C5a对组织因子表达的影响及潜在的机制研究
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摘要
脓毒症一直是医学界的一大挑战。尽管给予患者强有力的抗生素治疗和器官功能支持,其仍旧是ICU患者死亡的主要原因之一。据北美相关统计：脓毒症的发病率约为0.3%、在美国每年750,000脓毒症患者中约有215,000死亡；ICU病房中出现脓毒症的约占2%-11%、脓毒症和脓毒症休克的病死率分别为30%和60%。在过去的数十年,脓毒症的病死率并没有明显的改善。究其原因,与疾病本身复杂的病理生理过程和目前人们对它的认识程度有很大的关系。
脓毒症共同的病理生理特征是一系列炎症细胞被相继激活,呈“瀑布效应”释放出大量炎症因子,对全身组织器官造成损伤,最终引起多器官功能障碍综合征(multiple organs dysfunction syndromes, MODS),进一步发展为多器官功能衰竭(multiple organs failure,MOF)。在炎症反应的过程中,多种生物学活性系统被激活如：补体系统、凝血系统、免疫系统以及凋亡相关系统等。其中,弥散性血管内凝血(disseminated intravascular coagulation, DIC)是脓毒症患者凝血系统异常引发的一种严重并发症,一旦出现DIC,患者将因为全身微血管广泛微血栓的形成,造成组织器官缺血、缺氧从而加速MODS的进程。一项研究显示,在脓毒症特别是脓毒症休克的病例中,发生DIC的比例约为30-50%。凝血功能异常也俨然成为了脓毒症病理生理过程中的突出表现。在脓毒症或全身炎症反应过程中,多种促炎症细胞因子和炎症介质,如TNF-α、ILs、PAF、C5a等,均可引起凝血功能异常。其中,补体系统活化产物C5a在凝血中的作用日益受到学者的关注。
C5a是三条补体途径共同裂解产生的活性产物,具有广泛的生物学功能。在脓毒症治疗新策略中,拮抗C5a已经被人们在动物实验中所提及和证实,本课题组前期自主筛选合成了一种新型C5a拮抗多肽(C5a反义肽),相关实验证明了其能拮抗C5a的生物学活性,对改善小鼠脓毒症模型的炎症反应有一定的保护作用。但C5a作为一种促炎介质,其在凝血方面的作用也不容忽视。已有的研究显示,其对凝血的影响包括增加炎症细胞,如中性粒细胞、单核巨噬细胞等细胞膜表面组织因子(tissue factor, TF)的表达,从而使凝血激活亢进；降低AT-Ⅲ、血栓调节蛋白(thrombomodulin,TM)等物质的表达而使机体抗凝功能削弱；影响纤溶酶原激活物和抑制物(plasminogen activator,PA;plasminogen activator inhibitor, PAI)的表达而使纤溶和抗纤溶平衡失调。鉴于TF是外源性凝血途径的启动因子,在外源性凝血激活“网络模式”中处于中心地位。故现有的研究主要集中在C5a影响中性粒细胞、大血管内皮细胞TF的表达方面。但由于凝血异常主要发生在微小血管,尤其是肺作为MODS首发靶器官,故C5a对微血管内皮细胞表达TF的影响以及机制更值得关注。
在纵多参与炎症反应调控的转录因子中,核因子-κB (Nuclear factor kapp B,NF-κB)是研究的焦点。它存在于体内多种细胞中,是一种可调控多种功能蛋白分子表达而参与多种疾病发病过程的转录因子。激活的NF-κB二聚体通过核易位与目的基因启动区κB结合位点结合,调节目的基因的表达。它主要参与炎症反应与炎症损伤相关蛋白分子的表达调控,如促炎细胞因子(包括TNF-α、ILs等)、黏附分子、急性期蛋白及可诱导的效应期酶等。由于TF基因的启动区包含了包括NF-κB等多种转录因子结合位点。因此,NF-κB在TF基因表达调控中的作用值得探讨。
组织因子途径抑制物(tissue factor pathway inhibitor,TFPI)是TF启动机体凝血过程中的重要生理抑制物。机体凝血正常状态的维持有赖于TF与TFPI之间的平衡。人们在多种动物疾病模型如冠心病、脑梗塞、白血病等血标本中检测到TF和TFPI的异常,用二者的变化作为疾病病程中凝血异常的一种解释。由于凝血异常也是脓毒症的一种突出表现,是否在动物模型上也能发现TF和TFPI的异常的变化,我们合成的C5a反义肽能否在体内拮抗C5a引起凝血异常的生物学活性也有待验证。
为此：在广州市科委攻关引导项目(0723-E0121)的资助下,本实验拟进行以下研究：
1)C5a对HPMEC TF表达的影响：以微血管内皮细胞HPMEC株作为研究对象,观察不同时间(C5a浓度200ng/ml刺激细胞4h、8h、12h)、不同浓度(C5a浓度100ng/ml、200ng/ml、300ng/ml刺激细胞8h) C5a与TF表达的关系,利用适时荧光定量PCR和免疫印迹方法,检测TF基因和蛋白水平的变化情况。了解C5a刺激浓度和刺激时间对HPMEC表达TF的影响。
2)C5a促进TF表达可能的机制：选定C5a浓度200ng/ml刺激8h组为观察对象,分两组进行实验。干预组：加入C5a反义肽(2ug/ml)以阻断C5a与细胞膜表面C5aR的结合；对照组：不加反义肽。观察TF基因和蛋白变化。C5a刺激细胞后用免疫细胞化学方法观察胞内NF-κB p65表达变化,与正常HPMEC比较,观察p65活化情况。探讨TF表达上调的潜在机制。
3)脓毒症动物血液TF和TFPI的变化：对昆明小鼠进行盲肠结扎穿孔手术,复制脓毒症动物模型,分正常组、假手术组、模型组、反义肽干预组,在不同时间点(2h、4h、8h、12h)用ELISA方法,检测血液中TF和TFPI的浓度变化。了解脓毒症TF与TFPI的变化情况,以及C5a拮抗对TF与TFPI失衡的作用。
研究的主要结果和结论如下：
1、C5a对HPMEC TF表达的影响：
正常的HPMEC基本检测不到TF基因和蛋白的表达。C5a刺激时间与HPMEC的TF表达的关系,以200ng/ml浓度的C5a刺激HPMEC时,作用时间为4h、8h、12h的基因表达定量为(1.00±0.13)、(3.04±0.26)、(6.58±0.44),p<0.05；而其相应的时间点TF的蛋白合成定量为(0.15±0.05)、(0.27±0.06)、(0.45±0.07),p<0.05。
C5a刺激浓度与HPMEC的TF表达的关系,以C5a对HPMEC作用时间8h,C5a浓度分别为100ng/ml.200ng/ml.300ng/ml,TF基因表达定量分别为(1.00±0.14)、(1.80±0.16)、(3.09±0.09),p<0.05；而其蛋白合成定量分别为(0.29±0.05)、(0.41±0.73)、(0.53±0.40),p<0.05。
