| Objective: To observe the changes of cell apoptosis related p53 gene expression in different timepoint during is chemia-reperfusion injury in skin flap cellsby means of RT-PCR technique and immunohistochemial technique. To investigate effects of ischemia-reperfusion injury on cell apoptosis related p53 gene expression in skin flap cells for exploring a new method that can lessen even avoid ischemia-reperfusion injury and heighten the survival rate in clinical skin flap.Method:1 laboratry animal: 40 male and healthy Wister rats were weighted and ordered.2 The rats were divided into two groups randomly, control group (non-ischemia-reperfusion group) and Experimental group (ischemia-reperfusio group ), each group included 20 rats.3 The preparation of skin flap: The rats were anesthetized with 2% sodium pentobarbital(40mg/kg) by intraperitoneal injection, their limbs were fixed and their hairs on abdomen were sheared, the skin of abdomen was sterilized by 75% alcohol after the hairs were cut down. A right low abdominal island skin flap, which size was 6cm×3cm, was created according to the method of Petry JJ's. control group: There were no other treatments after creating the skin flap according to the method above. Experimental group: After creating the skin flap according to the method above, the proximal end of the point that the superficial epigastric artery arised from femoral artery, was occluded by a vascular clamp, the vascular clamp was taken out in another operation after 8 hours.4 To draw the materials from the skin flaps: control group: Full-thickness of the skin flap which size was 1cm×0.5cm was taken at the moment, 2 hours, 6 hours, 12 hours, 24 hours when the skin flap was created. Experimental group: The same sample of skin flap was taken respectively at the moment, 9 hours, 14 hours, 20 hours, 32 hours when after ischemia-reperfusion. One part of the obtain skin flap was conserved in 4% paraformaldehyde liquor, commit paraffin section in good time, in order to dyeing using method of immunohistochemistry and HE. The other part of the obtain skin flap (100mg) was conserved in Liquid nitrogen tank, in order to detected the the expression of P53 mRNA by RT-PCR technique.5 The method of detection and the observation target:(1) The paraffin sections were stained of HE, to observe the changes.(2) According to the specification of the test kit, dyed the paraffin section by immunohistochemistry, the pigmenting and the location of Bcl-2 were observed with a light microscope. The immunohisto- chemical scores (IHS) was calculated by using image analysis system.(3) The expression of P53 mRNA was detected by RT-PCR technique.6 Analysis of results: Different kinds of results were dealed with by using SPSS 13.0 software.Results:1 Histological examination of HE staining:In group at various time the subcutaneous tissue of flaps showed edema and light inflammation, Cells arranged in neat rows. IR group after 1h, tissue of congestion, edema, epidermal cuticle began to local damage, vasodilatation, seen scattered neutrophil infiltration, after 6h severe subcutaneous edema, skin layers of loose structure, disorder in the Seen a large number of perivascular neutrophil adhesion, aggregation, a large number of white blood cells, red blood cells leaking into the interstitial space; after 24h existing situation continues to grow, further increase in neutrophils, vascular integrity of the damage, severe skin necrosis and the structure is unclear.2 The expression of Bcl-2 mRNA was detected by RT-PCR technique: the experimental group after reperfusion immediately, 1h, 6h, 12h, 24h, the level of the relative expression of p53 mRNA respectively was (0.132±0.011), (0.262±0.0528), (0.371±0.057), (0.467±0.068) and (0.552±0.047). The experimental group after flap ischemia reperfusion injury, with ischemia and reperfusion time changes, the level of p53mRNA expression increased gradually, and achieved to peak after reperfusing 24 hours. The experimental group was significantly higher than control group (P <0.05).3 The results of immunohistochemistry staining: Light microscope, p53-positive cells were stained brown or tan. Non-IR group and IR group after Surgery immediately, p53 protein expression in vascular endothelial cells accidentally. Non-IR after 9h and 33h, p53 positive cells expressed in cytoplasm of epidermal basal cells, fibroblasts, vascular endothelia cells and the hair follicle, it was small. IR group after 1 h, p53 positive cells expressed in basal cells, fibroblasts, vascular endothelial cells, hair follicles, sebaceous glands and sweat glands. the expression position of p53 positive cells in 1h was the same with 6h, 12h, 24h, the number of positive cells was increased and the intensity of expression was enhanced(P <0.05), and regulation of skin flap from the genetic point of view to provide theoretical basis of apoptosis.Conclusion: The ischemia-reperfusion injury can increase the expression of P53 gene in skin flap. P53 gene has been concerned with the mechanism of ischemia-reperfusion injury. it can provide a theoretical basis on protecting and controling the flaps ischemia-reperfusion injure from theperspective of gene regulation.
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