【作者】 郭立；

【导师】 章蕴毅； 翁鸿博； 力弘；

【作者基本信息】 复旦大学 ， 药理学， 2008， 硕士

【摘要】 补体系统是体内非特异性免疫系统的重要组成部分之一,在机体的防御机能、免疫功能的调节以及免疫病理过程中起着重要作用。同时,补体系统也是重要的炎症媒介物质,其非正常激活会引发人体自身组织严重的病理损伤。急性肺损伤（acute lung injury,ALI）/急性呼吸窘迫综合征（acute respiratory distresssyndrome,ARDS）是临床上较为常见的危急重症,病人常由于急性低氧性呼吸功能不全或衰竭而导致死亡。有研究表明,补体系统的活化与该疾病的发展过程相关。柴胡是一种常用的中药,可用于治疗炎症疾病和自身免疫性疾病等,小叶黑柴胡（Bupleurum smithii,B.smithii）的根部在中药中也被当作柴胡使用。先前的实验表明,柴胡具有抗补体溶血的作用,但药物的作用位点仍有疑问。体内实验也表明,柴胡对大鼠急性呼吸窘迫综合征模型具有保护作用,但尚无直接证据表明柴胡在体内是通过抑制补体系统而产生作用的。本课题采用溶血实验,通过观察补体经典激活途径和旁路激活途径中的中药抑制溶血的情况,来确认柴胡的作用位点,并对现有模型中的大鼠肺部组织切片进行染色,确定是否存在补体沉积,然后对模型进行改进,增加血气分析指标,完善该模型,为以后的实验提供指导。第一部分柴胡总多糖对补体系统的抑制作用目的研究柴胡总多糖对补体系统的作用。方法将柴胡总多糖分别与补体溶血实验中的某一要素混合,作用一段时间后再加入其他组分,观测体系的溶血情况,探讨其对补体经典途径或旁路途径激活的作用。结果在经典激活途径中,柴胡总多糖与补体预先混合,能降低体系最终的溶血,而与溶血素或羊红细胞预先混合,体系的溶血无明显改变。在旁路激活途径中,柴胡总多糖与补体预先混合也能降低体系最终的溶血,而与兔红细胞预先混合,体系的溶血无明显改变。结论柴胡总多糖通过抑制补体,从而对溶血产生影响。第二部分大鼠急性肺损伤中补体在肺部组织的沉积目的建立大鼠ALI模型,观察补体在肺部组织的沉积及柴胡总多糖对动物的保护作用。方法通过缺血再灌注和内毒素气道滴注建立两次打击大鼠ALI模型,监测大鼠血清总补体活性和血液中CO₂浓度的变化,并采用H&E染色和免疫组化的方法观察动物肺部组织的病变情况及补体C3c沉积情况。结果与伪手术组血样CO₂浓度有所上升、补体活性略微下降相比,造模组和阴性对照组的血样CO₂浓度出现大幅度上升、补体活性小幅下降,而柴胡组和阳性药物组的血样CO₂浓度上升幅度相对较小、补体活性大幅下降。H&E染色结果显示伪手术组动物的肺部基本无损伤,造模组及阴性对照组的动物肺部损伤严重,而柴胡组和阳性药物组的动物肺部损伤情况较轻。免疫组化结果也显示造模组和阴性对照组的动物肺部有大量补体C3c沉积,柴胡组、阳性药物组和伪手术组动物的肺部基本看不到补体沉积。结论柴胡总多糖在体内具有抑制补体的作用,且能改善缺血再灌注和内毒素引起的肺部炎症,有缓解大鼠ALI的作用,同时伴有肺组织补体沉积的显著减少。第三部分大鼠急性肺损伤模型中的问题探讨目的改进现有的大鼠ALI模型,探讨模型制作过程中遇到的问题。方法通过缺血再灌注和内毒素气道滴注建立两次打击大鼠ALI模型,测定大鼠血清补体溶血活性,并用两种方法测定动脉血氧分压（PaO₂）和二氧化碳分压（PaCO₂）的变化。结果造模组与正常对照组相比,PaO₂随造模时间延长明显下降,PaCO₂有所上升,伪手术组与正常对照组血气分析值相当。采用全自动血气分析仪测定的值比实验室用相应氧电极和二氧化碳电极测定更为合理、准确且精密度高。补体溶血活性结果显示,正常对照组维持在较高水平,造模组随时间延长补体活性明显下降,伪手术组在2h时与正常对照组相当,在4h时也有明显下降。结论在大鼠ALI模型中,应采用全自动血气分析仪测定PaO₂和PaCO₂,研究补体活化时,需控制造模时间在4h内,避免非ALI/ARDS原因造成的补体活化。更多还原