以上说明,在C5a作用下,微血管内皮细胞TF在基因水平和蛋白水平的表达明显上升,在刺激的12h内随着C5a刺激浓度和刺激时间的增加TF随之表达上调。证明补体成分C5a能刺激HPMEC的TF表达,参与凝血激活亢进的过程。
2、C5a增加TF表达可能的机制：
C5a反义肽干预后,与不干预组相比,TF基因为(1.00±0.10)和(0.66±0.67),p<0.05；而蛋白定量为(0.33±0.34)和(0.27±0.31),p<0.05。
NF-κB p65免疫组化示：阴性对照组细胞胞浆无黄褐色颗粒,正常对照组胞浆有淡染的黄褐色颗粒；而200ng/ml C5a刺激8h组胞浆出现深染的黄褐色颗粒。正常细胞细胞核内无黄染颗粒,而经200ng/ml C5a刺激后细胞核内出现黄褐色颗粒。
上述结果说明,我们用新型C5a反义肽拮抗剂阻断C5a与其受体结合后,TF基因水平降至未干预组的68%,而蛋白水平也有下降至80%左右。由C5a刺激后NF-κB p65在胞浆出现深染,同时核内出现黄染颗粒,提示其在胞浆的量有增加的同时,包含活化的p65的二聚体发生核易位。由此推测C5a与微血管内皮细胞膜表面的C5a受体结合后,通过多种胞内信号途径激活了NF-κB,继而与TF基因调控序列上的相应位点结合,参与了其基因和蛋白表达的调控。
3、脓毒症动物血液TF和TFPI的变化：
时间点为2h时,正常组、假手术组、造模组和反义肽干预组小鼠血液TF的浓度分别为：(42.44±2.19)pg/ml.(54.12±3.47)pg/ml.(59.81±3.69) pg/ml.(54.23±3.73)pg/ml.时间点为4h时,各组小鼠血液TF的浓度分别为：(42.29±1.98)pg/ml.(53.70±4.14)pg/ml.(82.77±9.11)pg/ml.(70.91±7.19)pg/ml.时间点为8h时,各组小鼠血液TF的浓度分别为：(41.45±1.32)pg/ml.(56.33±2.68)pg/ml.(125.34±6.32)pg/ml.(103.36±6.72)pg/ml.时间点为12h时,各组小鼠血液TF的浓度分别为：(42.75±1.25)pg/ml.(60.73±1.43)pg/ml.(139.81±6.17)pg/ml.(121.41±7.21)pg/ml.TF浓度随着时间的增加呈现上升趋势,各时间点反义肽干预组与造模组相比,TF浓度均低于造模组,p<0.05。
时间点为2h时,正常组、假手术组、造模组和反义肽干预组小鼠血液TFPI的浓度分别为：(14.60±0.76)ng/ml.(16.67±2.19)ng/ml.(26.2±2.21)ng/ml. (26.18±3.3)ng/ml.时间点为4h时,各组小鼠血液TFPI的浓度分别为：(15.11±0.37)ng/ml、(18.94±0.39)ng/ml.(28.08±2.46)ng/ml.(28.72±1.46)ng/ml.时间点为8h时,各组小鼠血液TFPI的浓度分别为：(15.72±0.92)ng/ml.(18.01±2.92)ng/ml.(27.45±2.52)ng/ml.(29.20±1.61)ng/ml.时间点为12h时,各组小鼠血液TFPI的浓度分别为：(15.00±0.42)ng/ml.(19.1±0.96)ng/ml. (29.2±1.41)ng/ml.(29.4±1.32)ng/ml.2h之后,TFPI的水平没有继续增加。反义肽干预组与造模组相比,两组TFPI浓度没有明显的差异,p>0.05。
这说明,脓毒症早期便出现了凝血异常,表现在TF与TFPI的不平行升高。予以C5a反义肽治疗后,血液中的TFPI并没有明显的改变,故它影响凝血的过程可能主要是通过减少TF的产生,从而影响TF.TFPI之间的平衡来实现的。
本次实验从细胞和动物两个水平对C5a与TF表达的关系做了初步的研究。从细胞实验中,我们发现了TF在12h内随着C5a刺激时间和刺激浓度有明显的上调表达；C5a与受体结合介导胞内信号,通过NF-KB活化、核易位调控TF基因和蛋白表达可能是其重要途径；同时验证了合成的C5a反义肽在体外部分拮抗C5a生物活性的作用。
从动物实验中,我们发现脓毒症小鼠存在凝血与抗凝平衡失调,表现在TF与TFPI的不平行升高；用C5a反义肽进行干预后,小鼠的凝血紊乱有一定程度的纠正,证实了C5a对脓毒症发生凝血与抗凝失衡的影响,同时也验证了合成的C5a反义肽在体内具有部分拮抗C5a的生物学活性。当然,若能进一步优化实验条件更合理的进行实验设计,将更能完善和支持我们的结果和推论。
Sepsis is a major challenge in medicine.Despite enhanced antibiotics and the best available supportive care,sepsis remains one of the most common life-threatening complications in intensive care units.According to studies from North America,the incidence is a bout 3 cases per 1,000 population,an annual burden of 750,000 cases in the United States resulting in 215,000 deaths.It is estimated that severe sepsis is responsible for 2% to 11% of all admissions to intensive care units. The hospital mortality rate associated with severe sepsis and septic shock (30% and more than 60% ,respectively) has not changed much during the last decades.It maybe related to its complex pathophysiological process and the cognition of the diseases.
