【作者】 张亮；

【导师】 黄显凯；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 硕士

【摘要】 创伤、感染均可诱发全身炎症反应综合征（Systemic Inflammatory Response Syndrome,SIRS）,其中由感染引起的SIRS被称为脓毒症（Sepsis）。最近美国和欧洲的流行病学调查表明,脓毒症的发病率约为住院病人的2-11%,其中重症脓毒症和脓毒性休克患者死亡率则高达30-50%以上。由于目前脓毒症的治疗措施尚无显著进展,只有早期诊断、早期治疗才有助于降低其死亡率。内皮细胞既是脓毒症中的效应细胞和靶细胞,又在维持机体正常生理中扮演重要角色。因此,我们推测反映内皮细胞损伤的指标能间接反映脓毒症时组织损伤程度。监测内皮细胞状态可望能更早、更敏感地判断机体脏器功能损害程度。目的本研究通过检测脓毒症动物模型外周血液及肺、肾组织中内皮细胞特异性分子-1 （ESM-1）表达水平的变化,从而了解脓毒症时内皮细胞损伤的程度,同时观察肺、肾组织学和功能的改变,分析内皮细胞损伤与其功能损害间的关系。同时选择35例多发伤病例,研究其内皮细胞损伤情况与全身病情变化的关系,为多发伤患者脏器功能损害的评估提供实验依据。方法1、大鼠脓毒症模型的制作36只清洁级雄性SD大鼠按完全随机分组分为6组,即正常对照组,假手术（sham）组,盲肠结扎穿孔（cecal ligation and puncture,CLP）3h组,CLP6h组,CLP12h组和CLP24h组,每组6只。CLP大鼠结扎盲肠全长,16号针头穿孔3个;sham组仅做剖腹术。2、炎症反应（sirs）的检测36只大鼠（每组6只）,观察呼吸、体温变化,鼠尾采血计数全血白细胞数量,外周血检测IL-6水平变化。3、大鼠CLP后ESM-1表达变化的检测36只大鼠（每组6只）相应时间点采集血清ELISA法检测外周血ESM-1表达水平;通过免疫组化方法检测肺、肾组织ESM-1表达情况。4、大鼠CLP后肺、肾功能变化的检测应用全自动生化分析仪检测36只大鼠（每组6只）BUN和Cr的水平,血气分析仪检测氧分压、二氧化碳分压、PH等血气变化;同时HE染色观察肺脏、肾脏的病理组织学变化。5、分析大鼠ESM-1表达水平变化与肾、肺功能改变的相关性。6、多发伤及并发脓毒症患者内皮细胞损伤与病情变化的相关性分析选择多发伤并发脓毒症患者35例,在入院后第1、3、7天采血, ELISA法检测患者外周血ESM-1、VEGF表达水平,将其变化与ISS评分及并非脓毒症与否进行相关性分析。结果1、大鼠脓毒症模型的一般评价正常对照和sham组的白细胞数量、体温无显著差异。CLP3h组白细胞数量、体温显著升高（P<0.05）,之后逐渐下降;CLP各组呼吸频率随时相点的变化显著增快（P<0.01）;CLP组血清IL-6表达水平随时间的延长持续增长,但直到CLP后6h时IL-6增高幅度才具有统计学意义（P<0.05）,12h组、24h组IL-6表达水平进一步增加（P<0.01）。2、CLP后ESM-1表达变化与正常对照组和sham组比较,血清ESM-1水平在CLP后6h出现明显升高（P<0.05）,在CLP后12h、24h出现显著升高（P<0.01）;CLP3h组肺组织ESM-1免疫组化染色阳性表达增多,CLP12h、24h组肺组织ESM-1的积分光密度值和平均光密度值有显著增高（P<0.05）;CLP各组肾组织ESM-1的表达均明显增高（P<0.01）;随时相点的变化其表达呈进行性增高,与CLP3h、6h组比较,CLP12h、24h组肾组织ESM-1的积分光密度值和平均光密度值有显著差异（P<0.05）。3、大鼠CLP后肺、肾脏功能变化CLP3h时部分肺泡扩张或塌陷,6h和12h时可见较大面积的肺泡闭塞,且肺泡内红细胞渗出明显,有微血栓形成。CLP24h可见较广泛的肺泡内出血,肺间质水肿,局灶性肺不张,与正常对照组相比,CLP组3h即出现PaO₂、PH下降,且随时间变化呈进行性下降,而PaCO₂持续升高,均在CLP6h后具有统计学意义（P<0.05）。CLP后3h即可见肾小球毛细血管淤血,并在CLP6h与12h组逐渐加重。CLP后24h可见小管内有脱落的上皮细胞。BUN水平在CLP后12h出现显著升高（P<0.05）;Cr水平在CLP后24h出现显著升高（P<0.05）。4、脓毒症大鼠ESM-1表达与PaO₂、PaCO₂、BUN、Cr、肺及肾组织损伤的相关分析相关分析表明,血清ESM-1表达水平变化与PaO₂、PaCO₂和肺组织损伤呈显著相关性（r=-0.659,P=0.000;r=0.559,P=0.000;r=0.825,P=0.000）;肺组织中ESM-1表达水平变化与PaO₂、PaCO₂和肺组织损伤呈显著相关性（r=-0.994,P=0.000;r=0.996,P=0.000;r=0.978,P=0.001）;血清ESM-1表达水平变化与BUN、Cr和肾组织损伤呈显著正相关（r=0.799,P=0.000;0.514,P=0.002;r=0.802,P=0.000）;肾组织中ESM-1的表达水平变化与BUN、Cr和肾组织损伤呈显著正相关（r=0.946,P=0.000;r=0.860,P=0.028;r=0.980,P=0.001）。5、多发伤并发脓毒症患者ESM-1、VEGF表达水平的变化与正常对照组比较,血清ESM-1重伤组第7天表达水平显著增高,血清VEGF轻伤组及重伤组均显著增高,差异有统计学意义（P<0.01）;重伤组第7天较轻伤组的血清ESM-1浓度显著增高,差异有统计学意义（P<0.05）。重伤组较轻伤组的血清VEGF含量显著增高（P<0.01）。并发脓毒症组与非脓毒症组间比较,第3、7天血清ESM-1含量比较均有显著增高,第1、3、7天血清VEGF含量均显著增高,差异有统计学意义（P<0.01）。结论1、CLP后外周血中ESM-1表达水平持续性升高,大鼠肺、肾脏组织学水平进行性损伤加重,肺脏及肾脏组织中ESM-1表达持续增高。2、CLP后大鼠肺、肾脏组织学检查进行性损伤,PaO₂进行性降低,PaCO₂、BUN、Cr显著增高,功能出现明显异常,其损害程度与ESM-1表达水平呈显著正相关关系,提示ESM-1表达水平的变化对创伤后脏器功能损害的评估具有较重要意义。3、多发伤患者外周血ESM-1、VEGF的表达水平进行性升高,并发脓毒症患者外周血ESM-1、VEGF的表达水平增高更为显著,能间接反映脓毒症时内皮细胞损伤程度,其表达水平与ISS评分及并发脓毒症与否呈明显正相关关系,可能对脓毒症时器官损害有一定临床意义。更多还原

