| Background:Appropriate material is the crucial factor of the operation’s effect, and it is also the focus of many researches. Tissue-engineering blood vessel is an important ivestigation area of new style meterial in transplantation of blood vessel. There is a considerable mode to construct tissue-engineering blood vessel is to plant some "seeding-cells" such as bone marrow stem cells (BMSC) onto a tubiform supporter. But the intensity of immunogenicity is different because of different tubiform supporters or seeding-cells, which can severely affect the result of surgical treatment..Objective:The rats’MSCs were implanted to acellular guinea pig’s arteries to construct simple tissue-engineering blood vessels, and the blood vessels were then transplanted subcutaneouly. Blood samples and the transplanted blood vessels were harvested at special time after the operation. The concentrations of complement C3and IgG of anti-guninea pig’s arteries in the blood-serum and those deposited on the grafts’ surface were tested, respectively. The aim of this study was to explore the immunogenicity of of tissue-engineering blood vessels constructed by acellular guinea pig’s arteries planted with rat’s MSCs.Methods:The rat’s MSCs were harvested with density gradient centrifugation and modified adherence screening method and cultivated in vitro. Aafter identificated, the MSCs were then transplanted onto acellular guinea pig’s arteries treated by enzyme-detergent cell extraction to construct simple tissue-engineering blood vessels. Two hundreds healthy clonic masculine clean Sprague-Dawley (SD) rats weighing200-250g were selected, then were randomly devided into4teams to transplant different samples in the operation (team A:tissue-engineering blood vessels planted with rat’ s MSCs, n=50; team B:acellular guinea pig’s arteries, n=50; team C:fresh guinea pig’s arteries, n=50; team D:sham operation, n=50). Ten rats of each team were executed on the3rd、7th、15th、30th、60th days after the operation, and the rats’ blood-serum and the grafts were obtained. The acellular arteries and inflammatory reaction on the grafts’surface were observed with HE method. The concentrations of anti-guninea pig’s arteries IgG and complement C3in the blood-serum and deposited on the grafts’surface were tested with the methods of ELISA (enzyme linked immunosorbent assay) and IHC (immunohistochemistry), respectively.Results:1. HE stain:The fresh and acellular guinea pig’s arteries were both white, semiopaque and soft. There were no cells or cellular fragments in the acellular guinea pig’s arteries, but the fabric net structure was still complete. There were many cell nuclei dyed in deep blue and normal structure of guinea’s blood vessel but no obvious blank areas in the fresh guinea pig’s arteries.There was obvious inflammatory cell infiltration in the grafts in all teams, and it was significantly severer in team C than that in team A and B. Normal cells in guinea vessel gradually disappeared till15th days postoperatively and were replaced by large numbers of inflammatory cells.2. ELISA results of IgG of anti-guinea pig’s arteries:The IgG of anti-guinea pig’s arteries increased in team A、B and C. On the3rd day, postopratively, there were no significant differences among all those teams (team A102.59±8.87, team B100.04±8.48, team C103.49±12.85, team D101.09±13.79) μ g/ml (P>0.05). They were significantly higher on the7th、15th、30th days, postoperatively, in team A (116.63±9.72,123.79±9.12,110.76±12.44) μ g/ml and B (112.77±9.63,122.54±17.15,116.23±16.49) μ g/ml than those in team D (98.41±16.49,102.54±10.61,97.73±9.71) μ g/ml (P<0.05). There were no differences on the60th day among team A (108.47±12.70) μ g/ml、B (105.44±11.59) μ g/ml and D (100.55±16.10) μ g/ml (P>0.05). On the7th.15th、30th、60th days, the concentrations of team C (121.14±6.75,138.03±17.70,141.09±18.93,133.28±16.13) μ g/ml were higher than those in team D (p<0.05), and higher than those in team A and B on the15th、30th、60th days (P<0.05). There were no significant differences between team A and B (P>0.05).3. ELISA results of complement C3:The complement C3decreased in team A、B、 and C. On the3rd day, postopratively, there were no significant differences among all those teams (team A158.57±21.98, team B154.47±18.27, team C156.88±16.67, team D157.89±13.57) μ g/ml (P>0.05). On the7th、15th、30th、60th days, postoperatively, the concentrations in team C (130.38±16.85,106.58±11.62,105.25±10.71,113.84±11.05) μ g/ml were lower than those in team A (152.61±21.84,130.94±16.55,127.71±15.10,134.79±16.20) u g/ml、B (153.10±15.81,135.24±24.56,125.18±11.88,137.25±21.17) μ g/ml and D (154.45±19.69,161.74±23.78,153.33±24.41,156.90±22.20) μ g/ml (P<0.05). They were lower in team A and B than those in team D on the15th、30th、50th、60th days (P<0.05). There were no significant differences between team A and B (P>0.05).4. IHC results of IgG of anti-guinea pig’s arteries:There was much IgG of anti-guinea pig’s arteries deposited on the the grafts’surface. On the3rd day, the AO (average optical) in the grafts had no significant differences among team A (0.1506±0.0394)、B (0.1632±0.0340) and C (0.1668±0.0510)(P>0.05). The AO of team C (0.2157±0.0452,0.2563±0.0442,0.2752±0.0579,0.2545±0.0466) was higher than that in team A (0.1633±0.0388,0.1787±0.0532,0.1692±0.0406,0.1633±0.0282) and B (0.1555±0.0348,0.1740±0.0364,0.1698±0.0330,0.1621±0.0349) on the7th、15th、30th、60th days, postopratively (P<0.05). There were no significant differences between team A and B (P>0.05).5. IHC results of complement C3:There was obvious complement C3of rat deposited on the the grafts’surface. On the3rd、7th、15th days, the AO of the grafts had no significant differences among team A (0.1304±0.0313,0.1552±0.0425,0.1612± 0.0358)、B (0.1316±0.0254,0.1535±0.0396,0.1603±0.0591) and C (0.1671±0.0422,0.1766±0.0493,0.2401±0.0706)(P>0.05). The AO (average optical) in team C (0.2674±0.0788,0.2044±0.0494) on the30th、60th days after the operation was higher than that in team A (0.1597±0.0439,0.1382±0.0299) and B (0.1517±0.0404,0.1407±0.0288)(P<0.05). There were no significant differences between team A and B (P>0.05).Conclusions:1. The IgG of anti-guinea pig’s arteries and complement C3change in the blood-serum after the operation in rats, and they also deposit on the grafts’surface.2. Tissue-engineering blood vessels and acellular guinea pig’s arteries have lower immunogenicity than fresh guinea pig’s arteries.3. The method of planting allogeneic BMSC to acellular hetergeneic arteries cao decrease the immunogenicity significantly. This method may be an appropriate way to construct tissue-engineering blood vessels. |
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