【摘要】 目的:利用Ⅶ型胶原酶加肝素构建大鼠脑出血致多器官功能障碍综合征模型,为进一步探讨急性脑血管病致多器官功能障碍综合征的发病机制提供实验模型依据。方法:实验于2003-11/2004-02在山东省医学科学院完成。①选取清洁级5～8月龄健康雄性Wistar大鼠96只,随机数字表法分为4组:正常对照组6只、假手术组6只、Ⅶ型胶原酶6.668nkat+肝素钠组42只、Ⅶ型胶原酶13.336nkat+肝素钠组42只,后两组又分为4,8,12,24,36,48,72h7个时相点,6只/时相点。②除正常对照组外,其余组均采用立体定向术在大鼠尾状核注入Ⅶ型胶原酶加肝素建立脑出血致多器官功能障碍综合征模型。在立体定向仪的引导下,用微量注射器(针头直径约0.6mm)沿钻孔垂直进针6mm,Ⅶ型胶原酶6.668nkat+肝素钠组缓慢注入Ⅶ型胶原酶6.668nkat和3.5IU肝素钠混合液2μL;Ⅶ型胶原酶13.336nkat+肝素钠组注入VII型胶原酶13.336nkat和3.5IU肝素钠混合液2μL,留针10min后缓慢退出。假手术组注射等体积生理盐水。③观察术后各时相点大鼠症状及体征变化,检测外周血白细胞、肝肾功能及心肌酶学的改变,光镜下观察脑、肺、肝、肾和小肠组织病理变化,计算全身炎症反应综合征、多器官功能障碍综合征的发生率。结果:实验选取清洁级健康雄性Wistar大鼠96只,全部进入结果分析。①各组大鼠术后脑大体形态观察结果:假手术组未见明显血肿;Ⅶ型胶原酶6.668nkat+肝素钠组、Ⅶ型胶原酶13.336nkat+肝素钠组在术后4h均形成血肿,24h后血肿基本不再增大。Ⅶ型胶原酶13.336nkat+肝素钠组形成的血肿大于Ⅶ型胶原酶6.668nkat+肝素钠组。②各组大鼠术后神经功能的变化:假手术组神经功能评分为0分;Ⅶ型胶原酶13.336nkat+肝素钠组神经功能评分明显高于Ⅶ型胶原酶6.668nkat+肝素钠组[(3.32±0.49),(1.64±0.18)分,P<0.01]。③各组大鼠术后呼吸、心率、体温、外周血白细胞、肝功、肾功、心肌酶学的变化:与正常对照组比较,假手术组上述各项指标均无明显差异(P>0.05);Ⅶ型胶原酶6.668nkat+肝素钠组、Ⅶ型胶原酶13.336nkat+肝素钠组均明显升高(P<0.01),且Ⅶ型胶原酶13.336nkat+肝素钠组各指标升高幅度显著高于Ⅶ型胶原酶6.668nkat+肝素钠组(P<0.05)。④各组大鼠术后主要脏器病理的改变:正常对照组和假手术组各器官组织结构基本正常。Ⅶ型胶原酶6.668nkat+肝素钠组肺、肝、小肠、肾等脏器各时相点的病理改变以非特异性炎症反应为主。Ⅶ型胶原酶13.336nkat+肝素钠组肺、肝、小肠、肾等脏器各时相点的炎性病理改变均较Ⅶ型胶原酶6.668nkat+肝素钠组更加明显、持久。⑤脑出血后全身炎症反应综合征、多器官功能障碍综合征的发生率:Ⅶ型胶原酶6.668nkat+肝素钠组分别为75.4%和21.4%,Ⅶ型胶原酶13.336nkat+肝素钠组分别为100%和67.9%。结论:大鼠尾状核注入13.336nkatⅦ型胶原酶加肝素法可以成功构建脑出血致多器官功能障碍综合征模型。更多还原

【Abstract】 AIM: To establish rat models of multiple organ dysfunction syndrome (MODS) induced by cerebral hemorrhage with type Ⅶ collagenase and heparin,so as to provide basis of experimental models for the pathogenesis of acute cerebrovascular diseases(ACVD)induced-MODS. METHODS: The experiment was conducted in Shandong Academy of Medical Sciences from November 2003 to February 2004.①Ninety-six healthy male Wister rats of clean grade aged 5-8 months were selected and randomly divided into four groups: normal control group (n=6),sham-operation group (n=6),type Ⅶ collagenase 6.668 nkat + heparin group (n=42) and type Ⅶ collagenase 13.336 nkat + heparin group (n=42). The latter 2 groups were subdivided into 7 groups according to the time-points of the 4th,8th,12th,24th,36th,48th,72nd hours with 6 rats in each time-point group.②The stereotaxy was adopted to establish models of MODS induced by cerebral hemorrhage by injection of type Ⅶ collagenase and heparin into the caudate nucleus of rats. Under the guidance of stereotaxic apparatus,2 μL of mixed liquor of 6.668 nkat type Ⅶ collagenase and 3.5 IU of heparin were slowly and vertically injected for 6 mm with micro-injector (the diameter of pinhead was about 0.6 mm) in rats of type Ⅶ collagenase 6.668 nkat + heparin group,and rats in type Ⅶ collagenase 13.336 nkat + heparin group were injected with 2 μL of mixed liquor of 13.336 nkat type Ⅶ collagenase and 3.5 IU of heparin with 10-minute retention. Rats in the sham-operation group were injected with the same-volume of normal saline.