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 observe the effects of mechanical stretch on cytokines release from alveolar macrophages( AMs) and the expression of macrophage inflammatory protein-2( MIP-2) induced by lipopolysaccharide( LPS) . Methods AMs were divided into the following groups: ①AMs were subjected to 20% elongation by Flexercell 4000T cell stress system for 24 hours and the supernatant was collected to detect the levels of TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, macrophage inflammatory protein-1α( MIP-1α) , MIP-2, monocyte chemoattractant protein-1( MCP-1) , granulocyte /macrophage colony stimulating factors( GM-CSF) , interferon inducible protein-10( IP-10) , regulated on activation in normal T-cell expressed and secreted( Rantes) and keratinocyte chemoattractant( KC) , by using LiquiChip system. ② AMs were subjected to 5% , 10% , 15% and 20% elongation for 24 hours and the supernatant was collected to detect the levels of MIP-2. ③AMs were subjected to 20% elongation and MIP-2 in supernatant was detected 1, 3,6, 12, and 24 hours later. ④ AMs were subjected to 20% elongation and/ or LPS at a concentration of 10 ng/mL, and MIP-2 in supernatant was detected 24 hours later. Unstretched AMs were used as control in all kind of test. Results ①The levels of IL-1β, IL-6,MIP-2, MCP-1, IFN-γand IP-10 secreted by stretched AMs were 8. 7, 4. 3, 38. 6, 4. 8, 14. 2 and 5. 0 times those of the control group( all P lt; 0. 001) . ② The levels of MIP-2 secreted by AMs subjected to 10% , 15% and 20% elongation were ( 480. 5 ±93. 1) pg /mL,( 806. 3 ±225. 9) pg/mL and ( 1335. 7 ±18. 5) pg/mL respectively, all significantly higher than those oft he control group [ ( 34. 6 ±11. 4) pg/mL, all P lt;0. 001] . ③ Three hours after the stimulation of stretch the level of MIP-2 began to increase gradually. And 6, 12, and 24 hours after the stimulation the levels of MIP-2 secreted by the AMs were ( 819. 4 ±147. 5) pg/mL, ( 1287. 6 ±380 ±3 ) pg/mL and ( 1455. 9 ±436. 7) pg/mLrespectively, all significantly higher than those of the control group[ ( 33. 4 ±10. 2) pg/mL, all P lt; 0. 001] . ④When the AMs were stimulated individually by LPS( 10 ng /mL) or mechanical stretch ( 20% ) , the levels of MIP-2 increased to ( 1026. 3 ±339. 5 ) pg/mL and ( 1335. 7 ±318. 5 ) pg/mL respectively( both P lt; 0. 001) . When the AMs were costimulated by LPS and mechanical stretch, the level of MIP-2 increased to ( 2275. 3 ±492. 1) pg/mL, implicating a synergistic effect between mechanical stretch and LPS ( F = 121. 983, P lt; 0. 001) . Conclusions Mechanical stretch activates AMs to produce multiple inflammatory cytokines and induce AMs to secret MIP-2 in a strength- and time-dependent manner.Mechanical stretch also has synergistic effect with LPS in inducing MIP-2 release, which might play an important role in the development of ventilator-induced lung injury.
Objective To evaluate the clinical, radiological and pathological manifestations of lipoid pneumonia, to improve the diagnosis of this rare disease. Methods Retrospective analysis of clinical data of 19 cases with confirmed lipoid pneumonia was conducted. Results There was 13 male and 6 female patients with an average age of 54. 4 years ranging from 24 to 71 years. Lipoid pneumonia presented a chronic clinical course with few physical signs. The main complaints included cough, expectation, bloody phlegmand chest pain. Finding of chest mass in routine physical examination was also a common reason for doctor visiting. Radiological features of lipoid pneumonia could be non-specific, usually presenting mass shadow in the chest, with lymph nodes enlargement in some cases. Eighteen cases were diagnosed by the histopathology of pulmonary tissue and one case was diagnosed through a combination of bronchoscopy, thoracoscopy and CT-guided percutaneous lung puncture biopsy. Histopathological finding showed accumulation of foamy macrophages in the alveolar spaces. Conclusions The clinical and radiological manifestations of lipoid pneumonia are non-specific. Histopathological examination for diagnosis is essential. If the medical therapy is not responsive, pulmonary lobectomy might be necessary.
