【作者】 杨帆；

【导师】 白祥军；

【作者基本信息】 华中科技大学 ， 创伤外科， 2010， 博士

【摘要】 目的：观察负压封闭引流技术(VSD)及不同负压值对兔创面模型局部微环境、细胞因子表达、免疫细胞功能、炎症状态和创面修复的影响,以及VSD在临床治疗中预防挤压综合症、多器官功能障碍(MODS)和治疗GustiloⅢ型骨折的效果。方法：动物实验：12只兔建立创面模型并实施VSD手术(负压组),以不同负压值(-75 mmHg、-125 mmHg、-225 mmHg和350 mmHg)持续吸引。(1)应用组织氧分压测量仪监测创面局部组织氧分压(PtO2)的变化；应用游标卡尺测量并计算VSD敷料和创面大小的变化；应用细菌培养检测创面有无厌氧菌生长；应用组织切片HE染色观察创面修复情况。(2)应用实时荧光RT PCR法检测创面低氧诱导因子1-α(HIF-1α)mRNA的表达；组织匀浆后以ELISA法检测创面组织血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量。(3)创面组织HE染色后通过光镜观察并行血管内皮细胞(VEC)、新生毛细血管(NBC)巨噬细胞(MΦ)、中性多形核白细胞(PMN)和淋巴细胞(LC)计数。(4)抽取静脉血,应用全自动血细胞分析仪检测外周血白细胞(WBC)计数；应用BNⅡ特种蛋白分析仪检测C反应蛋白(CRP)含量；应用ELISA法检测外周血TNF-α和IL-6含量的变化。并设立常规换药实验兔(常规组)进行对照。临床研究：(1)随机筛选出我院创伤外科收治的102例挤压伤患者,并分为研究组(急诊VSD组50例、延迟VSD组22例)和对照组(非VSD组30例),分析各组间及组内VSD术后挤压综合征相关指标的变化。(2)回顾性分析我科2005年10月至2009年2月收治的90例合并GustiloⅢ型开放性骨折多发伤患者的临床资料,根据治疗方式分为急诊VSD组(53例)、延迟VSD组(28例)和非VSD组(9例),分析各组间缺损创面修复和骨折愈合的情况,以及预防创伤后“二次打击”的效果。(3)随机筛选出94例伴有严重皮肤软组织损伤的多发伤患者,分为研究组(56例)和对照组(38例),分析两组间“首次打击”和“二次打击”指标的差异和同MODS和预后的关联。结果：动物实验：(1)正常PtO2平均为5.79±0.50 Kpa,创面PtO2平均为3.06±1.20 Kpa,负压组PtO2较常规组和正常组显著性降低(P<0.01),随着负压值的增高,同一时间点的PtO2相应降低；7 d内PtO2逐步降低,术后5 s时降为起始PtO2的80.94%。VSD敷料经吸引面积显著性缩小(P<0.01),术后5 min时缩小为原敷料面积的65.36%%。经物理负压治疗,负压组创面面积较常规组显著性缩小(P＜0.01),术后7 d时缩小为起始创面面积62.82%。各组创面细菌培养均未见厌氧菌生长。(2)自30 m时间点起负压组的HIF-1αmRNA表达和VEGF含量较常规组显著性升高(P＜0.01)。自1 d时间点起两组HIF-1amRNA表达和VEGF含量均开始下调,但负压组仍持续高于常规组(P<0.05),同时负压组的创面VEC和NBC计数开始较常规组持续性显著性升高(P＜0.05)。(3)建模后自30 m时间点起两组创面TNF-α和IL-6含量均开始升高,自1d时间点起两组创面MΦ和PMN计数均开始升高。自6d时间点起两组创面MΦ计数均开始下降,负压组的创面MΦ计数显著性高于常规组(P<0.05)。负压组和常规组创面PMN计数分别自3d和4d时间点起开始下降,负压组创面PMN计数持续低于常规组(P＜0.01)。负压组创面LC计数于1d时间点时到达最峰值后持续下降且显著低于常规组(P<0.05)。两组创面TNF-α含量均于24h时间点达到峰值后开始下降,自1h时间点起负压组创面TNF-α含量显著低于常规组(P＜0.05)。两组创面IL-6含量于24 h时间点达到峰值后开始持续下降。自12 h时间点起负压组创面IL-6含量显著低于常规组(P＜0.05)(4)建模后两组指标含量均不同程度增高,自6 h时间点起负压组的外周血WBC计数、CRP和IL-6含量较常规组显著性降低(P<0.05)；自1 d时间点起负压组的外周静脉血TNF-α含量较常规组显著性降低(P＜0.01)。临床研究：(1)患者术后的生命征、尿量、实验室检查结果、挤压综合征发生率、并发症、病死率等评价指标在急诊VSD组及延迟VSD组分别较延迟VSD组和非VSD组显著好转(P<0.05)；挤压综合征患者中研究组术后并发症、住院时间、病死率等指标较对照组显著好转(P＜0.05)(2)VSD组术后的创面修复速度、骨折愈合速度、全身炎症反应综合征、创面感染、脓毒症、骨筋膜室综合征、骨髓炎等评价指标的结果均显著优于非VSD组(P<0.05),MODS发生率和病死率显著低于非VSD组(P＜0.01)(3)研究组术后的体温、休克、酸中毒、凝血功能、全身炎症反应综合征、感染、脓毒症、等评价指标的结果显著优于对照组(P<0.05),MODS发生率和死亡率显著低于对照组(P＜0.01)。结论：动物实验：(1)VSD技术可显著降低创面PtO2,且未见厌氧菌生长。同时可缩小VSD敷料协助收敛创面,促进创面的修复和愈合。(2)VSD技术可通过降低创面PtO2、上调HIF-1αmRNA的表达,促进VEGF的合成,增加VEC的分化和NBC的形成,血管化有利于创面愈合。(3)VSD技术通过持续吸引造成局部负压环境,可增加MΦ的数量,减少PMN和LC的数量,并下调TNF-α和IL-6的表达,以使炎症反应局限化,避免炎症细胞聚集和失控性炎症反应的发生。(4)VSD技术可通过修复创面、引流创面坏死组织和渗出液,早期降低外周血WBC计数、CRP、TNF-α和IL-6的含量,下调炎症反应,避免全身性炎症反应的发生临床研究：VSD技术可促进创面血管化和肉芽组织生长；可早期闭合GustiloⅢ型开放性骨折创面,促进创面愈合；减轻炎症反应,对预防和治疗感染、挤压综合征、MODS和降低死亡率具有显著效果。更多还原

【Abstract】 Objective:This study aimed to investigate the effect of vacuum sealing drainage (VSD) on partial micro-environment, cytokine expression, immune cell function, inflammatory status and wound healing of rabbit wound model by different negative pressure, and prevention and treatment of crush syndrome and multiple organ dysfunction syndrome, and treatment of Gustilo III fracture.Methods:Animal experiment:12 cases of rabbit’s wound models were undergoing the VSD (vaccum group)by different negative pressure(-75 mmHg, -125 mmHg,-225 mmHg and-350 mmHg)(1) Variation of oxygen partial pressure (PtO2) was measured by oxygen partial pressure admeasuring apparatus, area of VSD dressing and surface of wound were measured by vernier caliper, growth of anaerobic bacteria was detected by bacterial culture, progress of wound repair was examined by HE slice of tissue.