【Abstract】 Sepsis is a systemic inflammatory response syndrome induced by infection. Recently, American and European surveys indicated that the incidence of sepsis occupyed 2-11% of all inpatients. Despite improved supportive care, the hospital mortality reached to 30%-50% by severe sepsis and septic shock. As there is no significant improvement in treatment for sepsis, only early diagnosis and treatment are available to reduce the mortality of sepsis. Endothelial cell is a key involved in the pathogenesis of sepsis as both a target cell and an effector cell. Besides, endothelial cell plays important roles under physiologic conditions. Therefore, we hypothesized that marker of endothelial cell damage could indirectly reflect the severity degree of tissue damage in sepsis and monitoring endothelial cell status could more sensitively to detect organ function damage in early phase.ObjectiveIn this experiment, the damages of endothelial cells will be evaluated by ESM-1 in peripheral blood, kidney, and lung in rats with sepsis resulting from cecal ligation and puncture（CLP）. Meanwhile, the changes of histology and function of lung and kidney were observed to analyze their relationships with endothelial damages. Meanwhile,35 cases of multiple injury patients were applied to explore the correlation between endothelial damage and the wounded’conditions,which may supply the experimental basement for evaluation of multiple injury patient’s organ damage.Material and Methods1. Preparation the model of rat with sepsis 36 rats were randomly divided into 6 groups:normal control, sham operation, CLP3h, CLP6h, CLP12h and CLP24h, with 6 rats in each group. CLP was performed as ligating the cecum below the ileocecal valve and puncturing thrice with a 16-gauge needle.Sham surgery was only performed laparotomy.2. Determination of SIRS Body temperature and breathing frequency of 36 rats were monitored. Blood were collected from tail vein to count the number of leucocytes.The serum IL-6 was measured by enzyme linked immunosorbent assay（ELISA）simultaneously.3. ESM-1 were determined in sepsis rats The serum ESM-1 was measured by ELISA and the expression of ESM-1 in the kidney and the lung were determined by immunohistochemistry at every time point.4. Functional changes of the kidney and the lung The levels of BUN and Cr of all rats were measured by automatic biochemistry analyzer. PaO₂, PaCO₂ and PH were measured by blood gas analyzer. The histologic change of lung and kidney were observed with H.E.staining.5. The correlation analysis between ESM-1 and functional changes of the rats’kidney and lung.6. The correlation analysis between endothelial cells’damages and severity of multiple injury patients blood of trauma patients, who were divided into sepsis and non-sepsis group, were collected on day 1,3,7 after admission. The serum ESM-1 and VEGF were measured by ELISA. Then analyzed the correlation of ESM-1, VEGF, Injury Severity Score, and sepsis.Results1. General evaluation of rat CLP model There was no significant difference between the normal and sham group. The amount of leukocyte and body temperature rosed in CLP3h group （P<0.05）and then decreased gradually; the breath frequency of all CLP group were quicker than others（P<0.01）.The levels of IL-6 increased continuously with time passing after CLP operation.2. The change of ESM-1 after CLP Compared with the normal control and sham groups,the levels of serum ESM-1 increased after CLP 