Objective To construct gene silence adenovirus vector targeting both transglutaminase 2 （TG2） and Mer receptor tyrosine kinase （Mertk） synchronously and detect the gene silence function of it. Methods The interfering plasmids targeting TG2 protein and Mertk protein were constructed firstly， then the H1 promoter and RNA interfering （RNAi） sequence were cut and ligated to pAdTrack for constructing pAdTrack/TG2/Mertk. The pAdTrack/TG2/Mertk was transfected into BJ5183 bacterial cells which contained pAdEasy-1， then the plasmid was detected by enzyme digestion after recovery. Adenovirus were harvested after that pAdTrack/TG2/Mertk was infected into HEK293 cells. The virus titer was measured after repeated amplification. The RAW264.7 cells were infected by pAdTrack/TG2/Mertk， pAdTrack/TG2， pAdTrack/Mertk， and pAdTrack/green fluorescent protein （GFP）， respectively. Then the expression levels of TG2 protein and Mertk protein of mouse macrophages were detected by Western blot after infection. Results The virus titer of pAdTrack/TG2/Mertk plasmid was 6.13×1010GFU/mL. The pAdTrack/TG2/Mertk plasmid which contained 2 promoters and 2 RNAi sequences was identified successfully by enzyme digestion. Compared with pAdTrack/GFP group and pAdTrack/Mertk group （there was no significant differece between the 2 groups）， the expression levels of TG2 protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/TG2 decreased obviously （P＜0.01）， but there was no significant difference between the later 2 groups. Compared with pAdTrack/GFP group and pAdTrack/TG2 group （there was no significant difference between the 2 groups）， the expression levels of Mertk protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/Mertk decreased obviously too （P＜0.01）， but there was no significant difference between the later 2 groups. Conclusion Gene silence adenovirus vector plasmid targeting both TG2 and Mertk synchronously is constructed successfully， and the pAdTrack/TG2/Mertk can reduce the expressions of TG2 protein and Mertk protein of mouse macrophages obviously.
ObjectiveTo explore the effect of H2O2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice. MethodsMC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H2O2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I). ResultsThe results of MTS and SOD assay showed that the appropriate concentration and action time of H2O2-actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A (P < 0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P < 0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P < 0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05). ConclusionThe appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.
Objective To investigate the effect ofstaphylococcal lipoteichoic acid (LTA-sa) on RAW264.7 cells differentiation into osteoclasts. Methods RAW264.7 cells were cultured with LTA-sa of 100 ng/mL (group A), LTA-sa of 200 ng/mL (group B), LTA-sa of 400 ng/mL (group C), receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) of 100 ng/mL as positive control (group D), and equal volume of PBS as blank control (group E) respectively for 5 days. And then, tartrate resistant acid phosphatase staining (TRAP) was used to detect the formation of osteoclast-like cells, Image-Pro Plus 6.0 software to measure the areas of bone resorption pits in Corning Osteo Assay Surface (COAS) wells, and MTT assay to observe the proliferation activity of RAW264.7 cells in group A, B, C, and E. Results After cultured for 5 days, the formation of osteoclast-like cells and bone resorption pits were observed in all groups. The number of osteoclast-like cells and the area of bone resorption pits in groups A, B, C, and D were more than those in group E. And with the increased concentration of LTA-sa, the indexes in groups A, B, and C increased gradually, but were lower than those in group D, and differences were significant between groups (P<0.05). At 5 days after culture, there was no significant difference in absorbance value among the experimental groups (groups A, B, C, and E) (P>0.05). Conclusion LTA-sa has promoting effect on RAW264.7 cells differentiation into osteoclasts.