Sepsis is characterised by the activiation of a wide range of cells in succession, then release multiple inflammatory factors and cause tissue damage,multiple organs dysfunction syndromes,even multiple organs failure.During sepsis many biological systems has been activated,such as complement system,coagulation system, immunologic system,apoptosis related system et cetera.Disseminated intravascular coagulation is a severe complication due to coagulation disturbance.Once DIC emerge,a wide range of micro-thrombosis will cause ischemia and hypoxia in tissues and accelerate MODS progression.A study revealed that the proportion of DIC in sepsis especially septic shock is approximately 30%-50%. Coagulation disorder has already become an outstanding feature in sepsis pathophysiologic process.Many kinds of inflammatory mediators and cytokines can lead to coagulation disorders,for instance:LPS,TNF-a,ILs,et cal.Among these,Complement 5a has been drew more and more attentions.
C5a as a common cleave product of three complement activation pathways owns wide biological activities.In the new therapeutic strategies of sepsis,不得C5a-antagonist therapy has been mentioned and many studies demonstrate its effectiveness.Our team has screened and synthetized a new type of C5a-antagonist:C5a antisense peptide.Related experiment results show that it can antagonize C5a' functions and ameliorate inflammatory response in sepsis.While the role of C5a in coagulation disorders cannot be ignored.Previously experiment date showed that C5a can up-regulation tissue facotor(TF) on the membrance of vascular endothelial cells,neutrophils and monocytes/macrophages,cause hyper-coagulation activation;down-regulation antithrombin III and thrombomodulin,cause anticoagulation system impaired;effect the expression of tissue plasminogen activator and plasminogen activator inhibitor,cause fibrinolytic system unbalanced. since tissue factor is the initiator of extrinsic coagulation pathway and holds the central status in the modern activation of blood coagulation modle.More attention were paid to the relationship of C5a and TF.Because coagulation disoder was mainly occur in microvascular and lung was the first targeted organ,the effects and mechanisms of C5a on TF in pulmonary microvascular endothelial cells worthy to be focused on.
NF-κB is a hot spots of all the transcription factor associated with inflammations.It exists in various kinds of cells and plays a vital role in regulating many functional protein expression.Activiated NF-κB travel to the cell nucleus in the form of dipolymer,then combine to theκB binding site in the gene promoter region and regulate gene and protain expression such as pro-inflammation cytokines(TNF-α、ILs),adhesion molecules,acute phase proteins and enzymes.TF gene promoter region also has anκB binding site so the role of NF-κB in the regulation of TF gene expression deserve to be researched.