6h（P<0.05）and obviously in CLP12h and 24h（P<0.01）. Immunohistochemistry result displayed that positive staining of ESM-1 singnificantly higher in kidney and lung tissue in CLP3h、6h、12h、24h group than normal control group （ P<0.01）; in CLP12h、24h group ESM-1 integral luminosity, average luminosity singnificantly increased than CLP3h、6h group（P<0.05） .3. The function change of rat’s lung and kidney after CLP Part alveolar ectasia or collapse imerged at 3h after CLP, and large area atelectasis are found in CLP6h and CLP12h group with erthrocyte exdudation, and microthrombosis in pulmonary alveoli. Extensive intra-alveolar hemorrhage, pulmonary mesenchymal edema, and focal atelectasis were observed at 24h after CLP. Compared with the normal controls, the PaO₂ and pH decreased at 3h after CLP and decreased continuously, while PaCO₂ continuously increased.Renal glomerulus engorgement imerged at 3,6,12h after CLP and tuble epithelial cells cast were observed at 24h after CLP. BUN and Cr were first increased remarkably in CLP12h and 24h respectively（P<0.05）.4. Correlation between ESM-1 of rat with sepsis and PaO₂,PaCO₂,BUN,Cr,lung and kidney damage Serum ESM-1 had significantly negative correlation with PaO₂（P<0.01） and it had significantly positive correlation with PaCO₂ and lung tissue injury.The result of ESM-1 in the lung was as the same as that in serum. ESM-1 in the Serum was positively correlated with BUN、Cr, and kidney tissue injury. ESM-1 in the kidney was positively correlated with BUN、Cr, and kidney tissue injury.5. Changes of ESM-1 and VEGF in multiple trauma patients with sepsis Compared with the normal control group,the levels of serum ESM-1 increased in severe injury group at 7d, and serum VEGF increased in minor injury group and severe injury group（P<0.01）. Moreover, the levels of serum ESM-1 in severe injury group were obviously higher than that of minor injury group at 7d. However the levels of serum VEGF in severe injury group were obviously higher than that of minor injury group at every time point. Compared with non-septic trauma patients,the levels of serum ESM-1of trauma patients with sepsis increased remarkably at 3d and 7 d and the levels of serum VEGF increased at every time point.Conclusion1. After CLP, the level of ESM-1 in peripheral blood and in kidney and lung tissue increased continuously meanwhile lung and kidney tissue damages aggravated continuously.2. Lung and kidney tissue damages aggravated continuously after CLP with decreasing PaO₂ but increasing PaCO₂,BUN and Cr,moreover the level of ESM-1 was significantly positively correlated with tissue injury.Thus the results indicated that the changes of ESM-1 played important role in evaluation of organ function after trauma.3. The level of serum ESM-1and VEGF increased continuosly in multiple injury patients, moreover the level of serum ESM-1and VEGF was even higher in the trauma patients with sepsis. The level of serum ESM-1and VEGF may indirectly reflect the endothelial damages and they had significantly positive correlation with ISS and incidence of sepsis, so they were clinically available in organ injury in sepsis.更多还原