【作者】 石冰；

【导师】 陈绍宗；

【作者基本信息】 中国人民解放军第四军医大学 ， 外科学， 2003， 博士

【摘要】 慢性创面的治疗是整形外科的一个十分棘手的问题，给患者经济上、心理上都造成了极大的负担。封闭负压引流技术(Vacuum-Assisted Closure, VAC)的出现给慢性创面的治疗带来了新的希望，许多临床以及实验研究表明VAC可以提高肉芽创面血流量，促进创面细菌、坏死组织以及引流液清除，从而可以显著提高急、慢性创面愈合速度。目前VAC技术已在国外广泛应用，在国内正在逐渐推广，但国内外有关VAC促进慢性创面愈合的机理研究还刚刚起步，很不系统，相关报道较少，特别是对于VAC应用后ECM发生的变化还未见报道。而且就慢性创面本身而言，它的形成机制还远没有阐述清楚。本实验以ECM领域中的重要成分MMPs为主要研究内容，分析VAC前后MMPs的含量、定位、活性发生的变化，同时也对MMPs的几种调控因素的变化进行了观察，从此方面部分地阐明VAC促进慢性创面的机理，而且也可部分阐明慢性创面的形成机制。 本实验以我科收治的13例慢性创面患者为研究对象(见附表)，其中压力性溃疡5例，静脉性溃疡6例，外伤后皮肤缺损1例，乳癌术后伤口迁延不愈1例，分别给予—120mmHg的VAC治疗，从以下六个部分进行观察： 第一部分：对9例慢性创面患者给予VAC治疗(-120mmHg压力)，分别于治疗前和治疗后1、4、7天切取创面中央肉芽组织，提取总RNA， 第四军医大学博士学位论文利用 RT－PCR方法测定 MMP－l、MMP－2J 3和 TIW．l、TIMPZmRNA的表达情况，并对其半定量结果进行统计学分析。结果发现：MMP－l。13mRNA在VAC治疗后表达下降，以MW上 下降趋势尤为明显。h4MI）－2 mRNA表达呈现波动，但总体为下降趋势；在VAC治疗前，检测不到TIMP4、ZInRNA的表达，应用VAC后，出现了两者的表达，而且随时间的延长，TIMP－l、ZmRNA的表达呈现明显的上升趋势。第H部分：对9例慢性创面患者给予VAC治疗～120mmHg人分别于治疗前和治疗后l、4、7天切取创面创缘组织，用免疫组织化学方法观察 MW上、2、8、9、13，TI衅上、2的蛋白分布动态变化。结果发现：总体上随 VAC作用时间延长，MMP．1、2、8、9、13的表达渐弱，而TIMP－l、2的表达增多。VAC治疗后MMi－1在表皮角化层表达增加，真皮层变化不很显著；A4MP－8不仅在炎细胞周围表达，而且在表皮中也有表达，VAC治疗前后变化不很明显，真皮染色渐淡；VAC治疗前表皮角化层有MMPq 的染色，治疗后不仅表皮染色消失，而且主要是真皮弥漫性分布变淡，越接近创缘表达越减少；MMP．2则角化层染色变淡，基底层染色加深；h4Nl，－9则表皮染色在7天时加深明显；TM上、2在VAC治疗前基本无表达，治疗后表真皮均出现表达，最深在颗粒细胞层，血管壁周围染色加深。第三部分：收集4例急性创面在术后1，2，3天的创面引流液（乳癌术后）和 4例慢性创面在VAC治疗前以及治疗后 2，4，6天的创面渗出液，利用酶谱分析的方法，观察各时间点的渗出液对可溶性＊型胶原的降解情况，同时应用强力霉素抑制实验来分析渗出液中胶原酶的主要类型。结果发现：急性创面引流液可以部分降解＊型胶原，但降解量变化不大；而慢性创面渗出液中的胶原酶活性较高，VAC治疗前基本将＊型胶原全部降解，而且随时间推移降解减少，胶原酶活性下降，强力霉素抑制实验证明在 100w浓度时对胶原酶活性无抑制，在 4 第四军医大学博士学位论文600PM浓度时出现部分抑制。第四部分：取8例急性创面在术后1、2、3天的创面引流液（乳癌术后），同时收集 11例慢性创面在 VAC治疗前以及治疗后 1、3、5、7天的创面渗出液，利用夹心ELISA法，分别测定其中T’－a－．口的含量并进行动态的观察与比较。结果发现：急性创面引流液中TNF－口水平较低，第2天含量有所下降，第3天再度回升。静脉性溃疡及压力性溃疡的创面渗出液中在治疗前TNF－口水平较高（以前者更甚），治疗后均逐步下降，前者在治疗后第1、3、5天下降最为显著o功05人后者在治疗后第5和第7天下降最为显著o＜0刀5）。第五部分：对5例慢‘性创面患者给予VA C治疗～1201：Tll：lirlg压力人分别于吸引前以及吸引后1，4，7天切取创缘组织，固定、包埋、切片后应用免疫组织化学方法观察C－fos蛋白表达的动态变化。结果发现：负压吸引前，。－fos阳性细胞表达较多，主要分布在表皮基底以及真皮浅层中的成纤维细胞及炎性细胞的胞核中，少部分胞浆中亦存在。随负压时间的延长，C－fos阳性细胞渐少，主要分布在真皮层的成纤维细胞胞核中，至第7天己罕见c．fos蛋白的表达。第六部分：取8例急性创面在术后1，2，3天的创面引流液（乳癌术后），同时收集 11例慢性创面在 VAC治疗前以及治疗后 1，3，5，7天的创面渗出液，利用放射兔疫测定的方法，分别测定其中HA的含量并进行动态的观察?更多还原

