建立一种更稳定、可重复的小鼠皮瓣缺血再灌注损伤(IRI)模型，明确皮瓣缺血再灌注损伤机制，阐述皮瓣缺血时间、再灌注血流量与皮瓣损伤程度的关系。

共选用162只SPF级ICR健康雄性小鼠为本次实验对象。(1)实验一，选用90只ICR小鼠，采用抽签法随机分为3组(n＝30)：胸部皮瓣组、背部皮瓣组、腹部皮瓣组，再分别将各组小鼠按夹闭皮瓣蒂血管的时间(皮瓣缺血时间)分为6个亚组(n＝5)：Ⅰ、Ⅱ、Ⅲ、Ⅵ、Ⅴ、Ⅵ组，对应的缺血时间分别为0、1.5、3.0、5.0、8.0、10.0 h。胸部、背部、腹部皮瓣分别以左侧胸外侧动脉、胸背动脉、腹浅动脉为对称轴设计宽1.5 cm、长3.0 cm的轴型皮瓣。微型动脉夹夹闭血管蒂，建立缺血皮瓣模型。用全景激光灌注成像仪(FLPI)观察各组皮瓣基础血流量，再灌注7 d后观察皮瓣存活情况，计算坏死率，确定最佳小鼠皮瓣IRI模型。(2)实验二，选用另外72只ICR小鼠，建立上述胸部皮瓣IRI模型，按皮瓣缺血时间分为6组(n＝12)：ⅰ、ⅱ、ⅲ、ⅳ、ⅴ、ⅵ组，对应的缺血时间分别为0、1.5、3.0、5.0、8.0、10.0 h，用FLPI动态观察各组皮瓣的血流灌注量。再灌注7 d后观察皮瓣存活情况，计算各组皮瓣坏死率。分别取各组半数小鼠再灌注24.0 h后的皮瓣组织，用苏木精-伊红染色分析组织形态学改变，二氯氢化荧光素二乙酸酯探针法检测细胞内活性氧含量，末端脱氧核苷酸转移酶介导的d-UTP缺口末端标记(TUNEL)染色检测细胞凋亡并计算凋亡细胞阳性指数，酶联免疫吸附测定法(ELISA)检测再灌注24.0 h后肿瘤坏死因子-α(TNF-α)表达水平。对数据行单因素方差分析、t检验。

实验一结果显示：小鼠胸部皮瓣基础血流灌注量为(320.2±7.5) PU，背部皮瓣基础血流灌注量为(156.3±12.4) PU，腹部皮瓣基础血流灌注量为(78.5±5.5) PU，各部位差异有统计学意义(F＝174.8，P<0.05)。胸部皮瓣组不同亚组所致皮瓣坏死率比较差异有统计学意义(F＝261.7，P<0.05)，且背部、腹部皮瓣组皮瓣坏死率比较，差异均有统计学意义(F＝39.6、235.1，P值均小于0.05)。实验二结果显示：小鼠胸部皮瓣可耐受1.5 h以内缺血，再灌注血流量恢复快，ⅰ、ⅱ组皮瓣坏死率分别为(1.20±0.15)%、(6.50±0.24)%，均小于10.00%。而ⅰ组活性氧水平相对倍数、凋亡细胞阳性指数、TNF-α表达水平分别为1.00±0.12、(3.2±0.1)%、(2.09±0.93) μg/μL，ⅱ组分别为1.26±0.07、(4.3±0.1)%、(3.63±0.42) μg/μL，与ⅰ组相比均升高，差异均有统计学意义(t＝3.58、6.77、3.03，P值均小于0.05)；ⅲ、ⅳ组皮瓣再灌注血流量恢复缓慢，ⅳ组皮瓣坏死率、活性氧水平相对倍数、凋亡细胞阳性指数、TNF-α表达水平分别为：(48.2±3.57)%、2.34±0.17、(22.2±1.4)%、(4.79±0.72) μg/μL，与ⅱ组相比均升高，差异有统计学意义(t＝11.52、10.50、12.85、2.80，P值均小于0.05)。ⅴ、ⅵ组皮瓣再灌注血流量恢复极缓慢，苏木精-伊红染色显示：组织充血、血管内血栓形成，其中ⅴ组皮瓣坏死率、TNF-α表达水平分别为(75.7±3.30)%、(6.10±0.56)%，与ⅳ组相比均升高，差异均有统计学意义(t＝6.59、2.86，P值均小于0.05)；而ⅴ组活性氧水平相对倍数为1.47±0.21，较ⅳ组降低，差异有统计学意义(t＝5.55，P＝0.005)，凋亡细胞阳性指数为(20.5±2.2)%，与ⅳ组比较，差异无统计学意义(t＝0.15，P＝0.88)。

与小鼠背部、腹部皮瓣IRI模型相比，小鼠胸部皮瓣IRI模型基础血流量更大、更稳定，不同缺血时间所致皮瓣坏死率变化更有显著性。缺血再灌注可以引起小鼠胸部皮瓣的损伤，并随着缺血时间的延长，再灌注血流量明显降低，皮瓣的损伤逐渐加重。缺血再灌注致皮瓣损伤的机制可能是缺血再灌注后产生大量的活性氧，导致血管收缩，影响微循环；同时大量炎性细胞聚集，局部炎症反应加重，导致皮瓣损伤加重。

To design a mouse model of skin flap ischemia-reperfusion which is suitable and reproducible, clarify the mechanism of ischemia reperfusion injury (IRI) of the skin flap, and elaborate the relationship between the ischemia time, reperfusion blood flow and the degree of IRI.