Tissue factor pathway inhibitor is an endogenous inhibitor of TF-induced coagulation.Normal coagulation depends on the balance of TF and TFPI.The abnormal concentrations of TF and TFPI had been detected in many diseases:coronary heart disease,cerebral infarction,leukocythemia and so on.We consider them a reason of coagulation abnormality.As coagulation disoders also an outstanding clinical manifestation,weather there is a change in the concentration of TF and TFPI in sepsis animal model? Can the newly synthetized reagent antagonize C5a' functions in the aspect of coagulation disorder in vivo?
This study was funded by the Project of Science & Technology of Guangzhou City (07Z3-E0121).Experimental content is divided into three parts as follows:
First,we use C5a with different concentration (100ng/ml、200ng/ml、300ng/ml)and time (4h、8h、12h)to stimulate human pulmonary microvascular endothelial cells (HPMEN).Real-time fluorescent quantitative PCR and western blotting was used to detect the gene and protein changes in each groups.
Second,chose the group C5a 200ng/ml,stimulation time 8h to analyse the potential mechanism. Use C5a antisense peptide to blockade the link between C5a and C5aR and observe the TF changes.Simultaneously,we use immunohitochemistry techenique to survey NF-κB p65 changes.
Finally, establishe sepsis animal model with caecal ligation plus puncture (CLP).Kunming mouse were divided into normal groups,sham-operated groups,model groups and antisense peptide groups.At different time (2h、4h、8h、12h),we use ELISA to detect the concentration of TF, TFPI in the blood and estimate the antagonism of newly synthetized antisense peptides.
The main results and conclusions are as follows:
l.The effects of C5a on TF expression in HPMECs:
Normal HPMECs seemed not express TF.When the concentration of C5a is 200ng/ml,4h,8h,12h gene and protein is (1.00±0.13),(3.04±0.26), (6.58±0.44), p<0.05 and(0.15±0.05),(0.27±0.06),(0.45±0.07), p<0.05.
When the stimulation time is 8h,100ng/ml、200ng/ml、300ng/ml gene and protein is (1.00±0.14),(1.80±0.16),(3.09±0.09), p<0.05 and(0.29±0.05),(0.41±20.73), (0.53±0.40),p<0.05.
This prove that TF began to express after C5a stimulation.TF gene and protein expression increased dose-dependently and time-dependently in 12h.It suggests that C5a can up-regulate TF exprssion and play a role in hyper-coagulation activation.
2.The potential mechanism of C5a up-regulate TF exprssion.
With addition of C5a antisense peptide, TF mRNA decreased to(0.66±0.67)of the comparable group(1.00±0.10),p<0.05,protein decreased to(0.27±0.31)of the comparable group(0.33±0.34),p<0.05.
Immunohistochemistry images showed that no yellow-brown granular pigment in negative control;there is yellow-brown granular pigment in the cytoplasm of the normal group;in stimulated group,yellow-brown granular increased in cytoplasm and also emerged in cell nucleus.
It suggests that the newly synthetized has blocked C5a bind to C5aR. Gene expression decrased to 80% and protain 68%.Immunohistochemistry images showed yellow-brown granular increased in cytoplasm and also emerged in cell nucleus suggests that NF-κB had been activated.Results of these two part show that during 12h, TF increased dose-dependently and time-dependently after C5a stimulation.The protential mechanism may be C5a binds to its receptor then activiates NF-κB through specific signaling pathways and finally regulates TF expression.
3. Blood concentration of TF and TFPI.
When the time is 2h,the concentration of TF in each groups(normal group,sham-operative group,model group,C5a antisence peptide intervention group) are(42.44±2.19)pg/ml,(54.12±3.47)pg/ml,(59.81±3.69)pg/ml,(54.23±3.73)pg/ml.Wh en the time is 4h,the concentration of each groups are(42.29±1.98)pg/ml, (53.70±4.14)pg/ml,(82.77±9.11)pg/ml,(70.91±7.19)pg/ml,respectively. When the time is 8h,the concentration of each groups are:(41.45±1.32)pg/ml,(56.33±2.68) pg/ml,(125.34±6.32)pg/ml,(103.36±6.72)pg/ml respectively. When the time is 12h,the concentration of each groups are:(42.75±1.25)pg/ml,(60.73±1.43)pg/ml, (139.81±6.17)pg/ml,(121.41±7.21)pg/ml.The concentration of TF were gradually increased over time;C5a antisence peptide groups are lower than model group at any time points, p<0.05.
When the time is 2h,the concentration of TFPI in each groups(normal group,sham-operative group,model group,C5a antisence peptide intervention group) are:(14.60±0.76)ng/ml,(16.67±2.19)ng/ml,(26.2±2.21)ng/ml,(26.18±3.3)ng/ml.Whe-n the time is 4h,the concentration of each groups are:(15.11±0.37)ng/ml, (18.94±0.39)ng/ml,(28.08±2.46)ng/ml,(28.72±1.46)ng/ml,respectively. When the time is 8h,the concentration of each groups are:(15.72±0.92)ng/ml,(18.01±2.92) ng/ml, (27.45±2.52) ng/ml,(29.20±1.61)ng/ml,respectively. When the time is 2h,the concentration of each groups are:(15.00±0.42)ng/ml,(19.1±0.96)ng/ml,(29.2±1.41) ng/ml,(29.4±1.32)ng/ml. The oncentration of TFPI is not increased after 2h.There is no significant difference between model groups and C5a antisence peptide groups, p>0.05.
