| (1)Background Type 2 diabetes is a common and frequent disease,and also a global disease seriously affecting health.According to the existing statistics,there are 422 million people with diabetes in the world,which accounts for almost 8.5% of the human population.It is estimated that the number will increase to 640 million by 2040,while in China the proportion will reach 15.50%.Diabetic foot ulcers are the most common and serious complication associated with diabetes,with an incidence of up to 25 percent,with more than 15 percent of patients at risk of amputation or even death at any time.Treatment of diabetic foot ulcer is very difficult.Traditional treatment methods mainly include controlling blood sugar,resisting infection and local debridement.However,treatment is usually very difficult,and many patients still need amputation at the end.Exploring new therapeutic targets and strategies has become a hotspot and difficulty in modern medical research.Studies have shown that Ilizarov microcirculation reconstruction can not only promote the regeneration of microvessels between bones,but also promote the angiogenesis of skin and soft tissues,thus promoting the healing of diabetic foot ulcers.Currently,most of the research on Ilizarov microcirculation reconstruction techniques is conducted from angiography and color doppler ultrasound,and the mechanism of angiogenesis and vascular remodeling is rarely studied.(2)Objective To observe the effects of Ilizarov microcirculation reconstruction on VEGF,Notch1 and DLL4 m RNA proteins of diabetic foot ulcer injury tissue of New Zealand white rabbit and the expressions of VEGF and b FGF in peripheral blood of diabetic foot ulcer patients,and to explore the mechanism of Ilizarov microcirculation reconstruction on the healing of diabetic foot ulcer.(3)Methods The first part(Clinical research): 20 diabetic foot patients were selected and randomly divided into the Ilizarov group and the rb-b FGF group,among which there were 9 cases in the Ilizarov group and 11 cases in the rb-b FGF group.Wound healing was observed before and after treatment at 2 w,4 w and 8 w respectively;CTA was used to examine the situation of limb angiogenesis before and after surgery;to detect the levels of VEGF and b FGF through ELISA test.The second part(Experimental study):1)A lateral bone removal of tibia external fixation bracket was designed and placed on the medial left upper segment of the tibia of 4 New Zealand rabbits.At the 4th day after surgery,the bone mass was transferred,0.5 mm outward every day,and completed in two times.The rotation was 0.125 mm,0.25 mm per time,once per 12 h,twice per day,and the total migration was 7 days.The migration distance was 3.5 mm.The same method was used to move the bone mass back on the second day after the end of the out-moving,which was completed in a total of 7 days(accordion technique).The tibia bone mass was moved back to the original position and maintained for 2 weeks,and then the tibia external fixation bracket was removed.2)72 New Zealand rabbits were randomly divided into the blank control group,the model group,rb-b FGF group and the Ilizarov group,with 18 rabbits in each group.Models of diabetic foot ulcer were established in the model group,the rb-b FGF group and the Ilizarov group,a cute wound surfaces were established in the foot in the blank control group.In the Ilizarov group,the lateral movement of the tibia was performed according to the experimental method in the first part.Rb-b FGF group was given rb-b FGF external treatment,and the model group and the blank control group were treated with saline dressing treatment.Wound healing rate was analyzed in each group,histopathological changes were observed by HE staining,and the degree of vascularization was quantitatively analyzed.The granulation tissue at the same site of the wound was taken from each group at 3,10 and 17 days after intervention,respectively.The the expressions of VEGF,Notch1 and b FGF protein in the wound tissue were determined by Western Blot,the transcription levels of VEGF,Notch1 and DLL4 m RNA in the wound tissue were determined by real-time PCR.