深Ⅱ度烫伤创面愈合过程中转化生长因子β表达及微血管生成和胶原合成与解毒烧伤膏的干预作用

Interventional effect of Jiedu Shaoshang ointment on expression of transforming growth factor beta, microvessel generation and collagen anabolism during deep second-degree burn wound healing

目的:观察有一定促进浅Ⅱ度、深Ⅱ度创面愈合作用的解毒烧伤膏在烫伤创面愈合过程中对转化生长因子β、微血管生成和胶原合成的影响,探讨其可能的作用途径。方法:实验于2004-09/2004-12在中山大学动物中心和中山大学附属第一医院外科实验室完成。取10只小香猪,制作深Ⅱ度烫伤创面模型,随机分为解毒烧伤膏观察组和空白对照组,记录创面愈合时间;分别在伤后第3、5、7、9天取材,采用免疫组织化学链菌素生物素-过氧化物酶标记物法法检测转化生长因子β的表达,在400倍光镜下随机选取5个视野,每个视野观察100个细胞,以胞浆或(和)胞核着棕色者为阳性染色,计数阳性细胞数。以CD34一抗免疫组织化学标记血管内皮细胞,先后在低倍镜(10×10)下全面观察切片,以确定组织血管密度最高处,再在高倍镜(10×40)下计数并记录5个视野的微血管数,取其平均值,记数微血管密度。羟脯氨酸法检测每克组织胶原含量。比较实验组与对照组创面愈合时间,转化生长因子β的阳性表达率,创面微血管密度计数及胶原含量测定。结果:纳入动物10只,全部进入结果分析,无脱失值。①观察组烫伤愈合时间明显早于对照组[(11.0±1.5),(14.0±2.5)d,t=4.08,P<0.001]。②转化生长因子β表达主要在炎症细胞和毛囊上皮细胞,伤后第3、5天,观察组转化生长因子β的阳性表达率明显高于对照组[(25.0±7.5)%,(11.0±4.6)%;(45.7±8.3)%,(24.6±7.4)%,t=3.61,6.34,P<0.01];观察组转化生长因子β表达的高峰早于且强于对照组。③第5天微血管密度记数观察组显著高于对照组[(40.6±2.4),(32.6±2.4)个,t=2.69,P<0.05]。④观察组第7、9天胶原含量明显高于对照组[(2.45±0.72),(1.97±0.15)g/mg;(3.51±0.46),(2.85±0.13)g/mg;t=3.61,6.79,P<0.01]。结论:解毒烧伤膏能促进烫伤创面的愈合,可能与早期提高组织中转化生长因子β的表达、促进血管增生和后期胶原的合成有关。

AIM： To observe the reflect of Jiedu Shaoshang ointment, which has certain curative effect at superficial Ⅱ degree and deep Ⅱ degree, on transforming growth factor beta （TGF-β）, micro-vascularization and collagen synthesis, and explore its possible effect pathway. METHODS： The experiment was conducted in Animal Center of Sun Yat-sen University and surgical Laboratory of the First Affiliated Hospital of Sun Yat-sen University from September to December in 2004. Ten little pigs were used to make deep second-degree burn models and all these wounds were randomly divided into Jiedu Shaoshang ointment observational group and blank control group. Wound healing time was recorded; wound bed tissues were got on the 3^st, 5^th 7^th and 9^th days respectively after bums. Expression of TGF-β was detected using inmmunohistochemistry streptavidin biotin-peroxidase labeled method. Five sights were selected under 400 power light microscope, and 100 cells were observed in every sight. It was positive staining in plasma or （and） nucleus. Quantity of positive cells was counted. Vascular endothelial cells were labeled with CD34-anti-immunohistochemistry. Filleting was observed fully under low power microscope （10×10） so as to define highest density of tissue vessel, and then counting was done under high power microscope （10×40）and the number of microvessel was recoded at the 5 sights. Average was gained; Density of microvessel was counted. Content of tissue collagen was detected with hydroxyproline method. Wound healing time, positive expressive rate of TGF β , counting of microvessd density at wound and content of collagen were compared between experimental group and control group. RESULTS： Ten included animals were all involved in the result analysis, no drop-out animal. （1）Healing time in the observational group was earlier significantly than that in the control group [（11.0±1.5）, （14.0±2.5）days, t=4.08 ,P〈0.001].（2）The expression of TGF β was at inflammatory cell and epithelial cells of hair follicle. At the 3^st and 5^th days after injury, the positive expressive rate of TGF 13 was higher significantly than that in the control group [（25.0±7.5）%, （ 11.0±4.6 ）% ; （45.7±8.3）%, （24.6±7.4）%, t=3.61,6.34,P〈0.01]. The peak of expression of TGF β appeared earlier and stronger in the observational group than that in the control group. （3）The counting of microvessel density at the 5^th day was higher markedly in the observational group than that in the control group [（40.6±2.4）, （32.6±2.4） piece,t=2.69,P〈0.05]. （4）The content of collagen at the 7^th and 90^th days was higher remarkably than that in the control group [（2.45±0.72）,（1.97±0.15） g/mg;（3.51±0.46）,（2.85±0.13） g/mg;t=3.61, 6.79,P 〈 0.01]. CONCLUSION： The Jiedu Shaoshang ointment can promote wound healing, which may be related to the elevation of expression of TGF-β, acceleration of hyperplasia of vessel and the synthesis of collagen in later stage.