(2) Expression of hypoxia inducible factor-la (HIF-1α) mRNA was measured by real-time fluorescent quantitative PCR, content of vascular endothelial growth factor (VEGF), tumor necrosis factor-α(TNF-a) and interleukin-6 (IL-6) were measured by ELISA after tissuehomogenate.(3) Vascular endothelial cells (VEC), new blood capillary (NBC), macrophages (Mφ), polymorphonuclear neutrophils(PMN)and lymphocytes (LC) of HE slice of tissue were counted by using light microscope.(4) Number of peripheral white blood cells (WBC) was counted by automatic rlood cell analyzer, content of C-reactive protein (CRP) was measured by BNⅡspecial protein analyzer, content of peripheral TNF-a and IL-6 were measured by ELISA, and vaccum group were compared with conventional group.Clinical research:(1) 102 cases of crush injury patients were random sampled and divided into study group (emergency VSD group 50 cases and delayed VSD group 22 cases) and control group (non-VSD group 30 cases), then analyzed the variance of index of crush syndrome after VSD between and in each group.(2) 90 patients admitted to Wuhan Tongji hospital between Oct 2005 and Feb 2009 were included in this retrospective study and were classified according to treatment method. Overall,53 cases underwent emergency VSD operations and 28 cases underwent delay VSD operations and 9 cases without VSD operations. The groups of patients were compared in terms of parameters such as repair deficiency of raw surface, promote union of fracture, prevent of post traumatic secondary strike and treatment outcomes after operation.(3) 94 cases of multiple traumatic patients with serious skin and soft tissue injury were random sampled and divided into study group (56 cases) and control group (38 cases), then analyzed the variance of "primary strike" and "secondary strike" related to the MODS and prognosis between two groups.Results:Animal experiment:(1) Average normal value of PtO2 was 5.79±0.50 Kpa, PtO2 value of wound was 3.06±1.20 Kpa, and negative pressure group was significant lower than conventional group and normal group (P<0.01). PtO2 was drop with increasing of negative pressure at the same time-point. PtO2 was reducing within 7 days, it falle to 80.94% of basic value in 5 seconds. Area of VSD dressing was significantly decreased after drainage (P＜0.01). Area was reduced to 65.36% of basic area after 5 minutes, then 62.82% after 7 days, and negative pressure group was significant lower than conventional group and normal group(P<0.01). There was no evidence of growth of anaerobic bacteria.(2) Expression of HIF-la and content of VEGF of negative group were increased after 30 minutes and significant lower than conventional group(P＜0.01), and both of them were reduced after 24 hours (P<0.05). Counting numbers of VEC and NBC of negative group were increased at the same time-point and significant higher than conventional group (P<0.05)(3) Contents of TNF-a and IL-6 of two groups were increased after 30 minutes. Counting numbers of Mφand PMN of two groups were increased after 24 hours. Counting number of Mφof two groups were reduced after 6 days, and negative pressure group was significant