Objective To explore the expression of myeloid differentiation protein2 ( MD-2) in rat lung and its role in acute lung injury ( ALI) induced by lipopolysaccharide ( LPS) . Methods Twenty male SD rats were randomly divided into a LPS group and a control group. The wet/dry ratios of lung tissues were measured and the histological changes of lung tissues were observed under microscope. Alveolar macrophages were collected from bronchial alveolar lavage fluid ( BALF) . The MD-2 mRNA and protein expressions were detected by RT-PCR, Western blot, and immunohistochemistry respectively. The MD2-siRNA oligo were transfected into NR8383 cells and 1 μg/mL LPS was used to stimulate the cells. The expressions of MD-2 mRNA and protein were detected by RT-PCR and Western blot. The levels of TNF-αin rat serum and cell culture supernatant were detected by ELISA. Results Compared with the control group, the expressions of MD-2 mRNA and protein in alveolar macrophages and lung tissue were elevated ( P lt;0. 01) , as well as the level of TNF-αin rat serum. The expressions of MD-2 mRNA and protein in NR8383 cell and the level ofTNF-αin supernatant increased obviously after LPS stimulation ( P lt;0. 01) . There were no changes of MD-2 mRNA and protein expressions and TNF-α of NR8383 cells treated by MD-2 siRNA with or without LPS stimulation ( P gt;0. 05) . Conclusions The expression of MD-2 in lung increases obviously after challengedby LPS. KnockdownMD-2 gene of NR8383 cell byMD-2 siRNA can inhibit TNF-αsecretion induced by LPS stimulation.MD-2 may play an important role in rat ALI induced by LPS.
Objective To explore the role of macrophage-stimulating protein ( MSP) and receptor tyrosine kinase RON in the airway inflammation of chronic obstructive pulmonary disease( COPD) , and investigate its possible mechanism. Methods The rat COPDmodel was established by exposing the rats to cigarette smoke daily for three months. Rat alveolar macrophages ( AMs) were isolated in vivo and cultured,and then challenged with different concentrations of MSP for 24 hours. The concentrations of MSP in broncho-alveolar lavage fluid ( BALF) and serum, and the levels of IL-1β, TNF-α, IL-8, and IL-10 in the supernatants were measured by ELISA. The expression of RONmRNA in lung tissue was assessed by reverse transcription-polymerase chain reaction. The levels of RON protein in the lung tissue and AMs cultured in vitro were observed by immunohistochemistry. The activity of superoxide dismutase ( SOD) and malondialdehyde ( MDA) content in the culture solution were measured with chromatometry method. Results Compared with the control group, the concentrations of MSP in serum and BALF of the COPD rats were significantly higher ( P lt;0. 01) . The levels of RONmRNA and RON protein in the COPD rats were also upregulated significantly ( P lt; 0. 01) . MSP evoked the AMs isolated from the normal and COPD rats to generate more content of MDA and caused a reduction in activity of SOD. In addition, MSP stimulated TNF-α, IL-8, IL-1βand IL-10 release fromAMs of the normal and COPD rats dose-dependently. The levels of TNF-α, IL-8, and IL-1βwere higher, while the level of IL-10 and the SOD activity were lower in AMs of the COPD group than those of the control group in the same dose of MSP ( P lt;0. 01) . The more significant increase in the levels of TNF-α, IL-8, IL-1β, and the more notable decrease in the activity of SOD was found in the COPD group compared with the control group. But the degree of increasing MDA and IL-10 in the AMs of the COPD group was lower than that in the control group. Linear correlation analysis showed that the MSP concentration and the RON protein level in the COPD rats were positively associated with the total cellcounts and AM counts in BALF, and were related to the indexes for pulmonary emphysema. Conclusions There is a close correlation between the MSP and receptor tyrosine kinase RON with the airway inflammation of COPD. The mechanism might be that MSP promote the macrophages release inflammatory factors and increase the production of oxygen free radicals.
