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Objective: the application of molecular biology and immunohistochemical methods to study skin flap ischemia reperfusion (ischemia-reperfusion I / R) injury at different time points associated gene p53 expression variation explore ischemia-reperfusion injury of skin flap tissue apoptosis related genes p53 expression, from the genetic level, for clinical alleviate until the elimination of the flap tissue ischemia-reperfusion injury, improve skin flap survival rate of transplanted open up new therapeutic approach from the perspective of the regulation of apoptosis . Methods: Experimental animals: health Wister rats 40, weighed and numbered. 2 Animal groups: the rats were randomly divided into two groups, the control group (non-ischemic reperfusion group) and the experimental group (ischemia-reperfusion group), n = 20, each group is divided into five time points, from each time point 4. 3 flap preparation: 2% sodium pentobarbital (40mg/kg) anesthetized by intraperitoneal injection, the additional once every 3 hours (20mg/kg) to maintain anesthesia, rat supine, limbs fixed cut off chest hair, 75% alcohol disinfection, in accordance with the method described Petry JJ in the right lower quadrant design a shaft-shaped flap superficial epigastric vascular pedicle size 6cm × 3cm. Control group that is non-IR group: prepared as described above flap sutured after the formation of the flap, without other treatment; experimental group, IR group: flaps prepared as described above, after the formation of the flap, microvascular clip occlusion superficial epigastric artery to issue a point proximal femoral artery, after eight hours of surgery to remove vascular clips. 4 drawn: the control group, 20 rats were set off flap instantly, 9h, 14h, 20h, 32h, respectively, on the edge of the flap middle to take full-thickness skin flap 1cm x 0.5cm, the experimental group of 20 rats in postoperative instantly, 1h, 6h, 12h, for 24 h with the law is based. Of tissue placed in 4% paraformaldehyde solution fixed paraffin sections in a timely manner, HE staining and immunohistochemical staining to be done; another part of the organization (about 100mg) stored in liquid nitrogen tank, p53 mRNA. Detection . Detection methods and indicators: (1) the paraffin section, HE staining, observed changes in the organizational structure. (2) detected by immunohistochemistry and ratings: SP immunohistochemical staining of paraffin sections stained steps follow the kit instructions simultaneously light microscope to observe the distribution of p53 protein and coloring extent of positive and negative controls, positive signal brownish yellow or brown granules in the cytoplasm or membrane judgment. Immunohistochemical score (immunohistochemical scores IHS) method according to the literature, combining the two aspects of ratings of the percentage of positive cells and the staining intensity of positive cells: a percentage of positive cells (positive cells = 0, the positive cells accounted for 1-10% = 1, 11-50% = 2,51-80% = 3,81-100% = 4), b for the staining intensity of positive cells (negative = 0, weak positive = 1, moderate positive = 2, strongly positive = 3) , a, b two multiplied is the tissue IHS. (3) reverse transcription - polymerase chain reaction (reverse transcription, RT-PCR) semi-quantitative detection of p53 mRNA level: primers were designed based on the literature. Primer sequences: p53 upstream primer was: 5'-CAGCCAAGTCTGTGACTTGCACGTAC -3 ', p53 downstream primer 5'-CTGTGCCGAAAAGTCTGCCTGTCGTC -3'. β-actin upstream primer 5'-TCCTGACCCTGAAGTACCCCATTG -3 ', β-actin downstream primer material: the 5'-GGAACCGCTCATTGCCGATAGT -3'. Trizol RNA extraction kit manual extraction of total cellular RNA, RNA stored in liquid nitrogen, and input to the microcomputer after Electrophoresis after the UV transilluminator observation, digital camera photographic the application gel image analysis system for the purpose of electrophoretic band analysis in order to internal reference electrophoretic bands as a reference, the results expressed as the ratio of absorbance between integral. 6 Statistical analysis: with SPSS13.0 statistical processing software, different statistical treatment of the different types of data. Results: 1 HE stained histological examination: non-IR group period subcutaneous tissue edema light organization inflammatory cell infiltration rare cells arranged in neat rows. IR group after 1h, tissue congestion, edema, the stratum corneum started local damage, vasodilation, visible scattered focal neutrophil infiltration 6h subcutaneous tissue edema serious skin layers of loose structure, disorder, The perivascular visible massive neutrophil adhesion, aggregation, a large number of white blood cells, red blood seeping into the tissue space; 24h in the original situation continues to grow, further increase in neutrophils undermine the integrity of the blood vessels, and severe skin necrosis structure is unclear. (Figs. 1 to 4). Experimental group immediately after reperfusion for 1h, 6h, 12h, 24 h relative expression level of p53, respectively (Table 5): (0.132 ± 0.011) (0.262 ± 0.0528), (0.371 ± 0.057) (0.467 ± 0.068 ) and (0.552 ± 0.047). Flap ischemia-reperfusion injury, the experimental group after the change of p53 mRNA with prolonged ischemic reperfusion time, the expression of the gene transcription level gradually increased, reached a peak of 24 hours to reperfusion, significantly higher than that in the control group and the experimental group (P lt; 0.05). 3 immunohistochemical results: light microscopy of p53-positive cells were stained brownish yellow or tan. Immediately after the non-IR group and IR group, occasionally p53 protein expression in vascular wall cells, IR group after 9 h and 33 h see above basal underlying cellular subcutaneous tissue fibroblast cells, vascular wall cells, hair follicles, few positive cells expressed . IR group, reperfusion 1 h basal underlying cell fibroblasts, vascular wall cells, hair follicles, sebaceous glands and sweat glands are positive expression, subcutaneous tissue, reperfusion 6h, 12h site of p53 protein expression for 24 h reperfusion 1h same, but The upward trend in the number of positive cells, positive expression intensity increased, (P lt; 0.05). Conclusion: the skin flap ischemia-reperfusion injury induced p53 expression, apoptosis related genes p53 is involved in ischemia-reperfusion injury mechanisms, and provide a theoretical basis for the regulation of apoptosis of the skin flap from the genetic point of view.
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