肿瘤坏死因子α与基质金属蛋白酶2及胶原在大鼠压疮皮肤组织中的表达及意义

Expression and significance of tumor necrosis factor alpha, matrix metalloproteinase 2 and collagen in skin tissue of pressure ulcer of rats

目的观察TNF-α、基质金属蛋白酶2（MMP-2）及胶原在大鼠压疮局部皮肤组织中的表达，探讨压疮形成的可能机制。方法将40只SD雄性大鼠按随机数字表法分为正常对照组、受压3d组、受压5d组、受压7d组、受压9d组，每组8只。正常对照组大鼠不做任何处理；后4组大鼠应用缺血一再灌注磁片循环压迫的方式，在大鼠两侧后肢股薄肌处建立深部组织损伤模型（受压3d组）和压疮模型（另3组）。受压3d组大鼠仅受压3个循环；受压5d组、受压7d组、受压9d组大鼠在受压3个循环后，划破受压皮肤，后继续分别施压至5、7、9个循环。受压3d组大鼠于受压3d后（正常对照组大鼠于相同时间点），另3组大鼠分别于受压5、7、9d后，切取两侧后肢股薄肌中心部位处的皮肤组织。HE染色观察皮肤组织形态，Masson染色观察胶原纤维表达，免疫组织化学法检测Ⅳ型胶原、MMP-2表达，蛋白质印迹法检测TNF-α、磷酸化NF—κB表达。对数据行单因素方差分析、LSD检验。结果（1）正常对照组大鼠皮肤组织为复层鳞状上皮，皮肤结构清晰，无明显炎性细胞浸润；受压3d组大鼠上皮层次清晰，Fh较多，炎性细胞开始浸润；受压5d组、受压7d组、受压9d组大鼠随着受压时间延长，表皮增厚，Fb数量减少，炎性细胞浸润增强。（2）正常对照组大鼠皮肤组织中胶原纤维排列整齐，含量丰富。受压3d组大鼠皮肤组织中胶原纤维排列有序，表达量仍较高，与正常对照组大鼠相近（P〉0．05）。受压5d组、受压7d组大鼠皮肤组织中胶原纤维排列紊乱，表达量逐渐减少，均明显低于正常对照组（P值均小于0．01）；受压9d组大鼠皮肤组织中胶原纤维表达量较受压7d组少量增加，但仍明显低于正常对照组（P〈0．01）。（3）正常对照组、受压3d组、受压5d组、受压7d组、受压9d组大鼠皮肤组织中Ⅳ型胶原表达量分别为11．0±2．8、9．0±1．7、8．3±2．8、5．1±1．8、5．4±1．2。受压3d组大鼠皮肤组织中Ⅳ型胶原表达量与正常对照组相近（P〉0．05），受压5d组、受压7d组、受压9d组大鼠皮肤组织中Ⅳ型胶原表达量均较正常对照组明显降低（P〈0．05或P〈0．01）。受压3d组大鼠皮肤组织中MMP-2表达量与正常对照组相近（P〉0．05），受压5d组、受压7d组、受压9d组大鼠皮肤组织中MMP-2表达量均明显高于正常对照组（P〈0．05或P〈0．01）。（4）正常对照组大鼠皮肤组织中TNF—α表达量为O．48±0．11；受压3d组、受压5d组、受压7d组、受压9d组大鼠皮肤组织中TNF—α表达量分别为0．84±0．08、1．13±0．19、1．34±0．16、1．52±0．23，均明显高于正常对照组（P值均小于0．01）。受压3d组、受压9d组大鼠皮肤组织中磷酸化NF．KB表达量与正常对照组相近（P值均大于0．05），受压5d组、受压7d组大鼠皮肤组织中磷酸化NF—κB表达量明显高于正常对照组（P〈0．05或P〈0．01）。结论大鼠压疮局部皮肤组织中TNF-α表达增高介导的炎症反应引起MMP-2高表达和胶原减少可能是压疮形成的重要原因之一。

