ObjectiveTo find out an effective method for amplification and purification of dendritic cells(DC) from peripheral blood of patients with pancreatic carcinoma. MethodsPeripheral blood mononuclear cells were purified from peripheral blood of health volunteers(control group,10 cases) and patients with pancreatic carcinoma (experimental group,12 cases) with incubation of granulocyte/macrophage colonystimulating factor(GMCSF) and interleukin4(IL4).The quality of DC were detected by immumofluorescence method and the expression levels of HLADR and B72 on DC were detected by flow cytometer after and before DC incubation with GMCSF and the IL4. ResultsThe expression level of HLADR and B72 of DC in experimental group were significantly less than those in control group(P＜0.01).DC in experimental group was significantly proliferated in the presence of GMCSF and IL4(P＜0.01).On day 7,the expression level of HLADR and B72 of DC in experimental group were significantly increased(P＜0.01) and there was no difference versus control group(Pgt;0.05).ConclusionIt is suggested that combination of GMCSF and IL4 can selectively and effectively enhance proliferation and immune function of DC from peripheral blood of patient with pancreatic carcinoma.
Objective To investigate the effect of peroxisome proliferatoractivated receptor-γ coactivator-1α(PGC-1α) on early ischemic preconditioning (IPC) which may act as an important role in early IPC. Methods Building isolated working rat heart Langendorff model, thirty Wistar rats were divided randomly into three groups. Control group(CON group,n=10): a 120-min perfusion was performed without any intervension; ischemia and reperfusion group(I/R group,n=10): a 30-min equilibration period perfusion, a 30-min ischemia and a 60-min reperfusion were performed.; IPC group (n=10): a 10-min equilibration period perfusion was performed, then was elicited by two cycles of 5-min of ischemia interspersed with 5-min reperfusion prior to 30-min ischemia and a 60-min reperfusion. Frozen sections of myocardium at cardiac apex were made and immunohistochemical staining was used to detect expression and the intergrated optical density average (IODA) of PGC-1α. Ultrathin sections were made and the mitochondria under each specimen was evaluated according to Flameng score. Results PGC-1α expression in IPC group (IODA 10.94±5.23) was significantly higher than that in I/R group (IODA 3.88±1.72) and that in CON group (IODA 3.39±2.46; P=0.009, 0.007). The mitochondria changes in I/R group were significant edema and severe damage; but there were not so severe in CON group and IPC group.Flameng score of IPC group (0.44±0.13) and CON group (0.88±0.22) were lower than that in I/R group(1.78±0.14;P=0.003, 0.014) respectively. Conclusion IPC can protect myocytes mitochondria from ischemia and reperfusion.The cardioprotection may be related with the activation and the high expression of PGC-1α, which may act as one of the most important endogenous defence factors of the heart.
Abstract: Objective To investigate the effects of haemopoietic stem cell mobilization on vein graft patency and intimal hyperplasia of anastomosis. Methods Twentyfour New Zealand rabbits were randomly divided into experimental group and control group, 12 rabbits in each group. A double side of carotid arteryvein transplantation model was made in each rabbit. One side of vein graft was digested by 0.25% trypsin for complete endothelial denudation before transplantation. Recombinant human granulocyte colonystimulating factor was given by subcutaneous injection 24 hours after operation, once per day in successive 10 days in experimental group, saline was given in the same way in control group. Bone marrow stem cells mobilization was observed after operation, including karyote counts and mononuclear cell proportion in peripheral blood. The patency rate of vein grafts and the degree of anastomosis intimal hyperplasia were observed too. Results The karyote counts (t=8.406,P=0.000)and mononuclear cell proportion(t=31.267,P=0.000) in peripheral blood of experimental group increased significantly 5 days after operation than those in control group. The vein grafts with intact endothelium had higher patency rate in both groups. In the vein grafts with complete endothelial denudation, the patency rate were obviously lower, but it was higher in experimental group than those in control group (67% vs. 30%). In the end of experiment, the pulsatility index of the vein grafts anastomosis with complete endothelial denudation was lower in experimental group than that in control group(t=2.958,P=0.009). Pathological examination showed that various degrees of intimal hyperplasia in all anastomoses of vein grafts were observed 4 weeks after operation. The degree of anastomosis intimal hyperplasia was more severe in vein grafts with complete endothelial denudation. Compared with control group, re-endothelization occurred completely in vein grafts with complete endothelial denudation of experimental group and the degree of anastomosis intimal hyperplasia was relatively lower (Plt;0.05). Conclusion Haemopoietic stem cell mobilization can provide protective effects on vein grafts by accelerating reendothelization which might increase vein grafts patency rate in the near future after operation and reduce anastomosis restenosis caused by intimal hyperplasia.