It suggests that coagulation disorders emerged in the early stage of sepsis. Mainly expressed in the imbalance of TF and TFPI. C5a antisence peptide can not change the concentration of TFPI. So the primary mechanism of antisence peptide effects coagulation disorder maybe reducing the production of TF then striking the balance of TF and TFPI.
We have discussed the relationship of C5a and TF in cells and in animals.From cell experiments,we found that TF increased dose-dependently and time-dependently after C5a stimulation.The potential mechanism is C5a binds to its receptor then activiates NF-κB through specific signaling pathways and finally regulates TF expression and the newly synthetized reagent has the antagonism in vitro.
From animal experiments,we found that there is coagulation disorders in sepsis.Exhibited with persistent hyper-coagulation activation but relative insufficient anti-coagulation.After the treatment of C5a antisense peptide,the coagulation disorders have a certain degree of alleviate.It evidence that the newly synthetized reagent has the antagonism in vivo.If we can improve the experiment conditions and design the experiment more reasonable,potent evidences will support our hypothesis.
脓毒症共同的病理生理特征是一系列炎症细胞被相继激活,呈“瀑布效应”释放出大量炎症因子,对全身组织器官造成损伤,最终引起多器官功能障碍综合征(multiple organs dysfunction syndromes, MODS),进一步发展为多器官功能衰竭(multiple organs failure,MOF)。在炎症反应的过程中,多种生物学活性系统被激活如：补体系统、凝血系统、免疫系统以及凋亡相关系统等。其中,弥散性血管内凝血(disseminated intravascular coagulation, DIC)是脓毒症患者凝血系统异常引发的一种严重并发症,一旦出现DIC,患者将因为全身微血管广泛微血栓的形成,造成组织器官缺血、缺氧从而加速MODS的进程。一项研究显示,在脓毒症特别是脓毒症休克的病例中,发生DIC的比例约为30-50%。凝血功能异常也俨然成为了脓毒症病理生理过程中的突出表现。在脓毒症或全身炎症反应过程中,多种促炎症细胞因子和炎症介质,如TNF-α、ILs、PAF、C5a等,均可引起凝血功能异常。其中,补体系统活化产物C5a在凝血中的作用日益受到学者的关注。
C5a是三条补体途径共同裂解产生的活性产物,具有广泛的生物学功能。在脓毒症治疗新策略中,拮抗C5a已经被人们在动物实验中所提及和证实,本课题组前期自主筛选合成了一种新型C5a拮抗多肽(C5a反义肽),相关实验证明了其能拮抗C5a的生物学活性,对改善小鼠脓毒症模型的炎症反应有一定的保护作用。但C5a作为一种促炎介质,其在凝血方面的作用也不容忽视。已有的研究显示,其对凝血的影响包括增加炎症细胞,如中性粒细胞、单核巨噬细胞等细胞膜表面组织因子(tissue factor, TF)的表达,从而使凝血激活亢进；降低AT-Ⅲ、血栓调节蛋白(thrombomodulin,TM)等物质的表达而使机体抗凝功能削弱；影响纤溶酶原激活物和抑制物(plasminogen activator,PA;plasminogen activator inhibitor, PAI)的表达而使纤溶和抗纤溶平衡失调。鉴于TF是外源性凝血途径的启动因子,在外源性凝血激活“网络模式”中处于中心地位。故现有的研究主要集中在C5a影响中性粒细胞、大血管内皮细胞TF的表达方面。但由于凝血异常主要发生在微小血管,尤其是肺作为MODS首发靶器官,故C5a对微血管内皮细胞表达TF的影响以及机制更值得关注。
在纵多参与炎症反应调控的转录因子中,核因子-κB (Nuclear factor kapp B,NF-κB)是研究的焦点。它存在于体内多种细胞中,是一种可调控多种功能蛋白分子表达而参与多种疾病发病过程的转录因子。激活的NF-κB二聚体通过核易位与目的基因启动区κB结合位点结合,调节目的基因的表达。它主要参与炎症反应与炎症损伤相关蛋白分子的表达调控,如促炎细胞因子(包括TNF-α、ILs等)、黏附分子、急性期蛋白及可诱导的效应期酶等。由于TF基因的启动区包含了包括NF-κB等多种转录因子结合位点。因此,NF-κB在TF基因表达调控中的作用值得探讨。
组织因子途径抑制物(tissue factor pathway inhibitor,TFPI)是TF启动机体凝血过程中的重要生理抑制物。机体凝血正常状态的维持有赖于TF与TFPI之间的平衡。人们在多种动物疾病模型如冠心病、脑梗塞、白血病等血标本中检测到TF和TFPI的异常,用二者的变化作为疾病病程中凝血异常的一种解释。由于凝血异常也是脓毒症的一种突出表现,是否在动物模型上也能发现TF和TFPI的异常的变化,我们合成的C5a反义肽能否在体内拮抗C5a引起凝血异常的生物学活性也有待验证。
为此：在广州市科委攻关引导项目(0723-E0121)的资助下,本实验拟进行以下研究：
1)C5a对HPMEC TF表达的影响：以微血管内皮细胞HPMEC株作为研究对象,观察不同时间(C5a浓度200ng/ml刺激细胞4h、8h、12h)、不同浓度(C5a浓度100ng/ml、200ng/ml、300ng/ml刺激细胞8h) C5a与TF表达的关系,利用适时荧光定量PCR和免疫印迹方法,检测TF基因和蛋白水平的变化情况。了解C5a刺激浓度和刺激时间对HPMEC表达TF的影响。