A total of 162 SPF grade healthy male ICR mice were selected as the experimental subjects. (1)In the experiment one, 90 IRI mice were respectively drawn into 3 groups (n=30): thoracic skin flap group, dorsal skin flap group and epigastric skin flap group, and then the mice in each group were divided into 6 groups (n=5), Ⅰ, Ⅱ, Ⅲ, Ⅵ, Ⅴ, Ⅵ group according to the time of the vessel pedicled clamped(time of ischemia), the corresponding ischemia time respectively was 0, 1.5, 3.0, 5.0, 8.0, 10.0 h. The 3.0 cm×1.5 cm flaps were designed with symmetry axis of left lateral thoracic artery, thoracic dorsal artery and superficial abdominal artery. A model of ischemic flap was established by clamping pedicle with miniature artery clip. Basic blood flow of flap in each group were detected by full-field laser perfusion imager (FLPI), after 7 days reperfusion, percent necrosis of flap was measured. The optimal model of murine skin flap for ischemia/reperfusion was determined by statistical analysis.(2)In the experiment two, the remaining 72 male ICR mice were divided into 6 groups (n=12), ⅰ, ⅱ, ⅲ, ⅳ, ⅴ, ⅵ group according to the time of the vessel pedicled clamped(time of ischemia), the corresponding ischemia time respectively was 0, 1.5, 3.0, 5.0, 8.0, 10.0 h. Blood flow during ischemia and reperfusion were monitored and analysised using FLPI. After 7 days reperfusion, the survival of the flap was observed and the rate of skin flap necrosis was calculated. Flap tissues of half of the mice in each group were taken after 24.0 h of reperfusion. Histomorphological changes were analyzed by hematoxylin- eosin staining. Dichlorofluorescein diacetate probe method was used to detect the content of reactive oxygen species. Terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labing was used to detect apoptosis and calculate the positive index of apoptotic cells. The content of tumor necrosis factor-α(TNF- α)was determined by enzyme-linked immunosorbent assay (ELISA). Data were processed with one-way analysis of variance and t test.

In the experiment one, the results showed that the basal perfusion blood flow of the mice thoracic flap was (320.2±7.5) PU, the basal perfusion blood flow of dorsal skin flap was (156.3±12.4) PU, the basal perfusion blood flow of epigastric skin flap was (78.5±5.5) PU, and the differences was statistically significant (F=174.8, P<0.05). In the thoracic skin flap group, the necrosis rates of the flap were different in every ischemic time periods, the difference was statistically significant (F=261.7, P<0.05), meanwhile, the necrosis rates of the flap were different in every ischemic time periods in the dorsal skin flap group and in the epigastric flap skin group, the differences were statistically significant (F=39.6, 235.1; with P values below 0.05). In the experiment two, the results showed that the mice thoracic skin flap could tolerate ischemia within 1.5 hours, the flap reperfusion blood flow recovered fast. The necrosis rate of groupⅰ, ⅱ was (1.20±0.15)%, (6.50±0.24)%, which were less than 10.00%. The content of reactive oxygen species, the positive index of apoptotic cells, TNF-α in groupⅰwere 1.00±0.12, (3.2±0.1)%、(2.09±0.93) μg/μL, and in group ⅱ were 1.26±0.07, (4.3±0.1)%, (3.63±0.42) μg/μL, which were increased compared with groupⅰ, the differences were statistically significant(t=3.58, 6.77, 3.03; with P values below 0.05). In group ⅲ, ⅳ, the flap reperfusion blood flow recovered slowly, flap necrosis rate, relative multiples of reactive oxygen species, the positive index of apoptotic cells, TNF-α were(48.2±3.57)%, 2.34±0.17, (22.2±1.4)%, (4.79±0.72) μg/μL, which were increased significantly compared with group ⅱ, the differences were statistically significant(t=11.52, 10.50, 12.85, 2.80; with P values below 0.05). In groupⅴ, ⅵ, the flap reperfusion blood flow recovered very slowly. Hematoxylin - eosin staining showed tissue hyperemia and intravascular thrombosis. The necrosis rate and TNF-α were (75.7±3.30)%, (6.10±0.56)%, which were increased significantly compared with group ⅵ, the differences were statistically significant (t=6.59, 2.86; with P values below 0.05). Relative multiples of reactive oxygen species was 1.47±0.21 in groupⅴ, which was decreased significantly compared with group ⅵ, the difference was statistically significant(t=5.55, P=0.005); the positive index of apoptotic cells was (20.5±2.2)%, which was no significant difference compared with group ⅳ, the difference was statistically significant (t=0.15, P=0.88).

Compared with the model of mice dorsal skin flap and epigastric skin flap, the perfusion blood flow of mice thoracic skin flap model was higher and more stable, and the change of necrosis rate caused by different ischemic time was more statistically significant. Ischemia reperfusion can cause the injury of the thoracic skin flap in mice, with the prolongation of the ischemia time, reperfusion blood flow is more decreased, injury of the flap becomes worse. The mechanism of ischemia reperfusion injury to the skin flap may be that after ischemia reperfusion, a large amount of reactive oxygen species is produced, leading to vasoconstriction and affecting microcirculation. At the same time reactive oxygen species results in the accumulation of numerous neutrophils, local inflammatory reaction aggravation, leading to the aggravation of the flap injury.