Objective To observe the effect of transfer of immature mouse myeloid dendritic cells （DC） generated with low-dose granulocyte-macrophage colony-stimulating factor （GM-CSF） on cardiac allograft survival. Methods Mouse DC were generated with standard doses or low doses GM-CSF from bone marrow cells, the phenotype and functional properties of these DC were compared through fluorescence-activated cell sorting（FACS） analysis and mixed lymphocyte reaction（MLR）, 1. 0 × 106 DC generated with low doses GM-CSF were administered to the recipients 7 days before transplantation, and the cardiac allograft survival were observed. Results In contrast to DC generated with standard doses, DC generated with low doses were phenotypically immature DC （CD11c＋, CD80- , CD86- , MHCⅡlow）, and induced allogeneic T cell unresponsiveness, and administration of these DC to recipients prolonged cardiac allograft survival from 6.3±1.2 days to 14.3±1.9 days. Conclusions DC generated from mouse bone marrow progenitors in low doses of GM-CSF are phenotypically and functionally immature, and prolong cardiac allograft survival when they are administered 7 clays before transplantation.
Objective To investigate the mechanisms of local application of granulocyte macrophage- colony stimulating factor (GM-CSF) on healing of colonic anastomoses impaired by intraperitoneal oxaliplatin in rats. Methods Sixty 10-week-old male Wistar rats were made the colonic anastomosis model and randomized into 3 groups, 20 rats in each. The rats received intraperitoneal injection of 5% dextrose in group A, and intraperitoneal injection of 5% dextrose and 10 mL oxaliplatin (25 mg/kg) in group B at 1 day; and 50 μg GM-CSF was injected into the perianastomotic area immediately after operation and 10 mL intraperitoneal oxaliplatin (25 mg/kg) was given at 1 day. The general situation of rats was observed after operation. Anastomotic healing was tested by measuring the bursting pressure in vivo at 2, 3, 5, 7 days. Anastomotic healing score was evaluated by histological staining. Immunohistochemical staining of the anastomotic site was used to determine the amount of collagen type I content. Results All animals survived to the experiment end. There was no significant difference in the bursting pressure among 3 groups at 2 and 3 days (P gt; 0.05); the bursting pressure of group B was significantly lower than that of groups A and C (P lt; 0.05). There was no significant difference in mononuclear cells infiltration, mucosal epithelialization, submucosa-muscle layer connection degree, and granulation tissue formation between groups A and C at different time points (P gt; 0.05); groups A and C were significantly better than group B in mucosal epithelialization and granulation tissue formation (P lt; 0.05). Groups A and C were significantly better than group B in mononuclear cells infiltration at 2 and 3 days, and in submucosa-muscle layer connection degree at 5 and 7 days (P lt; 0.05). There was no significant difference in collagen type I content among 3 groups at 2 and 3 days (P gt; 0.05); the content of collagen type I in groups A and C were significantly higher than that in group B (P lt; 0.05) at 5 and 7 days. Conclusion Local administration of GM-CSF may enhance colonic anastomotic healing by early stimulating infiltration of macrophages and increasing collagen deposition.
Objective To investigate the effect of combined therapy of granulocyte colony stimulating factor (G-CSF) and bone marrow mesenchymal stem cells (BMSCs) carrying hepatocyte growth factor (HGF) gene on the angiogenesis of myocardial infarction (MI) in rats and the mechanisms of the synergistic effect. Methods BMSCs were aspirated from the femur and tibia of 3-week-old Sprague Dawley (SD) male rats. The third generation of BMSCs were harvested and transfectedwith Ad-HGF. The MI models were establ ished in 44 SD male rats (weighing 200-250 g) by l igating the left coronary artery. At 4 weeks after l igation, the shorting fraction (FS) of the left ventricle being below 30% was used as a criteria of model success. The BMSCs (5 × 107/ mL) transfected with Ad-HGF were transplanted into the infarct zone of 12 SD rats, and the expression of HGF protein was detected by Western blot method at 2, 7, and 14 days after transplantation. At 4 weeks, the other 32 SD rats were randomly divided into 4 groups (n=8). The 0.1 mL normal sal ine was injected into the infarct zone in control group; 0.1 mL normal sal ine was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in G-CSF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in combined therapy group. At 2 weeks after transplantation, heart function was detected by cardiac ultrasound and hemodynamic analysis, and then myocardial tissue was harvested to analyse the angiogenesis of the infarct zone, and the expression of VEGF protein by immunofluorescence staining. Results The expression of HGF protein in vivo was detected at 2 days and 7 days of BMSCs transfected with Ad-HGF transplantation. There was no significant difference in left ventricular systol ic pressure (LVSP), left ventricular end-diastol ic pressure (LVEDP), dP/dtmax, and FS between G-CSF group and control group (P gt; 0.05). When compared with the control group, LVEDP decreased significantly; LVSP, FS, and dP/dtmax increased significantly (P lt; 0.05) in HGF group and combined therapy group. When compared with HGF group, FS and dP/dtmax increased significantly in combined therapy group (P lt; 0.05). Immunofluorescence staining showed that the vascular endothel ial cells were observed in myocardial infarction border zone. The vascular density and the expression of VEGF protein were significantly higher in combined therapygroup than in other 3 groups (P lt; 0.05). Conclusion The combined therapy of G-CSF and BMSCs carrying HGF gene has a synergistic effect and can enhance infarct zone angiogenesis through inducing the expression of VEGF protein.