higher than conventional group (P＜0.05). Counting number of PMN of negative pressure group was reduced after 3 days, and it significant lower than conventional group which was reduced after 4 days (P<0.01). Counting numbers of LC of negative pressure group reached the peak value at 24 hours, then it started to decrease and significant lower than conventional group (P＜0.05). Contents of TNF-a of two groups reached the peak value at 24 hours, then they started to decrease. Negative pressure group was significant lower than conventional group after 1 hour (P<0.05). Contents of IL-6 of two groups reached the peak value at 24 hours, then they started to decrease. Negative pressure group was significant lower than conventional group after 12 hours (P＜0.05)(4) Number of WBC, content of CRP and IL-6 of negative pressure group were significant lower than conventional group after 6 hours (P<0.05). Content of TNF-a of negative pressure group was significant lower than conventional group after 24 hours (P< 0.01)Clinical research:(1) The evaluate index of emergency VSD group and delayed VSD group after operation, such as vital sign, urine volume, laboratory examination results, the incidence rate of crush syndrome, complications, fatality rate and so on were better than delayed VSD group and non-VSD group (P<0.05), the evaluate index of crush syndrome patients after operation in study group, such as complications, length of stay, fatality rate and so on were better than control group (P<0.05)(2) The speed of repair deficiency of raw surface, speed of union of fracture, systemic inflammatory response syndrome, infection of raw surface, sepsis, osteofascial compartment syndrome, ostemyelitis, ncidence rate of MODS and mortality rate in the both VSD groups were significant different from non-VSD group respectively (P<0.05)(3) The evaluate index of study group after operation, such as body temperature, shock, acidosis, blood clotting function, systemic inflammatory response syndrome, infection, sepsis and so on were significant better than control group (P<0.05), incidence rate of MODS and mortality rate were significant lower than control group (P<0.01)Conclusion:Animal experiment:(1) PtO2 could be reduced significantly by VSD without growth of anaerobic bacteria, and area of wound was reduced for promoting the wound healing.(2) Expression of HIF-la and content of VEGF were increased by VSD for enhanceing differentiated state of VEC and construction of NBC, which were better for vascularization and wound healing.(3) Microenvironment of negative pressure caused by VSD could increase the number of Mφ, and reduced the numbs of PMN and LC, decreased the contents of TNF-a and IL-6. It compartmentalized inflammation and avoided aggregation of inflammatory cell to manage uncontrolled inflammatory response.(4) Number of WBC, content of CRP, TNF-αand IL-6 were reduced by suction of nflammatory fluids, then reduce inflammatory response to avoid the systemic inflammatory response.Clinical research:VSD can promote the vascularization and granulation tissues of wound, improve to close raw surface of Gustilo III fracture early and wound healing, reduce the inflammatory response and was helpful to prevent and treat the infection, crush syndrome, MODS and reduce the mortality rate.更多还原