③Changes of syndromes and physical signs in rats were observed and changes of peripheral blood leucocyte (PBL),function of liver and kidney as well as zymologic changes in myocardium were detected. Pathological changes of brain,lung,liver,intestines and kidney were observed under light microscope,and the incidences of systemic inflammatory response syndrome (SIRS) and MODS were calculated. RESULTS: A total of 96 enrolled healthy male Wistar rats were involved in the analysis of results.①General observation on brain of rats in each group after operation: no obvious hematoma was seen in the sham-operation group,while hematoma was found in type Ⅶ collagenase 6.668 nkat + heparin group and type Ⅶ collagenase 13.336 nkat + heparin group,but the hematoma did not enlarge after 24 hours. The hematoma was greater in type Ⅶ collagenase 13.336 nkat + heparin group than in type Ⅶ collagenase 6.668 nkat + heparin group.②Neurofunctional changes in rats of each group after operation: the score of sham-operation group was zero,and that in type Ⅶ collagenase 13.336 nkat + heparin group was obviously higher than type Ⅶ collagenase 6.668 nkat + heparin group (3.32±0.49),(1.64 ±0.18) points,P < 0.01.③Changes of respiration,heart rate,body temperature,PBL,function of liver and kidney as well as zymologic changes in myocardium of rats in each group after operation: there were no significant differences in above-mentioned indicators between normal control group and sham-operation group (P > 0.05),while those in type Ⅶ collagenase 6.668 nkat + heparin group and type Ⅶ collagenase 13.336 nkat + heparin group were obviously increased (P < 0.01). Moreover,those were remarkably increased in type Ⅶ collagenase 13.336 nkat + heparin group than in type Ⅶ collagenase 6.668 nkat + heparin group (P < 0.05).④Pathological changes of main organs in rats of each group after operation: the structure of each organs in rats of the sham-operation group and normal control group were essentially normal. The pathological changes of lung,liver,intestine and kidney etc. in rats of type Ⅶ collagenase 6.668 nkat + heparin group were characteristics of non-specific inflammatory reaction,and the pathological changes of lung,liver,intestine and kidney etc. at each time-point were more permanent and lasting in type Ⅶ collagenase 13.336 nkat + heparin group than in type Ⅶ collagenase 6.668nkat + heparin group.⑤The incidences of SIRS and MODS after cerebral hemorrhage: those in type Ⅶ collagenase 6.668 nkat + heparin group were 75.4% and 21.4% respectively,and those in type Ⅶ collagenase 13.336 nkat + heparin group were 100% and 67.9% respectively. CONCLUSION: The rat model of MODS induced by cerebral hemorrhage can be successfully established by injecting 13.336 nkat of type Ⅶ collagenase and heparin into the caudate nuclear of rats.更多还原