| Objective To evaluate the effects of local delivery of HSP90?,“F-5”,exogenous heat shock protein on survival of the ischemia-reperfusion injury of venous-blood-congested flap respectively in rats.In this study,a model of venous congested flaps in rats was established,to test the HSP90?,protein as an intervention therapy to alleviate ischemia –reperfusion injury.Methods A recombinant plasmid p ET15b-F-5 carrying the HSP90? gene was constructed and the induced protein was purified from bacterial cell cultures.With nitrogen Sichuan three nickel acetate(Ni-NTA)metal affinity chromatography;through the Centricon YM-50 device and then concentrated by fast protein liquid chromatography(FPLC)purification again;by Coomassie brilliant blue method(Bradford method)was used to quantify the protein purification,to obtain the final concentration of 1mg/ml exogenous HSP90? “F-5”the expression of protein.The rats in the study were divided into three different intervention groups: group A rats were treated with normal saline prior to flap establishment,group B rats were treated with exogenous HSP90? prior to flap establishment,and group C rats were treated with the same exogenous protein after the surgical procedure.Additionally,2 reperfusion time points,namely,ischemia for 6 or 8 h(10 rats each),were set up in each group.After set periods of time,the flaps were observed for general aspect,blood flow,survival rate and histological changes including neovascularization and re-epithelialization.Results By Western blotting assay,SDS-polyacrylamide gel electrophoresis in 87 KD band,that extracts for the purpose of protein,HSP90?,“F-5” protein gene expression by RT-PCR and Southern;blotting assay,agarose gel electrophoresis of 550 bp and 332 bp in the band,the molecular weight of m RNA and plasmid vector and HSP90? gene with "F-5" for the purpose of protein extracts demonstrated "F-5" gene.Change of blood perfusion:Blood perfusion of the abdominal flap in rats under normal circumstances(397.4 + 26.7),the blood perfusion of each flap venous occlusion after 1 hours were significantly lower in the blank control group and HSP90? flaps in treatment group dropped to around 40%,blocking 6 hours reduced to 21%,block 8 hours dropped to below 15%.HSP90? pretreatment group in venous occlusion during and after reperfusion of blood perfusion flap group compared with control group and HSP90? control treatment group.Changes of blood perfusion that axial flap ischemia reperfusion injury model was successfully constructed.Pretreatment of flap perfusion in the group of ischemia and reperfusion at different time nodes of HSP90? was significantly higher than that of blank control group is better than that of HSP90? treatment group,the comparison between the two groups had significant difference(P<0.05).Gross observation:After first days of blank control group flap in the distal flap swelling significantly,varying degrees of purple or dark purple.On the 3 day after operation,there was still swelling in the flap of the blank control group,and the distal necrosis area became black,and there was an inflammatory reaction.The swelling of the skin flap began to subside in HSP90? preconditioning group and HSP90? treatment group.After 7 days of three groups of flap necrosis clear boundaries,HSP90? pretreatment group and HSP90? treatment group the survival range and nutritional status were compared with blank control group.After fourteenth days,the blank control group,the necrotic tissue black crusts fall off,the tissue healing is poor,the granulation tissue formation;HSP90? pretreatment group and HSP90? treatment group flap wound healing,skin soft,warm,flap surface new hair formation.The capillary filling time is prolonged or disappeared,with striae disappear,some blisters;compared with blank control group,mild HSP90? pretreatment group and HSP90? treatment group,edema,capillary filling phenomenon,time is basically normal.In the same group,the indexes in the 6 hours group were better than those in the 8 hours group,in which the blank control group was blocked for 8 hours,and 2 cases showed complete necrosis of the flap.The viability of the flap:7 days after,the flap survival rate of blank control group was(19.3±3.8)%,the flap survival rate of pretreatment group was(85.6±17.6)%,the flap survival rate of treatment group was(78.9±20.5)%,the rate of treatment group was the highest one(P<0.05).Path-histology changes:After 1,3,7,10,14 days of flap tissue were stained with H-E were observed.First days after operation,the flap group can be seen in infiltrating inflammatory cells,telangiectasia,platelet adhesion to the vessel wall,with cell swelling,especially in the blank control group.It was also observable to microvascular structure fuzzy.After 3 days,each flap cell swelling did not subside,the vessel wall is still a large number of inflammatory cell infiltration;blank control group showed intravascular thrombosis,cell swelling degree of HSP90? pretreatment group and HSP90? treatment group.7 days after operation,HSP90? prtreatment group and HSP90? treatment group showed normal structure of skin flap,the stratum corneum of the skin is more complete,the organizational structure basically normal,the stratum reduced,the number of fibroblasts increased capillary density increased formation of new epithelial tissue;structure of blank control group flap is not complete,part of the cuticle shedding,intravascular thrombosis,there are still many inflammatory cells infiltrated in the vicinity of the blood vessel wall,vascular endothelial cell The cell and fibroblast proliferation are rare angiogenesis.After 10 days,HSP90? pretreatment group and HSP90? treatment group flap structure returned to normal,at the edge of the necrotic area under the infiltration of inflammatory cells decreased,new capillaries increased and with new tissue formation;the control group number the vascular wall inflammatory cells decreased,angiogenesis and tissue was not significant.After 14 days,HSP90? pretreatment group and HSP90? treatment group showed that the number of inflammatory cells decreased further,new capillaries and mature fibroblasts increased,cells arranged in neat,new epithelial tissue at the same time increased,visible basal cell hyperplasia;blank control group the number of inflammatory cells was further reduced,but the local tissue angiogenesis,proliferation of epithelial cells is not active.Microvessel count:After 1,3,7,10,14 days of the flap were performed immunohistochemical staining and counting on microvessel density.Brown was found after the 7 day of each flap in vascular endothelial cells increased more than before the pretreatment group and heat shock protein,HSP90? treatment group than that of the control group increased significantly.The microvessel density in comparison with statistical significant difference between the groups(P<0.05).In addition,6 hour ischemia parameters of sub group were more than 8 hours of ischemia group,significant difference(P<0.05).After 10 days,HSP90? pretreatment group and HSP90? treatment group near the wound visible new epithelial tissue,even visible neovascularization.After 14 days,HSP90? pretreatment group and HSP90? treatment group distal edge of the flaps healed well,the local black scab off,you can see a complete epidermal and dermal junction The structure is accompanied by an increase in neovascularization.The flaps in the rats pre-treated with exogenous HSP90? performed better than the flaps of rats in the other two groups:blood flow was higher,inflammatory cell infiltration was decreased and angiogenesis increased,and new skin structure was better completed by the end of the experiment.Finally,all the parameters studied were better across all the groups when the ischemia time was 6 h instead of 8 h.Conclusion The recombinant plasmid,amplification,purification method can obtain active exogenous HSP90? “F-5”gene expression protein.The establishment of rat model of ischemia-reperfusion injury of venous-blood-congested flap was successful,and the control of the time of venous occlusion was 6 hours.Exogenous HSP90? intervention prior to flap establishment was shown to be beneficial in our model of ischemia-reperfusion injury in venous congested flaps by increasing flap reperfusion,reducing cell permeability,promoting the formation of new blood vessels.Early intervention was more effective. |
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