(4)Results The first part:In Ilizarov group,fresh granulation tissue began to grow on the wounds of all 9 patients 2 weeks after the lateral tibial transfer,and the wounds were significantly reduced after 4 weeks.At the 4 w and 8 w,the healing speed of the experimental group was significantly faster than that of the control group,and the difference was statistically significant(P<0.05).Finally,all foot ulcers were healed,The average healing time was 9.0±1.9 w,significantly shorter than the control group(12.7±3.7 w),and the difference was statistically significant(P<0.05),and there was less scar tissue after the healing.At 8 weeks after the operation,angiography showed a significant increase in the collateral arteries of the lower leg and foot,as well as in local small arteries;The ankle-brachial index measured at 12 w after surgery showed that the ankle-brachial index in the experimental group was better after treatment than before treatment(P<0.00).Peripheral blood VEGF and b FGF in Ilizarov group were significantly increased at 1 w,2 w and 4 w after treatment compared with that before surgery,and the difference was statistically significant(P<0.01),Which was increased at 2 w compared with that before surgery,and no significant changes were observed at the remaining time points(P> 0.05).The second part: 1)The lateral bone removal of tibia external fixation bracket was successfully placed inside the upper tibia segment of the New Zealand rabbit.The upper segment of the tibia can be effectively moved laterally by adjusting the device,with good stability and performance.2)The repair tissue scar tissue in Ilizarov group was less,hair growth was good,close to in situ regeneration;Comparison of healing rate among groups: On day 7,there was no significant difference in healing rate between groups(P>0.05);On the 14 th day,the healing of the Ilizarov group and the blank control group was significantly faster than that of the b FGF group and the model group,with statistically significant differences(P<0.01),there was no significant difference between the Ilizarov group and the blank control group(P>0.05).On the 21 st day,there was a statistically significant difference between the model group and the other three groups(P<0.01),and there was no statistically significant difference in the healing rate between the remaining groups(P>0.05).Number of positive blood vessels: On day 7,the number of positive blood vessels in the Ilizarov group,the blank control group and the b FGF group were all higher than those in the model group,and the difference was statistically significant(P< 0.00).On the 14 st day,the number of positive blood vessels in the blank control group was more than that in the other three groups,and the difference was statistically significant(P<0.00),while there was no statistically significant difference between the remaining groups(P>0.05).3)Real Time PCR results showed that the expression levels of VEGF,Notch1 and DLL4 m RNA in the granulation tissues of the wounds of the Ilizarov group and the b FGF group showed an up-down trend at three time points,while the expression levels in the blank control group gradually decreased,and there was no significant change in the model group.On the 10 th day of intervention,the elevation of Ilizarov group was more obvious,and the difference was statistically significant compared with that of the b FGF group(P<0.05).Compared with the third day,the difference was statistically significant(P<0.05).West blot results showed that the expression levels of VEGF,Notch1 and b FGF in the marginal tissues of large white rabbits in Ilizarov group and b FGF group showed an up-down trend at three time points,while the blank control group showed a high to low trend,while the model group showed no significant change.VEGF protein expression level in Ilizarov group was higher than that in the b FGF group at day 10,and the difference was statistically significant(P<0.05).The Notch1 and b FGF protein expression levels in Ilizarov group were higher than those in b FGF group at day 10 and day 17,and the difference was statistically significant(P<0.05).(5)Conclusion1.Ilizarov microcirculation reconstruction technology can stimulate the angiogenesis of distal ischemic tissue in the limbs of patients with diabetic foot ulcer,promote the repair of diabetic foot ulcer.2.This lateral tibial transfer system is simple to make,light in weight,easy to operate and cheap in price.It can effectively carry out the transversal bone movement experiment of the upper segment of the tibia.It is suitable for wide application in animal experiments and convenient for related researchers to carry out experimental research on the lateral bone movement of the tibia.3.Ilizarov microcirculation reconstruction technology may through enhanced VEGF,Notch1 and b FGF and VEGF protein expression level,Notch1 and DLL4 m RNA transcription,boosting the corresponding protein expression,the function of promote angiogenesis,rebuild microcirculation,and promote healing of diabetic foot ulcers,improve wound healing rate,shorten the healing time,reduce the scar hyperplasia. |
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