GM-CSF在小鼠放射性复合切割伤中的异常表达及干预治疗实验

目的 对比观察小鼠放射性复合切割伤与单纯切割伤愈合过程中粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony stimulating factor，GM-CSF）动态表达水平的差异以及 GM-CSF对小鼠放射性复合切割伤的干预治疗效果，进一步探讨 GM-CSF 对放射性复合切割伤愈合的影响。方法 56 只雌性昆明种小鼠（20～22 g）随机分为辐照组和对照组，各28只，辐照组小鼠在 6 Gy 60Co γ 射线全身一次性均匀辐照后，即刻于背部皮肤制作全层缺损伤口，构建放射性复合切割伤模型；对照组小鼠伤口制备部位、方法同实验组，但小鼠不作辐照处理。伤后 1、3、5、7 d 时分别处死小鼠7 只，取创面周围皮肤及下方的薄层肌肉组织，通过Real-time PCR和免疫组化方法检测伤口愈合过程中GM-CSF的mRNA及蛋白表达水平。另将 40 只同品系、同体质量小鼠随机分为两组（实验组和对照组，各20只），均按上述相同方法构建放射性复合切割伤小鼠模型，于伤后 0、1、3、4、5、7、9、11、14 d 时分别给予rhGM-CSF （6 000 ng/mL）凝胶或空白凝胶涂抹，通过检测创面残余面积与胶原纤维水平，评价 rhGM-CSF 凝胶对放射性复合切割伤口的治疗效果。结果 致伤后 1～3 d 辐照组GM-CSF的mRNA及蛋白水平均低于对照组（P<0.05或P<0.01）；5～7 d 时，对照组 GM-CSF的mRNA及蛋白表达逐渐下降，而辐照组GM-CSF水平无明显改变。凝胶涂抹4～11 d内，rhGM-CSF凝胶治疗组中小鼠皮肤伤口残余面积低于空白凝胶组（P<0.05或P<0.01）；Masson 染色显示：与空白凝胶组相比，rhGM-CSF 凝胶治疗后放射性复合切割伤口中可见显著增生、广泛分布，排列紧密的胶原纤维。结论 放射性复合切割伤口早期愈合过程中 GM-CSF的mRNA及蛋白表达水平较正常伤口低，而持续给予6 000 ng/mL rhGM-CSF 刺激可明显加快放射性复合切割伤的愈合进程。
Objective To observe the changes of granulocyte-macrophage colony stimulating factor(GM-CSF) expression in mice with radiation and incision combined injury during wound healing and analyze its therapeutic effect, paving a way for exploring the effect of GM-CSF on healing of radiation combination injury. Methods A total of 56 Kunming mice (20-22 g) were randomly divided into two groups: the experimental group and control group (n=28 for each group). Mice in the experimental group were made into irradiated injury plus skin wound by incising the back immediately after exposing to 6 Gy 60Co γ radiation. Pure incision injury without radiation exposure was made in the mice in control group. Mice were sacrificed on day 1, 3, 5, and 7 (7 mice for each time) after model establishment, and skin and thin layer of muscle tissues around the wound were collected. Real-time PCR and immunohistochemical analysis were used to examine the mRNA and protein expression of GM-CSF. Moreover, radiation and incision combined injury models were also established in 40 species-, body mass-matched mice using same method described above and the model mice were divided into two groups: treatment group and blank control group (n=20 for each group). The wounds were treated with rhGM-CSF gel (6 000 ng/mL) in the treatment group at 0, 1, 3, 4, 5, 7, 9, 11, and 14 d after model establishment, while blank gel was given to the blank control group. Then the therapeutic effects of rhGM-CSF gel on wound healing were evaluated based on the wound residual area and the level of collagenous fiber around the wound. Results The expression levels of GM-CSF protein and mRNA were significantly lower in the radiation