Objectives To investigate the effects of the distribution of tumor associated macrophages (TAMs) on prognosis in the patients with non-small cell lung cancer. Methods The number of CD68+ macrophages in 136 lung cancer nest and stroma was counted simultaneously by labelled streptavidin biotin method(LSAB）,and its correlation with patient postoperation prognosis was analyzed. Results CD68 macrophas were observed in both inside and around the cancer tissue,The mean TAMs in cancer stroma (36.00/HFP) was higher than that in cancer nest (23.80/HFP,Plt;0.05). Mean TAMs in nest of stage Ⅰ＋Ⅱ cancer was significantly higher than that of stageⅢ＋Ⅳ cancer（32.60/HFP vs. 14.80/HFP,Plt;0.05）,and mean TAMs in stroma of stage Ⅰ＋Ⅱ cancer was significantly lower than that of stage Ⅲ＋Ⅳ cancer（24.30/HFP vs. 47.60/HFP,Plt;0.05）.The number of TAMs in cancer nest and the ratio of nest TAMs /stoma TAMs were both positively correlated with the patient survival time (rs=0.510, 0.633, respectively). Otherwise the number of TAMs in cancer stroma was negatively correlated with the patient survival time (rs=-0.187). Five-year survival rate in patients with high density TAMs in cancer nest was significantly higher than that in patients with low density TAMs (51.4% vs. 11.1%, Plt;0.05), while reverse correlation between TAMs in cancer stroma and patient 5-year survival rate was observed (18.9% vs. 44.4%,Plt;0.05). And 5 year suvival rate in patients with high ratio of nest/stroma TAMs was higher than that with low ratio (58.1% vs.4.2%,Plt;0.01). Conclusion Cox regressive prognostic analysis showed that the higher the nest/stroma TAMs ratio, the higher probability of the patients survival time. While the higher number of TAMs in the cancer stroma, the lower probability of the patients survival time. Our results showed that distribution pattern of TAMs in cancer nest and cancer stroma could possibly be used to estimate the prognosis of patients with non-small cell lung cancer.
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 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 observe the changes in the number and function of bone marrow-derived endothel ial progenitor cells (EPCs) after bone-marrow stimulation, and to investigate the possible mechanism of improving ischemicl imb disease after bone-marrow stimulation through autologue bone-marrow stem cell implantation. Methods Twelvemale Lewis rats, weighing 200-250 g, were classified into the bone marrow stimulation group (n=6) and the control group(n=6). In the stimulation group, the bone marrow of each rat was stimulated by injection of recombinant human granulocytemacrophage colony-stimulatory factor. Mononuclear cells were harvested from bone marrow and cultured in EBM-2 medium. After 7-day culture, EPCs were stained by 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labbled acetylated low density l ipoprotein/fluorescein isothiocyanate-ulex europaeus agglutinin 1, and the double positive cells were counted by the fluorescent microscope. The adhesive abil ity of EPCs was determined by counting the number of re-cultured EPCs. The unilateral ischemia hindl imb model was made with 12 Lewis rats. Three days later, EPCs were transplanted into the ischemic tissues. According to different sources of EPCs, the 12 rats were divided into 2 groups: the stimulation group (n=6) and the control group (n=6). At 3 weeks after EPCs transplantation, the quantity of the collateral vascular was observed by digital subtraction angiography (DSA). Results After 7-day culture, the number of EPCs in the stimulation and control groups was (145.2 ± 37.0)/HP and (95.2 ± 39.4)/HP, respectively, and there was significant difference between the two groups (P lt; 0.05). Meanwhile, the number of adhesive EPCs in the stimulation and control groups was (21.8 ± 4.3)/HP and (15.0 ± 5.2)/HP, respectively, and the difference between the two groups was significant (P lt; 0.05). At 3 weeks after the EPCs implantation, the number of the collateral vascular was significantly larger in the stimulation group (4.2 ± 1.2) compared with the control group (2.7 ± 0.8), (P lt; 0.05). Conclusion Bone marrow stimulation increases the number of EPCs and improves the function concurrently, which may be the reason why autologue bone-marrow stem cell implantation improves the curative effect of ischemic l imb diseases after bone-marrow stimulation.