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 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.
ObjectiveTo investigate the effect of succinate induced polarization of MH-S murine alveolar macrophage cells on hyperoxia-induced epithelial-mesenchymal transition (EMT) of MLE-12 mouse alveolar epithelial cells. Methods Determine the exposure time: MLE-12 cells was cultured in an incubator with 95%O2 for different time to establish a cell model of acute hyperoxia-induced lung injury. The relative expression of EMT-related proteins (E-cadherin, N-cadherin, vimentin) was determined by Western blotting. Co-culture of MLE-12 and MH-S to explore the influence of MH-S on EMT: MLE-12 was divided into hyperoxia group for 0h, hyperoxia group for 48h and co-cultured with MH-S hyperoxia group for 48h (Co). The relative expression of EMT-related proteins was determined by Western blotting. Determination of succinate concentration and its effect on MH-S polarization and succinate receptor GPR91: MLE-12 was cultured in different concentrations of succinate medium for 24h, and the cell viability was determined by CCK-8. MH-S was divided into control group (C) and succinate group (S). Group C was cultured for 24h, and group S was added with succinate at the above concentration. The relative expression of GPR91 and polarization-related factor mRNA in MH-S was measured by RT-qPCR, and the expression of macrophage polarization-related proteins (CD11b, CD206, CD86) was measured by flow cytometry. Study on the effect of succinate on EMT by cell co-culture: MLE-12 and MH-S were co-cultured in a Transwell chamber and divided into control group (Co), succinate group (SUC) and GPR91 inhibitor group (I). Results Expression of EMT-related proteins in four groups of MLE-12 at different times: Compared with 0h, the expression of vimentin and N-cadherin in 24h and 48h increased, while the expression of E-cadherin in 48 h and 72 h decreased (P<0.05), and there was no significant difference in other groups. The follow-up experiment was conducted under hyperoxia conditions for 48h. Influence of MH-S on EMT: The expression of vimentin and N-cadherin in Co group was higher than that in 48h, and the expression of E-cadherin was lower than that in 48h (P<0.05). After 24 h of intervention with different concentrations of succinate on MLE-12, compared with the 0mmol/L, the cell viability of 2.5mmol/L, 1mmol/L and 500 μmol/L increased (P<0.05), and there was no significant difference in other groups, so the 1mmol/L succinate concentration was selected for subsequent experiment. Compared with group C, the expression of GPR91 mRNA in group S increased, and the expression of iNOS and CD86 mRNA in group S increased (P<0.05), but there was no significant difference in other groups. The analysis of flow cytometry showed that 1mmol/L succinate could increase the number and proportion of CD86+CD206– alveolar macrophages. Compared with Co group, the expression of vimentin and N-cadherin in SUC group increased, while the expression of E-cadherin decreased. Compared with SUC group, the expression of vimentin and N-cadherin in group I decreased, while the expression of E-cadherin increased (P<0.05). Conclusion Succinate can induce mouse alveolar macrophages polarization to M1 through GPR91, enhance EMT of mouse alveolar epithelial cell injury model under hyperoxia, and promote the formation of pulmonary fibrosis.
ObjectiveTo investigate the effect of miR-190a-5p on the polarization of bone-marrow-derived macrophage (BMDM) induced by lipopolysaccharides to M1- and M2-types.MethodsBMDM (M1-type) induced by bacterial lipopolysaccharide was a M1 group. The macrophage M1-type interfered with negative control miRNA mimics was a NC group. miR-190a-5p mimics interfered with the M1-type of macrophages in the miR-190a-5p group. Morphological changes of macrophages were observed under a microscope, and the proportion of M2-type macrophages (CD206+, F4/80) was detected by flow cytometry. The mRNA expression levels of argininase-1 (Arg1), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), target gene C/EBPα and PU.1 were detected by fluorescence quantitative PCR to verify whether C/EBPα and PU.1 were potential target genes of miR-190a-5p. The expression of pathway proteins C/EBPα and PU.1 were detected by Western blotting.ResultsAfter miR-190a-5p mimics interfered with macrophage M1-type, the antenna of macrophages elongated and showed long cord M2-type cell morphological characteristics. miR-190a-5p mimics interfered with M1-type macrophages for 24 h, and the percentage of M2-type macrophages increased significantly (P<0.05). Effects of miR-190a-5p simulator on mRNA expression levels of M1-type macrophages included: the expression of iNOS and TNF-α was significantly decreased (P<0.05), the expression of Arg1 marked by M2 macrophages was significantly increased (P<0.05), and the mRNA expression levels of target genes C/EBPα and PU.1 were significantly decreased (P<0.05). Western blotting results showed that the overexpression of miR-190a-5p significantly inhibited the protein expressions of C/EBPα and PU.1, while the miR-190a-5p inhibitor increased the expressions of both proteins.ConclusionmiR-190a-5p can promote the polarization of BMDM from M1-type to M2-type.
ObjectiveTo investigate the effect of graphene oxide (GO)-carboxymethyl chitosan (CMC) hydrogel loaded with interleukin 4 (IL-4) and bone morphogenetic protein 2 (BMP-2) on macrophages M2 type differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsGO solution was mixed with CMC, then the phosphate buffered saline (PBS), IL-4, BMP-2, or IL-4+BMP-2 were added to prepare different GO-CMC hydrogel scaffolds with or without different cytokines under crosslinking agents. The characteristics of pure GO-CMC hydrogel were characterized by gross observation, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR), and the CMC hydrogel was used as control. The sustained release of GO-CMC hydrogels with different cytokines was also tested. Macrophages were isolated and cultured from female Sprague Dawley rats aged 4-5 weeks, and then cultured with GO-CMC hydrogels with and without different cytokines, respectively. CD206 immunofluorescence staining was used to detect the differentiation of macrophages after 24 hours. The 3rd generation of rats BMSCs were cultured with GO-CMC hydrogels with and without different cytokines respectively for osteogenic induction. The early osteogenesis was observed by alkaline phosphatase (ALP) staining after 10 days, and the late osteogenesis was observed by alizarin red staining after 21 days.ResultsGenerally, GO-CMC hydrogel was brown and translucent. SEM showed that the pore diameter and wall thickness of GO-CMC hydrogel were similar to that of CMC hydrogel, but the inner wall roughness increased. FTIR test showed that CMC polymerized to form hydrogel. In vitro, the sustained release experiments showed that the properties of GO-CMC hydrogels loaded with different cytokines were similar. CD206 immunofluorescence detection showed that GO-CMC hydrogels could induce macrophages differentiation into M2-type. ALP and alizarin red staining showed that GO-CMC hydrogels could induce BMSCs osteogenic differentiation, in which GO-CMC hydrogel loaded with IL-4+BMP-2 showed the most significant effect (P<0.05).ConclusionThe GO-CMC hydrogel loaded with IL-4 and BMP-2 can induce macrophages differentiation into M2-type and enhance the ability of BMSCs with osteogenic differentiation in vitro, which provide a new strategy for bone defect repair and immune regulation.