2)C5a促进TF表达可能的机制：选定C5a浓度200ng/ml刺激8h组为观察对象,分两组进行实验。干预组：加入C5a反义肽(2ug/ml)以阻断C5a与细胞膜表面C5aR的结合；对照组：不加反义肽。观察TF基因和蛋白变化。C5a刺激细胞后用免疫细胞化学方法观察胞内NF-κB p65表达变化,与正常HPMEC比较,观察p65活化情况。探讨TF表达上调的潜在机制。
3)脓毒症动物血液TF和TFPI的变化：对昆明小鼠进行盲肠结扎穿孔手术,复制脓毒症动物模型,分正常组、假手术组、模型组、反义肽干预组,在不同时间点(2h、4h、8h、12h)用ELISA方法,检测血液中TF和TFPI的浓度变化。了解脓毒症TF与TFPI的变化情况,以及C5a拮抗对TF与TFPI失衡的作用。
研究的主要结果和结论如下：
1、C5a对HPMEC TF表达的影响：
正常的HPMEC基本检测不到TF基因和蛋白的表达。C5a刺激时间与HPMEC的TF表达的关系,以200ng/ml浓度的C5a刺激HPMEC时,作用时间为4h、8h、12h的基因表达定量为(1.00±0.13)、(3.04±0.26)、(6.58±0.44),p<0.05；而其相应的时间点TF的蛋白合成定量为(0.15±0.05)、(0.27±0.06)、(0.45±0.07),p<0.05。
C5a刺激浓度与HPMEC的TF表达的关系,以C5a对HPMEC作用时间8h,C5a浓度分别为100ng/ml.200ng/ml.300ng/ml,TF基因表达定量分别为(1.00±0.14)、(1.80±0.16)、(3.09±0.09),p<0.05；而其蛋白合成定量分别为(0.29±0.05)、(0.41±0.73)、(0.53±0.40),p<0.05。
以上说明,在C5a作用下,微血管内皮细胞TF在基因水平和蛋白水平的表达明显上升,在刺激的12h内随着C5a刺激浓度和刺激时间的增加TF随之表达上调。证明补体成分C5a能刺激HPMEC的TF表达,参与凝血激活亢进的过程。
2、C5a增加TF表达可能的机制：
C5a反义肽干预后,与不干预组相比,TF基因为(1.00±0.10)和(0.66±0.67),p<0.05；而蛋白定量为(0.33±0.34)和(0.27±0.31),p<0.05。
NF-κB p65免疫组化示：阴性对照组细胞胞浆无黄褐色颗粒,正常对照组胞浆有淡染的黄褐色颗粒；而200ng/ml C5a刺激8h组胞浆出现深染的黄褐色颗粒。正常细胞细胞核内无黄染颗粒,而经200ng/ml C5a刺激后细胞核内出现黄褐色颗粒。
上述结果说明,我们用新型C5a反义肽拮抗剂阻断C5a与其受体结合后,TF基因水平降至未干预组的68%,而蛋白水平也有下降至80%左右。由C5a刺激后NF-κB p65在胞浆出现深染,同时核内出现黄染颗粒,提示其在胞浆的量有增加的同时,包含活化的p65的二聚体发生核易位。由此推测C5a与微血管内皮细胞膜表面的C5a受体结合后,通过多种胞内信号途径激活了NF-κB,继而与TF基因调控序列上的相应位点结合,参与了其基因和蛋白表达的调控。
3、脓毒症动物血液TF和TFPI的变化：
时间点为2h时,正常组、假手术组、造模组和反义肽干预组小鼠血液TF的浓度分别为：(42.44±2.19)pg/ml.(54.12±3.47)pg/ml.(59.81±3.69) pg/ml.(54.23±3.73)pg/ml.时间点为4h时,各组小鼠血液TF的浓度分别为：(42.29±1.98)pg/ml.(53.70±4.14)pg/ml.(82.77±9.11)pg/ml.(70.91±7.19)pg/ml.时间点为8h时,各组小鼠血液TF的浓度分别为：(41.45±1.32)pg/ml.(56.33±2.68)pg/ml.(125.34±6.32)pg/ml.(103.36±6.72)pg/ml.时间点为12h时,各组小鼠血液TF的浓度分别为：(42.75±1.25)pg/ml.(60.73±1.43)pg/ml.(139.81±6.17)pg/ml.(121.41±7.21)pg/ml.TF浓度随着时间的增加呈现上升趋势,各时间点反义肽干预组与造模组相比,TF浓度均低于造模组,p<0.05。
时间点为2h时,正常组、假手术组、造模组和反义肽干预组小鼠血液TFPI的浓度分别为：(14.60±0.76)ng/ml.(16.67±2.19)ng/ml.(26.2±2.21)ng/ml. (26.18±3.3)ng/ml.时间点为4h时,各组小鼠血液TFPI的浓度分别为：(15.11±0.37)ng/ml、(18.94±0.39)ng/ml.(28.08±2.46)ng/ml.(28.72±1.46)ng/ml.时间点为8h时,各组小鼠血液TFPI的浓度分别为：(15.72±0.92)ng/ml.(18.01±2.92)ng/ml.(27.45±2.52)ng/ml.(29.20±1.61)ng/ml.时间点为12h时,各组小鼠血液TFPI的浓度分别为：(15.00±0.42)ng/ml.(19.1±0.96)ng/ml. (29.2±1.41)ng/ml.(29.4±1.32)ng/ml.2h之后,TFPI的水平没有继续增加。反义肽干预组与造模组相比,两组TFPI浓度没有明显的差异,p>0.05。
这说明,脓毒症早期便出现了凝血异常,表现在TF与TFPI的不平行升高。予以C5a反义肽治疗后,血液中的TFPI并没有明显的改变,故它影响凝血的过程可能主要是通过减少TF的产生,从而影响TF.TFPI之间的平衡来实现的。
本次实验从细胞和动物两个水平对C5a与TF表达的关系做了初步的研究。从细胞实验中,我们发现了TF在12h内随着C5a刺激时间和刺激浓度有明显的上调表达；C5a与受体结合介导胞内信号,通过NF-KB活化、核易位调控TF基因和蛋白表达可能是其重要途径；同时验证了合成的C5a反义肽在体外部分拮抗C5a生物活性的作用。
从动物实验中,我们发现脓毒症小鼠存在凝血与抗凝平衡失调,表现在TF与TFPI的不平行升高；用C5a反义肽进行干预后,小鼠的凝血紊乱有一定程度的纠正,证实了C5a对脓毒症发生凝血与抗凝失衡的影响,同时也验证了合成的C5a反义肽在体内具有部分拮抗C5a的生物学活性。当然,若能进一步优化实验条件更合理的进行实验设计,将更能完善和支持我们的结果和推论。
