| Objective Millions of people suffer from the skin and soft tissue defects caused by trauma,chronic wounds,tumor resection every year.Repair and reconstruction surgery is the most important and basic treatment method for these patients.Our team has carried out a series of research and exploration on the applications of the slender narrow pedicle flaps,and reached a good clinical benefit and has a fairly spread value.However,the necrosis rate of the flap used on diabetic patients is very high,and confined its clinical applications.So how to improve the survival rate of the special random flap under the condition of diabetes,and to expand the applieation range of this flap has a great clinical signifieance.So the purpose of this experiment is as follows:CD The biological characteristics of adipose-derived stem cells were studied by culturing adipose-derived stem cells;To explore the intervention effect of adipose-derived stem cells on the healing process of slender narrow pedicle flaps by an animal model.To measure the expression and changes of CD34,HIF-1α,VEGF and the changes of microvessels of the flap during the survival process,in order to further explore the mechanism of improving the survival mechanism of the slender narrow pedicle flaps;② Through the construction of rat model of type 1 diabetes(T1DM),the biological differences and survival rates of T1DM diabetic slender narrow pedicle flaps and common slender narrow pedicle flaps were studied.Improvements in the survival of the pedicle flap and related mechanisms;③ Clinical application of slender narrow pedicle flaps on diabetics.Methods 1.(a)Culture and identification of adipose stem cells:Rabbit inguinal fat was taken for the isolation and culture of adipose-derived stem cells.The adipose-derived stem cells were induced differentiation by three-way(osteogenesis,adipogenic,chondrogenic)and identification by flow cytometry;(b)Making animal models of the rabbit slender narrow pedicle flaps.Twelve New Zealand white rabbits were randomly divided into a control group and experimental group,then were made with slender narrow pedicle flaps on both sides of the back,and the length-width ratio of the pedicles was 4cm:2cm,carrying a circular flap with a diameter of 6 cm(like the appearance of a table tennis racket).Two narrow pedicle flaps were formed on each of the two sides of the rabbit,and the pedicle was 2 cm from the midline of the back.the flap of control group(n=24),and the flap of the experimental group(n=24).(c)P4 rabbit adipose-derived stem cells were injected under the flaps at days 1,2 and 3 after surgery.One week after the operation,when the boundary of the flap necrosis was clear,the survival rate of the flaps was measured.The full-thickness tissue specimens were cut down on the edge of each flap’s remote,which size was long(0.2cm)x width(0.2cm),at days 0,5,7,and 14 after operation respectively.As a specimen By HE staining,tissue microarray,immunohistochemical,enzyme-linked immunosorbent(ELISA)techniques,and so on,for the qualitative and quantitative analysis of CD34,HIF-1α and VEGF in the flap tissue.2.Thirty SD rats were selected for intraperitoneal injection of streptozotocin 60 mg/kg for T1DM diabetes model construction.Twenty T1DM rats were randomly selected to make a model of diabetic slender narrow pedicle flaps and were randomly divided into a model group and experimental group.Another 10 normal SD rats were selected as the control group.The slender narrow pedicle flap was made on the back of 20 diabetic rats and 10 normal rats.The length-width ratio of the pedicles was 2cm:1cm,carrying a circular flap with a diameter of 4cm.Normal saline was injected on the flap as the control group(n=20),and ADSCs was injected as the experimental group(n=20).The survival rate of the flaps was measured one week after operation.The expression of HIF-1α and VEGF in each group was detected by immunohistochemistry.RT-PCR and WESTERN blot were used to detect the expression of HIF-1α and VEGF mRNA and protein in each group.HE staining was performed to observe the histological changes of the flap.The statistical significance and statistical significance were analyzed by statistical methods and SPSS software.3.Clinical application:30 cases of diabetes with chronic ulcer wounds and skin soft tissue tumors were collected and repaired by slender narrow pedicle flaps.Results 1.Adipose-derived stem cells can be obtained by isolation and purification,surface antigens CD29,CD44 positive expression,CD45 and CD31 negative expression,and can be identified into adipogenic,chondrogenic,osteogenic three-way differentiation;2.Adipose stem cells can promote flap survival and improve the revascularization of the slender narrow pedicle flaps by up-regulating the HIF-VEGF pathway.Histological analysis showed that HIF increased in the process of intraoperative tissue injury and tissue ischemia and hypoxia during the survival of slender narrow pedicle flaps.Adipose stem cells can up-regulate the HIF-VEGF pathway and promote blood vessels.Freshrnen and CD34 increase.With the appearance of angiogenesis,tissue blood supply improved,wound healing,HIF-1α and VEGF gradually decreased in the tissue,and gradually became normal after 14 days.3.The HIF-VEGF pathway was up-regulated in the diabetic flap.The flap survival rate is low and the angiogenesis disorder is directly related.ADSCs can improve the survival of the slender narrow pedicle flaps by improving the up-regulation of HIF-VEGF pathway.The swelling of the experimental side flaps and the degree of edema are significantly reduced on the experimental side,and the congestion is significantly improved compared with the control side.HIF-1α and VEGF can be seen by histological analysis.The expression of CD34 was up-regulated.4.The slender narrow pedicle flap is an ideal method for repairing various skin and soft tissue defects in diabetic patients.The use of vasodilator drugs and vacuum sealing drainage can improve the survive rate.5 of 30 patients had blood stasis at the distal end of the flap,and the epidermal detachment healed in the second stage,the remaining all survived and the wound healed well.It has a certain clinical application value and aesthetic valueConclusions(1)Adipose-derived stem cells can up-regulate the HIF-VEGF pathway,promote the revascularization of the narrow and narrow pedicle flaps to promote flap survival and improve survival rate;(2)HIF-VEGF pathway up-regulation can be seen in diabetic flaps.ADSCs can up-regulate the expression of HIF,promote the level of VEGF,and improve the vascular regeneration disorder of diabetic flaps;(3)The narrow and narrow pedicle flap is an ideal method for repairing soft tissue defects in diabetic patients. |
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