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 effect of human tooth bone graft materials on the proliferation, differentiation, and morphology of macrophages, and to understand the biocompatibility and cytotoxicity of human tooth bone graft materials. Methods Fresh human teeth were collected to prepare human tooth bone graft materials, the adhesion of mouse mononuclear macrophages RAW264.7 to human bone graft materials was observed under confocal microscopy. Scanning electron microscopy was used to observe the morphology of human tooth bone graft materials, OSTEONⅡ synthetic highly resorbable bone grafting materials, and untreated tooth powder (dental particles without preparation reagents). Different components of the extract were prepared in 4 groups: group A (DMEM medium containing 10% fetal bovine serum), group B (human tooth bone graft materials), group C (OSTEONⅡ synthetic highly resorbable bone grafting materials), group D (untreated tooth powder without preparation reagents). The 4 groups of extracts were co-cultured with the cells, and the cytotoxicity was qualitatively determined by observing the cell morphological changes by inverted microscope. The cell proliferation and differentiation results and cell relative proliferation rate were determined by MTT method to quantitatively determine cytotoxicity. The cell viability was detected by trypanosoma blue staining, and tumor necrosis factor α (TNF-α ) and interleukin 6 (IL-6) expressions were detected by ELISA. Results Scanning electron microscopy showed that the surface of the human tooth bone graft material and the OSTEONⅡ synthetic highly resorbable bone grafting materials had a uniform pore structure, while the untreated tooth particle collagen fiber structure and the demineralized dentin layer collapsed without specific structure. Confocal microscopy showed that the cells grew well on human tooth bone graft materials. After co-culture with the extract, the morphology and quantity of cells in groups A, B, and C were normal, and the toxic reaction grades were all grade 0, while group D was grade 3 reaction. MTT test showed that the cytotoxicity of groups B and C was grade 0 or 1 at each time point, indicating that the materials were qualified. The cytotoxicity was grade 2 in group D at 1 day after culture, and was grade 4 at 3, 5, and 7 days. Combined with cell morphology analysis, the materials were unqualified. The trypanosoma blue staining showed that the number of cells in groups A, B, and C was significantly higher than that in group D at each time point (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). ELISA test showed that the levels of TNF-α and IL-6 in groups A, B, and C were significantly lower than those in group D (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). Conclusion The human tooth bone graft materials is co-cultured with mice mononuclear macrophages without cytotoxicity. The extract has no significant effect on cell proliferation and differentiation, does not increase the expression of inflammatory factors, has good biocompatibility, and is expected to be used for clinical bone defect repair.
Objective To investigate the role of calcium- and integrin-binding protein-1（CIB1） in oxidized lowdensity lipoprotein（OX-LDL） inhibiting migration of mouse macrophages. Methods To silence CIB1 express of mouse macrophages by RNA interference, then incubating mouse macrophages with OX-LDL, cell migration and cell spreading of mouse macrophages were analyzed. Results At 24-72h after macrophages transfected CIB1 siRNA, the express of CIB1 protein was restrained obviously. To silence CIB1 express could increase migration and spreading of mouse macrophages significantly. Conclusions CIB1 plays the important role in intracellular modulating mechanism of OX-LDL inhibiting mouse macrophages migration.