Sepsis is a major challenge in medicine.Despite enhanced antibiotics and the best available supportive care,sepsis remains one of the most common life-threatening complications in intensive care units.According to studies from North America,the incidence is a bout 3 cases per 1,000 population,an annual burden of 750,000 cases in the United States resulting in 215,000 deaths.It is estimated that severe sepsis is responsible for 2% to 11% of all admissions to intensive care units. The hospital mortality rate associated with severe sepsis and septic shock (30% and more than 60% ,respectively) has not changed much during the last decades.It maybe related to its complex pathophysiological process and the cognition of the diseases.
Sepsis is characterised by the activiation of a wide range of cells in succession, then release multiple inflammatory factors and cause tissue damage,multiple organs dysfunction syndromes,even multiple organs failure.During sepsis many biological systems has been activated,such as complement system,coagulation system, immunologic system,apoptosis related system et cetera.Disseminated intravascular coagulation is a severe complication due to coagulation disturbance.Once DIC emerge,a wide range of micro-thrombosis will cause ischemia and hypoxia in tissues and accelerate MODS progression.A study revealed that the proportion of DIC in sepsis especially septic shock is approximately 30%-50%. Coagulation disorder has already become an outstanding feature in sepsis pathophysiologic process.Many kinds of inflammatory mediators and cytokines can lead to coagulation disorders,for instance:LPS,TNF-a,ILs,et cal.Among these,Complement 5a has been drew more and more attentions.
C5a as a common cleave product of three complement activation pathways owns wide biological activities.In the new therapeutic strategies of sepsis,不得C5a-antagonist therapy has been mentioned and many studies demonstrate its effectiveness.Our team has screened and synthetized a new type of C5a-antagonist:C5a antisense peptide.Related experiment results show that it can antagonize C5a' functions and ameliorate inflammatory response in sepsis.While the role of C5a in coagulation disorders cannot be ignored.Previously experiment date showed that C5a can up-regulation tissue facotor(TF) on the membrance of vascular endothelial cells,neutrophils and monocytes/macrophages,cause hyper-coagulation activation;down-regulation antithrombin III and thrombomodulin,cause anticoagulation system impaired;effect the expression of tissue plasminogen activator and plasminogen activator inhibitor,cause fibrinolytic system unbalanced. since tissue factor is the initiator of extrinsic coagulation pathway and holds the central status in the modern activation of blood coagulation modle.More attention were paid to the relationship of C5a and TF.Because coagulation disoder was mainly occur in microvascular and lung was the first targeted organ,the effects and mechanisms of C5a on TF in pulmonary microvascular endothelial cells worthy to be focused on.