Objective To investigate the effectiveness and mechanism of recombinant human granulocyte-macrophage colony-stimulating factor (rhGMCSF) gel on wound debridement and healing of deep II thickness burn. Methods Between December 2008 and December 2010, 58 patients with deep II thickness burn, accorded with the inclusive criteria, were collected. There were 36 males and 22 females with an average age of 32.4 years (range, 12-67 years). The causes were hot liquid in 38 cases and fire in 20 cases. The time from injury to treatment was 1-3 days (mean, 2.1 days). In this randomized, double-blind, and self-control study, all patients were randomly divided into 2 groups, wounds were treated with rhGMCSF gel (test group) or gel matrix (control group). There was no significant difference in wound area between 2 groups (P gt; 0.05). The time of completed removal eschar and the percentage of removal-area of eschar were recorded at 2, 6, 10, 14, and 18 days during healing process. The time of wound healing was also recorded. Results Compared with control group, the necrotic tissues on the burn wound got soft in test group at 4 days after treatment. At 6 days, they loosened and eventually sloughed off. The wound bed presented in red and white, followed by rapidly growing granulation tissues. Except 18 days after treatment, there were significant differences in the percentage of removal-area of eschar between 2 groups (P lt; 0.05). The time of completed removal eschar in test group [(7.71 ± 2.76) days] was significantly shorter than that in control group [(14.71 ± 3.63) days] (t=13.726, P=0.000). The time of wound healing in test group was (18.41 ± 2.47) days, which was significantly shorter than that in control group [(23.58 ± 3.35) days] (t=15.763, P=0.000). Conclusion Compared with the gel matrix, the rhGMCSF gel may promote wound debridement and healing in deep II thickness burn.
Objective To investigate the effects of granulocyto-colony stimulating factor (G-CSF) on the mobil ization of endothel ial progenitor cells (EPCs) in the rats with myocardial infarction (MI), to observe the density of neovascularization and the mRNA expressions of vascular endothel ial growth factor (VEGF) and its receptor (Flk-1) in the border area of MI. Methods Thirty-six adult male rats (weighing 250-280 g) were divided randomly into control group, MI group, and G-CSF group. In MI group and G-CSF group, the models of MI were establ ished by left anterior descenting coronary artery l igation and were treated with intraperitoneal injection of sal ine (0.3 mL/d) or G-CSF [30 μg/(kg•d)] for 5 days. In control group, after open chest operation, chest was closed without treatment. The level of EPCs was surveyed and the plasma concentrations of VEGF and C-reaction protein (CRP) were measured at 7 days. The mRNA expressions of VEGFand its receptor Flk-1 in the border area of infarct myocardium were determined through RT-PCR. Results Compared withcontrol group, the number of circulating white blood cell (WBC) and EPCs levels, and the serum concentrations of VEGF and CRP were all significantly increased in MI group and G-CSF group (P lt; 0.05); when compared with MI group, the number of circulating WBC and EPCs levels, and the serum concentrations of VEGF were increased and the concentration of CRP was decreased in G-CSF group (P lt; 0.05). Compared with control group, the mRNA expressions of VEGF and Flk-1, and the density of neovascularization in the border area of infarct myocardium were increased in MI group and G-CSF group, whereas those in G-CSF group were significantly augmented compared with MI group (P lt; 0.05). Conclusion In the rats with MI, G-CSF could promote EPCs mobil ization, increase the mRNA expressions of VEGF and Flk-1, and augment the density of neovascularization in the border area of infarct myocardium.