEN
To study the effects of different pH HEPES-KH reperfusate solution on immature myocardial protection, isolated perfused Langendorff model from immature rabbit hearts were developed formed. Control group (C) was perfused only with pH 7.4 HEPES-KH solution for 90 min. Ischemia/reperfusion group (group I/R) was perfused with pH 7.4 HEPES-KH solution before ischemia or after ischemia. Experimental group (group E), after ischemia, was perfused with pH 6.8, pH 7.1 and pH 7.4 HEPES-KH solutions for 5 min, 5 min, and 20 min, respectively. The left ventricular function recovery, MWC, LDH and CK leakage, MDA, ATP content, and SOD activity were determined. Our results showed that the left ventricular function recovery, ATP content and SOD activity in group E were higher than those of group I/R (P < 0.05). MWC, MDA content, LDH and CK leakage in group E were lower than those of group I/R (P < 0.05). These findings suggested that pH paradox might be one of important mechanisms for immature myocardial ischemia-reperfusion injury, and acidic perfusate, at the beginning of reperfusion, might attenuate pH paradox and ameliorate functional recovery in isolated perfused immature rabbit hearts.
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Miocardio/metabolismo , Distribución Aleatoria , Superóxido Dismutasa/metabolismoRESUMEN
Objective To study the changes of ICAM-1 expression in transplanted lung at the early stage after rat pulmonary transplantation.Methods The expression level of ICAM-1 protein and its mRNA in rat transplanted lung after 4 h of ventilation and reperfussion were detected by using immunohisto chemical method(SABC method)and semiquantitative reverse transcriptase polymerase reaction method in the experimental group(n=6).When donor lungs had been flushed with low potassium solution(LPDS) and preserved in 4.C LPDS for 12 h,left orthotopic pulmonary transplantation was performed.In control group (n=6), left pulmonary artery,pulmonary vein and bronchi were fully freed to be naked.Immunohistochemical test results were recorded in negative(-),suspicion(±),faint positive(+),positive(++)and intensive positive(+++).PCR products were separated by agrose gels and the density of the bands were determined by density scanning.Results The stained color of alveolar epithelial cells and pulmonary vascular endothdial cells in the transplanted lung was significantly more intensive in experimental group than in control group(P＜0.01) and the relative density values(ICAM-1/βactin)were also significandy higher in experimental group(0.837±0.044) than in control group (0.442±0.037),P＜0.01. Conclusions ICAM-1 expression in the transplanted lung was up-regulated in the early stage after pulmonary transplantation,which was related with the enhancement of ICAM-1 mRNA.
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ObjectiveTo study the dynamic changes of rat donor myocardial cell apoptosis during hypot hermic preservation and the effect of melatonin.MethodsEighty rats were randomly divided into two groups: experimental group with the i solated rat hearts being preserved in 4?℃ St Thomas solution with melatonin ( 0.1?mmol/L ); control group with the isolated rat hearts being preserved in 4?℃ St Thomas solution only. After preservation for 4, 8, 12, 24 and 36?h, a poptotic index was evaluated by the percentage of mycardial cells using TUNEL po sitive staining.ResultsWith the hypothermic preservation time prolonging, more myocardial cell apoptosi s was found (control group, P