【Abstract】 The complement system is one of the major innate immune system,and plays an important role in body protection,immune function adjustment and immunopathogenesis.Some components of complement system are also inflammatory mediators that will cause serious damages to the human body by excess activation.Acute lung injury（ALI） and acute respiratory distress syndrome（ARDS） are common critical illness in clinic.The patients often die of acute hypoxic respiratory insufficiency or dysfunction.Experiments have informed that the activation of complement and the pathogenesis of ALI/ARDS are correlated.Radix Bupleuri,known as ’Chai Hu’,is one of the most frequently prescribed crude herbs in the prescriptions of Traditional Chinese Medicines for the treatment of inflammatory diseases and auto-immune diseases.The roots of Bupleurum smithii（B.smithii） are also used as Radix Bupleuri.Our previous experiment has informed that B.smithii has anti-complementary hemolysis effect,but the effect site is still in doubt.In vivo experiment has also shown that B.smithii has protective effect to ALI/ARDS rats.But there is no direct evidence that the protective effect of B.smithii is through the complement system.In our experiments,we studied the effect site of B.smithii in vitro by measuring the inhibitory effect through complement classical and alternative activating pathway using hemolytic assay.The lung tissue sections of the rats were stained to identify the deposits of complement C3c.The method was improved by adding blood gas analysis. AIM To investigate the effect of total polysaccharides of Bupleurum smithii（B. smithii） on the complement system.METHODS Various dilutions of B.smithii were pre-incubated with each component in the complement hemolytic assay.After that, other components were added to the mixture.The hemolysis was measured to assess the effect of B.smithii to the complement system through classical pathway or alternative pathway.RESULTS In classical pathway,the hemolysis was decreased after pre-incubation of B.smithii with complement while there was no change when B. smithii was pre-incubated with sheep red blood cell（SRBC） or anti-SRBC antibody. In alternative pathway,similar results were found that after pre-incubation of B. smithii with complement,the hemolysis was decreased while no change was seen when B,smithii was pre-incubated with rabbit red blood cell（RRBC）. CONCLUSION The total polysaccharides of B.smithii have effect in hemolytic assay by inhibiting the complement system.Part 2 Complement deposits in lung tissues of ALI ratAIM To establish a rat ALI model and to investigate complement deposits in lung and the protective effect of total polysaccharides of B.smithii to the animals.METHODS A "two-hit" lung injury model characterized by hemorrhagic shock with resuscitation, followed by intratracheal LPS administration was established.Serum total complement activity and total CO₂ concentration were measured.H&E and immunohistochemistry staining were used to determine the pathological changes and complement C3c deposits in lung tissues.RESULTS Compared to the CO₂ concentration of sham group increased slightly and the complement activity decreased a little,the CO₂ concentration of model group or vehicle group increased greatly and the complement activity had a moderate decreasing.The increase of CO₂ concentration of B.smithii group or positive control group was less than that of model or vehicle group but more than that of sham group.The complement activity of these two groups decreased greatly.H&E staining results showed that there was little injury in lungs of sham group,serious injury in model group or vehicle group and mild injury in B.smithii group or positive control group.The immunohistochemistry results showed that plenty of complement C3c deposits were found in lung tissues of model group or vehicle group while there was almost no deposit in lung tissues of B. smithii group,positive control group or sham group.CONCLUSION The total polysaccharides of B.smithii had anti-complementary effect in vivo.It could relieve the inflammations in ALI rats which were induced by ischemia/reperfusion and intratracheal LPS administration,and with complement deposits reduced in lung tissues.Part 3 Discussion of problems in rat ALI modelAIM To improve the rat ALI model and discuss the problems in establishing the model.METHODS A "two-hit" lung injury model characterized by hemorrhagic shock with resuscitation,followed by intratracheal LPS administration was established.Serum total complement activity was measured by hemolytic assay.Two blood gas analysis methods were used to determine the change of PaO₂ and PaCO₂. RESULTS Compared to those of normal group,the PaO₂ significantly decreased while the PaCO₂ increased slightly along with time in model group.The results of sham group were similar to those of normal group.The values measured by automatic blood gas analysis machine were more exact and accurate than those by using corresponding O₂ or CO₂ electrodes in the laboratory.The results of complement hemolytic assay indicated that the complement activity of normal group remained high level while the activity of model group greatly decreased along with time.The activity of sham 2h group was similar to that of normal group but it decreased in sham 4h group.CONCLUSION Automatic blood gas analysis machine should be of choise in measuring the PaO₂ and PaCO₂ in rat ALI model.The experiment should not be over 4h in order to avoid the non-ALI/ARDS induced complement activation.更多还原