ObjectiveTo investigate the changes of peritoneal macrophages function of mice with gastric cancer in the CO2 pneumoperitoneum environment, as well as its effect on the peritoneal metastasis of gastric cancer. MethodsAn orthotopic implantation model of mouse forestomach cancer was established using the 615 mouse. The mice bearing tumors were randomly divided into five groups (n=30): anesthesia alone, laparotomy, and 2, 4, and 6 mm Hg CO2 insufflation groups. Peritoneal macrophages were collected from six mice in each group and cultured. The macrophage phagocytic function on neutral red and the levels of NO and TNF-α produced by macrophages were measured after 12, 24, 48, and 72 h of culture. The remaining mice were observed after two weeks for the rate of peritoneal metastasis of forestomach cancer cells and the total weight of implanted nodules. ResultsNo death and ascites were found and the difference of weight body was not significant in all mice (Pgt;0.05). The uptake of neutral red by peritoneal macrophages and the levels of NO and TNF-α secreted by peritoneal macrophages in the laparotomy group after 12 h of culture were all significantly higher than those in other four groups (Plt;0.05). The corresponding values in the 2, 4， and 6 mm Hg CO2 insufflation groups after 12 h were all significantly lower than those in the anesthesia alone group (Plt;0.05). Among three insufflation groups, the corresponding values in the 2 mm Hg after 12 h were significantly higher than those in the 4 and 6 mm Hg CO2 insufflation group, though the difference in the two latter was not significant (Pgt;0.05). The uptake of neutral red by peritoneal macrophages and the levels of NO and TNF-α secreted by peritoneal macrophages in the 6 mm Hg CO2 insufflation group after 24 h of culture were all significantly lower than those in other four groups (Plt;0.05), while the difference in the four groups was not significant (Pgt;0.05). The uptake of neutral red by peritoneal macrophages and the levels of NO and TNF-α secreted by peritoneal macrophages after 48 h and 72 h were not significantly different in the five groups (Pgt;0.05). The rate of peritoneal metastasis of mice was significantly lower in the 6 mm Hg insufflation CO2 group (75.0%, 15/20) than that in the anesthesia alone group (100%, 24/24), Plt;0.05, but higher than other three groups(Plt;0.05), which was not different in 2 mm Hg (47.8%, 11/23), 4 mm Hg insufflation group (45.45%, 10/22) and laparotomy group (50.0%, 10/20), Pgt;0.05. The total weight of implanted nodules of mouse forestomach cancer was (1.24±0.48) g, (1.02±0.38) g, (0.96±0.33) g, (0.93±0.45) g, and (1.18±0.37) g in the anesthesia alone group, the laparotomy group, and 2, 4, and 6 mm Hg CO2 insufflation group, and the difference was not significant (Pgt;0.05). ConclusionHigh pressure (6 mm Hg) CO2 pneumoperitoneum can constantly inhibit the phagocytosis and cytokine secretion functions of peritoneal macrophages in gastric cancer-bearing mice and promote peritoneal implantation of gastric cancer.
Objective To explore the association of macrophages with carcinogenesis and development of gastric cancer. Method The related literatures at home and abroad were consulted and reviewed. Results The microenvironment of gastric cancer could induce the polarization of macrophages，and then the activated macrophages，especially the tumor associated macrophages，could in turn motivate the growth，invasion，and metastasis of tumor cells by secreting a series of active substances. Conclusions Macrophages，especially the tumor associated macrophages play an importantrole in the carcinogenesis and development of gastric cancer. Investigating the macrophages and their interaction with gastric cancer may lead to a profound understanding of carcinogenesis of gastric cancer as well as opening up a new prospectfor treatment.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
Objective To understand the role and mechanism of tumor associated macrophages (TAM) on the occurrence and development of primary liver cancer, and its application in the treatment. MethodThe related literatures about the researches of relation between TAM and primary liver cancer at home and abroad in recent years were collected, sorted out, and made a review. Results Under different stimulating factors, TAM could be polarized to anti-tumor type 1 TAMs or tumor-promoting type 2 TAMs, and type 2 TAMs was the main part in the tumor microenvironment. Through some mechanisms such as vascularity-promoting, invasion-promoting, and immunosuppression to promote the occurrence and development of tumors, and potential treatment plans for primary liver cancer could be found by targeting TAM from different perspectives. Conclusion TAM has a wide range of effects on primary liver cancer, and their mechanisms are complex, understanding the relation between them and make an effective control of TAM could provide new therapeutic ideas and plans for clinical treatment of primary liver cancer.
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 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 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.
