| Objective To investigate the effects and mechanism of hypoxia-inducible factor-1ɑ in vivo on the Vascular Endothecial Growth Factor(VEGF)expression in Cross-boundary flap,in order to promote the early vascularization and rate of survival of the clinical Cross-boundary flap to provide new theoretical ideas.Methods Sixty healthy adult male Sprague-Dawley rats weighing 250-300 g were randomly divided into 3 groups: agonist group,antagonist group and control group(group of Normal saline).The rats in antagonist group were given intraperitoneal DMOG(dimethylglyoxyglycine)in a single dose of 40 mg / kg,and the rats in the antagonist group were injected with intraperitoneal YC-1 [3-(5-hydroxymethyl-2-furyl)-1-benzylindazole],10mg/kg,and the control group was given an equal volume of normal intraperitoneal saline in2 hours before operation and 1,2,3 days after operation.In the left side of the back of rats,model of length 12 cm,width 3cm rectangular Cross-boundary flap was established which includes perforator vesselof iliolumbar artery a iliac,across the intercostal artery and thoracodorsal artery.This flap is based on the subscapular angle at the upper boundary,two posterior superior iliac spine connecting at the lower boundary and the posterior midline at the inside border,axillary line at the outer border.Observations include:(1)At 7 days after surgery,the survival of the flaps was observed by naked eyes.(2)The survival rate of the flap was calculated by the M2 Image Analysis system image analysis system on the 7th postoperative day(flap survival rate = flap survival area / total flap area).(3)Angiogenesis of ChokeⅡwas detected 7th postoperative day by angiogram.(4)Microscopic observtion of vascular diameter and density by HE staining.(5)HIF-1ɑ,VEGF expression in 7 postoperative days was detected by enzyme-linked immuno sorbent assay(Elisa).(6)HIF-1ɑ,VEGF expression in 7 postoperative day was detected by western blot.(7)HIF-1ɑ,VEGF expression in 7 postoperative days was detected Immunohistochemistry.Results1.On the 7th day after surgery,the extent of distal necrosis of the flap in the three groups of flaps was significantly smaller than that of the control and antagonist groups.In the agonist group,vasodilator and hyperplasia occurred in the area of Choke II,and direct access to the distal flap was observed.However,there was no significant change in the vessels in the region of Choke II of the control group,and the distal flap was slightly dark in color.In the antagonist group,disruption of vascular was occurred in Choke II,necrosis of distal flap tissue2.Necrosis area: On 7 days postoperatively,HD digital camera was taken and the data of images were calculated by M2 Image Analysis system.The survival rates(%)of the control group and the antagonist group were respectively calculated as 90.38±1.36,85.83±1.59,84.28± 1.45.Compared with the control group,the agonist group was significantly higher than the control group(P <0.05),with statistical significance.There was no significant difference between the inhibitor group and the control group(P> 0.05),with not statistically significant.3.Arteriography: On 7 days postoperatively,the structure of the choke II area in the agonist group was dense and clear,and a large area of vascular network was clearly visible.However,the vascular structure of the vascular area of Choke II in the control and antagonist groups was indistinctly and less vague vascular network.4.HE staining used to measure numbers of blood vessels: Observation of 5 visions were randomly selected in a 10×20 optical microscope microscope.According to the experimental results,it was found that the agonist group had a significant growth trends of blood vessel and numbers of blood vessels On 7 days postoperatively.The control group also showed growth trends fresh blood vessels On 7 days postoperatively,but no significant vascular proliferation was observed.In antagonist groups,postoperative vascular proliferation was poor,and there was no significant new vascular proliferation.The average numbers of blood vessels in the choke II area were found to be 24.00±5.52 in the agonist group,7.60±7.71 in the antagonist group,14.00±2.34 in the choke II area of the control group,and the agonist group>the control group>the antagonist group.The difference was statistically significant(P<0.05).5.Elisa method used to detect the expression of HIF-1αand VEGF:On 7 days postoperatively,the expression of HIF-1α in choke II was as agonist group > control group>antagonist group,(2.87±0.19,1.85±0.41,0.89±0.29,the unit as ng / mg).The expression of VEGF in choke II was as agonist group > control group> antagonist group,(34.87±3.05,19.64±1.13,14.41±1.34,the unit as ng / mg).Compared with the control group,the two were significantly different,with statistical significance(P <0.05).6.Western blot used to measure proteins expression levelof HIF-1ɑand VEGF On 7 days postoperatively:The gray value of proteins expression level of HIF-1ɑ and VEGF/β-acting was used to express the expression intensity of HIF-1ɑ and VEGF protein.the agonist group>the control group>the antagonist group,the difference between the two groups and the control group was statistically significant(P <0.05).7.Immunohistochemistry used to measure proteins expression level of HIF-1ɑ and VEGF:Observed in a 40×10 electron microscope,it was found that the expression of HIF-1ɑ and VEGF in the blood vessels of the agonist group was significant at the 7th day after operation,and the expression of HIF-1ɑ and VEGF were also observed in the blood vessels of the control group and the antagonist group,but the expression level was obvious less than the agonist group.Conclusion1.The protein expression of HIF-1α can significantly reduce the degree of early ischemia in rat choke vessles II.2.After the flap transplantation,the high expression of HIF-1α induced in vivo can promote the expression of VEGF in Cross-boundary flap,and then promote the angiogenesis in the flap of Choke II,further accelerating the process of early vascularization and promoting the skin.The blood supply to the distal end of the flap improves the survival of Cross-boundary flap on the back of the rat.In the early stage after operation,promoting the expression of HIF-1α is expected to be a new way to promote the survival of Cross-boundary flap. |
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