Experimental Study Of Lentiviral Vector Mediated "F-5" Gene In HUC-MSCs On The Treatment Of Skin Flaps Ischemia-reperfusion Injury

Objective: In order to improve the efficacy of flap ischemia-reperfusion injury,this experiment bound the functional fragment of heat shock protein 90?(Hsp90?)with adenovirus,and then transferred them into human umbilical cord mesenchymal stem cells(h UC-MSCs)through virus transfection.The point injection methods were homogeneously applied to the flaps of rat ischemia-reperfusion injury.The macroscopic phenomena such as flap color,hair growth and necrosis area,and the microscopic phenomena such as HE staining and immunohistochemistry were observed.The aim of this paper was to provide theoretical basis and experimental basis for follow-up experimental study by investigating the therapeutic effect of h UC-MSCs with functional fragment of Hsp90? on flap ischemia-reperfusion injury.Methods: In this experiment,we used three plasmids(including the objective plasmid vector)to transfect 293 T cells,and harvested the virus(that is,unpurified cell supernatant)within a certain time after transfection.According to the different experimental requirements,we got the high titer lentivirus preservation solution by adopting the corresponding concentration and purification methods.And then,we determined the various indicators of lentivirus,according to stringent quality standards.Next,h UC-MSCs were cultured and passaged in DMEM(low glucose)medium containing 10% fetal bovine serum.And then,the prepared adenoviruses were added.After a period of time,the optimal MOI values for transfection were determined under the electron microscope.The toxicity of adenovirus on h UC-MSCs and the proliferation of h UC-MSCs were detected by MTT and q PCR.The migration effect of h UC-MSCs carrying the target gene was detected by cell scratch assay.Then,the rat abdominal flap ischemia-reperfusion model was established.A 3×6cm rat abdominal island flap was prepared under aseptic condition.The flap was deep up to fascia and separated by the blunt dissection method.The abdominal arteries and veins(femoral artery branches and femoral vein branches)were found.The glass minute hand was used to carefully separate the abdominal arteries and veins from the skin.Finally,the abdominal skin was completely dissociated,and an island flap with only abdominal arteries and veins blood supply was prepared.The blood vessels were clamped with small blood vessel clips for 6 hours.Finally,h UC-MSCs containing the target gene,h UC-MSCs carrying no-load adenovirus,and normal h UC-MSCs were prepared into cell suspensions with the concentration of 4×10^5/ml by using conventional cell culture methods.When the animal model was successfully prepared,10 injection points on the abdominal perforator flap were selected for skin injection.Each injection point was 1 cm apart and the injection volume per point was 0.1 ml.In order to reduce the influence factors of the experiment,the control group injected with the same amount of saline was set.At 1 day,3 days,5 days and 7 days after operation,One rat was randomly selected from each experimental group for general anesthesia treatment.The changes in flap color and hair growth at each time were recorded with a digital camera.And the Image-Pro-plus software was used to assess the area of flap necrosis,HE staining was used to observe the skin tissue layer and capillary density,and immunohistochemistry(IHC)was used to detect CD31 expression around the skin flaps.Result: The color and hair growth of the flaps carrying the "F-5" gene were better than those of other experimental groups,and the area of flap necrosis was significantly reduced.The results of immunohistochemistry showed that the content of CD31 around the blood vessels of the flaps carrying the "F-5" gene was much higher than that of the no-load virus group,the untransfected group and the non-injected group.Conclusion: The experiment adopted a new method called “stem cell-gene combination therapy”.The "F-5" gene fragment in Hsp90? was transfected into h UC-MSCs by tissue engineering techniques to improve the rat abdominal flap ischemia-reperfusion injury.The data showed that h UC-MSCs carrying the "F-5" gene fragment can effectively improve the flap ischemia-reperfusion injury.