| Objectives Explore that whether the resveratrol(RSV)could inhibit CD4~+T cells from producing inflammatory factor to improve the microenvironment of bone repair,and then promote the bone repair of h UC-MSCs.Methods Randomly dividing 72 Balb/c nude mice which were 6 weeks' healthy mice into three groups respectively:blank group,control group,drug group,24 each group.h UMSCs were cultured in vitro,and the third generation of the cells were grew on sponge surface trained for three days.Separation and screening the Balb/c mice spleen CD4~+ T lymphocytes in vitro,the control group and drug group all nude mice were injected CD4~+T lymphocyte by caudal vein,1×106 each mouse,only after 2 days 4mm×4mm skull full-thickness bone defect model was established at the right side of all nude mice cranial parietal,at the same time gelatin sponge with the third generation of h U-MSCs were implanted to the defect areas,the blank group and the control group were given resveratrol blank solvent(5% DMSO saline solution)by intraperitoneal injection after building defect model,abdominal injection of 0.2 ml each mice,once a day.Drug group was given RES(RES was dissolved in DMSO,diluted with normal saline),dosage of 30 mg/kg/d,by intraperitoneal injection of 0.2 ml each mouse,once a day.Getting 6 nude mice randomly to draw blood by behead after 1 week,2 weeks,3 weeks,with ELISA to detect the serum concentration of IFN-??TNF-??IL-6.After 8 weeks killing the 6 nude mice remain in each group,getting the full skull.The Micro-CT and tissue pathological slices method to observe the defect part of the new bone growth,measuring the defect area,quantitative analysis of the defect in the parts of the bone for mation ability.The Micro-CT and pathology biopsy method to observe the new bone growth on the edge of the defect,to measuring the area size,to quantitatively analyze the new bone formation ability.detect Runx2 and OCN protein expression in the new bone defect parts by the IHC method.Results 1.h U-MSCs morphological characteristics under the inverted phase contrast microscope:h U-MSCs grew well,which were fusiform short rod adherent cells.Single cell was fibrous.Cells confluence was arranged parallel and grew rotaryly.2.h U-MSCs surface antigen expression: They uniformly expressed CD73,CD90,CD105,not expressed CD19,CD11 b,C D34,CD45,MHC-II molecules HLA-DR,in line with the phenotypic characteristics of mesenchymal stem cells.3.Scanning electron microscopy results: h U-MSCs adhered to Yu Mingjiao sponge surface or deep pore surface,gelatin sponge scaffolds inoculated stem cells and developing three days was slightly degraded but structure remained intact.4.T lymphocytes flow cytometry instrument test res ults: CD4~+ T lymphocytes accounting for the proportion of magnetic bead filter cells was 97.45%±0.05%,of which,the proportion of CD4~+CD25~+ cells was 8.05%±1.82%.5 ELISA results:at 1 week,2 weeks,3 weeks three time points,the control group level of IFN-? was significantly higher than that of blank group and drug group(P<0.05).Three time points the control group level of TNF-? was significantly higher than the blank group and drug group,the difference of them was statistically significant(P<0.05).At the three time points the three groups levels of IL-6 gradually reduced at the three time points,the control group level of IL-6 was significantly higher than the blank group and drug group(P<0.05).6.Micro-CT results:after 8 weeks the nude mice skull defect of the three groups weren't fully repaired,the defect edge was not neat,but the nude mice skull defect of three sets had grew,bone tissue present from the defect edge to the inside of the defect area stretching type growth and new bone tissue grew fro m the defect edge gradually to the inside defect area.Micro-CT testing results: TV value:Compared with the control group(40.76±0.95),the callus total volume of the blank group(46.04±3.53)and drug group(43.35±0.87)increased significantly,the difference was statistically significant(P<0.05).BV values:Compared with the control group(1.93±0.19),the mineralized osseous tissue volume of the blank group(2.93±0.35)and the drug group(2.58±0.33)increased significantly(P<0.05).The ratio of BV/TV: Compared with the control group(4.72±0.47),the relative bone volume of blank group(6.39±0.95)and drug group(5.94±0.70)was significantly increased,the difference was statistically significant(P<0.05).7.HE staining and Masson staining:HE staining showed: after skull defect,osteogenesis precursor cells migrated from the skull and the skull of membrane and the dura mater to the defect edge and subdural where they gathered,then differentiated into osteoblasts and secreted osteoid.we could find osteoblasts in the osteoid pouch,under the subperiosteal reactive periosteal new bone was visible.The Central of skull defect was filled with connective tissue,and no obvious bone tissue formed.O n the defect edge new bone further increased,and like "medullary cavity" structure was formed inside the new bone which was on the edge of the defect or under the dura mater,in which numerous aizen nuclear MSCs were.Masson staining showed: at 8 weeks,three groups all had new bone formation,besides aizen nuclear bone cells and bone tissues were visible and mature bony collagen fibers arranged compact and neatly,and a part of lamellar bone formed.Part of the more mature new bone tissue was dyed red.8.IHC staining: RUNX2 results:There were a small amount of positive expression cells of RUNX2 in new bone tissue of the control group.Compared with the control group,the positive cell number of RUNX2 dramaticly increased in the blank group,the IOD value was significantly increased(P<0.05).Compared with the control group,the positive expression of RUN X2 in the medicine group cells was significantly increased,the IOD value increased significantly(P<0.05).OCN results:The positive expression of OCN was small in the new bone tissue cells of the control group.Compared with the control group,the positive cell number of OCN dramaticly increased in the blank group,the IO D value was significantly increased(P<0.05).Compared with the control group,the positive cell number of OCN dramaticly increased in the drug group,the IOD value was significantly increased(P<0.05).Conclusions The resveratrol(RSV)could inhibit C D4+T cells from producing inflammatory factor to improve the microenvironment of bone repair,and then promoted the bone repair of human umbilical cord mesenchymal stem cel s(h UC-MSCs). |
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