Bibenzyl is a type of active compounds abundant in Dendrobium. In the present study, we investigated the inhibitory effects of six bibenzyls isolated from Dendrobium species on vascular endothelial growth factor (VEGF)-induced tube formation in human umbilical vascular endothelial cells (HUVECs). All those bibenzyls inhibited VEGF-induced tube formation at 10 micromol x L(-1) except tristin, and of which moscatilin was found to have the strongest activity at the same concentration. The lowest effective concentration of moscatilin was 1 micromol x L(-1). Further results showed that moscatilin inhibited VEGF-induced capillary-like tube formation on HUVECs in a concentration-dependent manner. Western blotting results showed that moscatilin also inhibited VEGF-induced phosphorylation of VEGFR2 (Flk-1/KDR) and extracellular signal-regulated kinase 1/2 (ERK1/2). Further results showed that moscatilin inhibited VEGF-induced activation of c-Raf and MEK1/2, which are both upstream signals of ERK1/2. Taken together, results presented here demonstrated that moscatilin inhibited angiogenesis via blocking the activation of VEGFR2 (Flk-1/KDR) and c-Raf-MEK1/2-ERK1/2 signals.

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.

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.

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.

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.
Cardiotonic Agents/*pharmacology ; Creatine Kinase/*metabolism ; L-Lactate Dehydrogenase/metabolism ; Metallothionein/biosynthesis ; Metallothionein/*pharmacology ; Myocardium/*metabolism ; Myocardium/ultrastructure ; Random Allocation ; Rats, Wistar ; Superoxide Dismutase/metabolism ; Zinc Sulfate/pharmacology

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.

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.

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.
Antigens, CD29/analysis ; Antigens, CD44/analysis ; Bone Marrow Cells/*cytology ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Flow Cytometry ; Mesenchymal Stem Cells/*cytology

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.

OBJECTIVETo 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.

METHODSThese 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.

RESULTSIn 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.

CONCLUSIONSThese 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.

Animals ; Antibodies, Monoclonal ; therapeutic use ; Cytokines ; genetics ; Graft Survival ; Heart Transplantation ; immunology ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Receptors, Interleukin-2 ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Homologous