【Abstract】 Effect of the Vacuum-Assisted Closure on matrix metalloproteinases and its regulated factors in humanchronic wound Doctoral candidate ShiBingAdvisor Chen Shao-zongTherapy to chronic wound, which results in very heavier burden both in economy and psychology to patients, is a very sophisticated problem of plastic surgery.The appearance of VAC brought about a new hope toward the treatment of chronic wound. Many experimental and clinical studies suggested that VAC could promote acute and chronic wound healing owing to its increasing the blood flow in granulation wound, facilitating the remove of wound bacteria, necrosis tissue and drainage fluid. At present, VAC has been applying exclusively in foreign countries and it is being popularized gradually in our country. However, study on mechanisms of VAC promoting chronic wound healing has not been carried through systematically and relative reports are very seldom, especially variation of ECM components before and after VAC therapy had no report. Furthermore, as far as chronic wound itself, the potential formative mechanism has so far been disclosed clearly. MMP, a very important component of ECM was used as main content in our experiment. We observed the changes of value, localization and activity of MMPs, also we observed the changes of some regulated factors relative to MMPs, so asto partly explain the mechanisms of VAC promoting wound healing and of chronic wound formation.13 patients (5 cases pressure sore, 6 cases venous ulcer, 1 case skin defect after trauma, 1 case non-healing wound after operation of breast cancer) hospitalized in our department were treated with VAC (-120mmHg). We studied as follows in six parts:Parti: Using RT-PCR, we determined the expressions of MMP-1, 2,13and TIMP-1, 2 mRNA in granulation tissues from 9 patients with chronic wounds before and l,4,7days after treatment with VAC .We found that MMP-1, 13 mRNA took on an obvious tendency of decrease with the steepest variations in MMP-13. Although there were contradictory results in MMP-2mRNA, the decrease tendency was the same. There was no expression of TIMP-1, 2 mRNA before VAC. But TIMP-1, 2mRNA began to express after VAC as well as taking on obvious tendencies of increase with the time going.Part2: Using immunohistochemistry, we observed the variations ofMMP-1, 2, 8, 9, 13, TIMP-1, 2 protein in margin tissues from 9 patients with chronic wounds before and after 1, 4, 7 days after treatment with VAC. We found that in general, expression of MMP-1, 2, 8, 9, 13, became less and of TIMP-1, 2 became strong with time going after VAC. More MMP-1 expressions were found in keratinocyte layer with no obvious changes in dermis after VAC. There was less stain in keratinocyte layer and more stain in basal layer in MMP-2 and distinctly more stain in epidermis in MMP-9 after VAC. Not only can we observe the expressions of MMP-8 around inflammatory cells, but also can observe the expressionsin epidermis with no changes before and after VAC. But less expression can be found in dermis after VAC. Before VAC, MMP-13 stain can be found in keratinocyte layer, but becoming disappear after VAC. Mainly changes were that stain became less in dermis after VAC with becoming less and less approaching the wound margin. Before VAC, no TIMP-1, 2 expressions can be found, but expressions became stronger after VAC as well as stain being found around blood vessels.Part3: Collecting exudates of 4 acute wound (postoperative breast cancer)at day 1, 2, 3 postoperatively and 4 chronic wounds before and 2,4,6 days after treatment of VAC, we observed the type-Ill collagen degradation from exudates of every timepoint with zymography as well as the type of collagenase in exudates using doxycycline inhibition test. We found that drainage fluids from acute wound might degrade type-Ill collagen partly with no changes at day 1, 2, 3. There was high collagenase activity in chronic woud exudates. Exudates from patients before treatment with VAC co更多还原