Objective To observe the expressions of tumor necrosis factor alpha （TNF-α） , matrix metalloproteinase 2 （MMP-2） and collagen in local skin tissue of pressure ulcer of rats, and to explore the possible mechanism of the pathogenesis of pressure ulcer. Methods Forty male SD rats were divided into normal control group, 3 d compression group, 5 d compression group, 7 d compression group, and 9 d compression group according to the random number table, with 8 rats in each group. The rats in normal control group did not receive any treatment, whereas the rats in the latter 4 groups were established the deep tissue injury model （3 d compression group） and pressure ulcer model （the other 3 groups） on the gracilis muscle on both hind limbs using a way of cycle compression of ischemia-reperfusion magnet. The rats in 3 d com- pression group received only three cycles of compression, while the compressed skin of the rats in 5 d com- pression group, 7 d compression group, and 9 d compression group were cut through and received pressure to 5, 7 and 9 cycles after three cycles of compression, respectively. The rats in 3 d compression group were sacrificed immediately after receiving compression for 3 d （ the rats in normal control group were sacrificed at the same time） , and the rats in the other 3 groups were respectively sacrificed after receiving compression for 5, 7, and 9 d, and the skin tissue on the central part of gracilis muscle on both hind limbs were harvested. The morphology of the skin tissue was observed with HE staining. The expression of collagen fiber was ob- served with Masson staining. The expressions of collagen type Ⅳ and MMP-2 were detected by immunohisto- chemical method. The expressions of TNF-α and phosphorylated NF kappa B （NF-κB） were determined by Western blotting. Data were processed with one-way analysis of variance and LSD test. Results （1） In normal control group, the skin tissue of rats was stratified squamous epithelium, with the clear skin structure, and there was no obvious infiltration of inflammatory ceils. In 3 d compression group, the skin layers of rats were clear, with quite a few fibroblasts, and the inflammatory ceils began to infiltrate. In 5 d compression group, 7 d compression group, and 9 d compression group, the epidermis of rats thickened, with the number of fibroblasts reduced, and the infiltration of inflammatory cells enhanced with the compressed time prolonging. （2） In normal control group, the collagen fibers in skin tissue of rats were arranged in order, with rich content. In 3 d compression group, the collagen fibers in skin tissue of rats were arranged orderly, with high expression level, which was similar to that in normal control group （ P 〉 0.05）. In 5 d compression group and 7 d compression group, the collagen fibers in skin tissue of rats were arranged in disorder, with the expression level gradually reduced, which were significantly lower than that in normal control group （ with P values below 0.01 ）. In 9 d compression group, the expression of collagen fiber in skin tissue of rats was a little higher than that in 7 d compression group, but it was still significantly lower than that in normal control group （ P 〈 0.01 ）. （3） The expressions of collagen type Ⅳ in skin tissue of rats in normal control group, 3 d compression group, 5 d compression group, 7 d compression group, and 9 d compression group were respectively 11.0 ±2.8, 9.0 ±1.7, 8.3±2.8, 5.1± 1.8, and 5.4 ± 1.2. The expression of collagen type IV in skin tissue of rats in 3 d compression group was similar to that in normal control group （ P 〉 0. 05 ）. The expressions of collagen type IV in skin tissue of rats in 5 d compression group, 7 d compression group, and 9 d compression group were significantly lower than that in normal control group （ P 〈 0.05 or P 〈 0.01 ）. The expression of MMP-2 in skin tissue of rats in 3 d compression group was similar to that in normal control group （ P 〉0. 05）. The expressions of MMP-2 in skin tissue of rats in 5 d compression group, 7 d compression group, and 9 d compression group were significantly higher than that in normal control group （ P 〈 0.05 or P 〈 0.01 ）. （4） The expression of TNF-α in skin tissue of rats in normal control group was 0.48 ± 0. 11, and the expressions of TNF-α in skin tissue of rats in 3 d compression group, 5 d compression group, 7 d compression group, and 9 d compression group were respectively 0. 84 ± 0.08, 1.13 ± 0.19, 1.34 ± 0. 16, and 1.52 ± 0.23, which were all significantly higher than that in normal control group （with P values below 0.01 ）. The expressions of phosphorylated NF-κB in skin tissue of rats in 3 d compression group and 9 d compression group were similar to that in normal control group （ with P values above 0.05） , and the expressions of phosphorylated NF-κB in skin tissue of rats in 5 d compression group and 7 d compression group were significantly higher than that in normal control group （ P 〈 0. 05 or P 〈0. 01 ）. Conclusions The high expression of MMP-2 and reduction of collagen induced by inflammatory reaction mediated by the high expression of TNF-α in local skin tissue of pressure ulcer of rats may be one of the important reasons for the formation of pressure ulcer.