NF-κB is a hot spots of all the transcription factor associated with inflammations.It exists in various kinds of cells and plays a vital role in regulating many functional protein expression.Activiated NF-κB travel to the cell nucleus in the form of dipolymer,then combine to theκB binding site in the gene promoter region and regulate gene and protain expression such as pro-inflammation cytokines(TNF-α、ILs),adhesion molecules,acute phase proteins and enzymes.TF gene promoter region also has anκB binding site so the role of NF-κB in the regulation of TF gene expression deserve to be researched.
Tissue factor pathway inhibitor is an endogenous inhibitor of TF-induced coagulation.Normal coagulation depends on the balance of TF and TFPI.The abnormal concentrations of TF and TFPI had been detected in many diseases:coronary heart disease,cerebral infarction,leukocythemia and so on.We consider them a reason of coagulation abnormality.As coagulation disoders also an outstanding clinical manifestation,weather there is a change in the concentration of TF and TFPI in sepsis animal model? Can the newly synthetized reagent antagonize C5a' functions in the aspect of coagulation disorder in vivo?
This study was funded by the Project of Science & Technology of Guangzhou City (07Z3-E0121).Experimental content is divided into three parts as follows:
First,we use C5a with different concentration (100ng/ml、200ng/ml、300ng/ml)and time (4h、8h、12h)to stimulate human pulmonary microvascular endothelial cells (HPMEN).Real-time fluorescent quantitative PCR and western blotting was used to detect the gene and protein changes in each groups.
Second,chose the group C5a 200ng/ml,stimulation time 8h to analyse the potential mechanism. Use C5a antisense peptide to blockade the link between C5a and C5aR and observe the TF changes.Simultaneously,we use immunohitochemistry techenique to survey NF-κB p65 changes.
Finally, establishe sepsis animal model with caecal ligation plus puncture (CLP).Kunming mouse were divided into normal groups,sham-operated groups,model groups and antisense peptide groups.At different time (2h、4h、8h、12h),we use ELISA to detect the concentration of TF, TFPI in the blood and estimate the antagonism of newly synthetized antisense peptides.
The main results and conclusions are as follows:
l.The effects of C5a on TF expression in HPMECs:
Normal HPMECs seemed not express TF.When the concentration of C5a is 200ng/ml,4h,8h,12h gene and protein is (1.00±0.13),(3.04±0.26), (6.58±0.44), p<0.05 and(0.15±0.05),(0.27±0.06),(0.45±0.07), p<0.05.
When the stimulation time is 8h,100ng/ml、200ng/ml、300ng/ml gene and protein is (1.00±0.14),(1.80±0.16),(3.09±0.09), p<0.05 and(0.29±0.05),(0.41±20.73), (0.53±0.40),p<0.05.
This prove that TF began to express after C5a stimulation.TF gene and protein expression increased dose-dependently and time-dependently in 12h.It suggests that C5a can up-regulate TF exprssion and play a role in hyper-coagulation activation.
2.The potential mechanism of C5a up-regulate TF exprssion.
With addition of C5a antisense peptide, TF mRNA decreased to(0.66±0.67)of the comparable group(1.00±0.10),p<0.05,protein decreased to(0.27±0.31)of the comparable group(0.33±0.34),p<0.05.
Immunohistochemistry images showed that no yellow-brown granular pigment in negative control;there is yellow-brown granular pigment in the cytoplasm of the normal group;in stimulated group,yellow-brown granular increased in cytoplasm and also emerged in cell nucleus.
It suggests that the newly synthetized has blocked C5a bind to C5aR. Gene expression decrased to 80% and protain 68%.Immunohistochemistry images showed yellow-brown granular increased in cytoplasm and also emerged in cell nucleus suggests that NF-κB had been activated.Results of these two part show that during 12h, TF increased dose-dependently and time-dependently after C5a stimulation.The protential mechanism may be C5a binds to its receptor then activiates NF-κB through specific signaling pathways and finally regulates TF expression.
3. Blood concentration of TF and TFPI.
When the time is 2h,the concentration of TF in each groups(normal group,sham-operative group,model group,C5a antisence peptide intervention group) are(42.44±2.19)pg/ml,(54.12±3.47)pg/ml,(59.81±3.69)pg/ml,(54.23±3.73)pg/ml.Wh en the time is 4h,the concentration of each groups are(42.29±1.98)pg/ml, (53.70±4.14)pg/ml,(82.77±9.11)pg/ml,(70.91±7.19)pg/ml,respectively. When the time is 8h,the concentration of each groups are:(41.45±1.32)pg/ml,(56.33±2.68) pg/ml,(125.34±6.32)pg/ml,(103.36±6.72)pg/ml respectively. When the time is 12h,the concentration of each groups are:(42.75±1.25)pg/ml,(60.73±1.43)pg/ml, (139.81±6.17)pg/ml,(121.41±7.21)pg/ml.The concentration of TF were gradually increased over time;C5a antisence peptide groups are lower than model group at any time points, p<0.05.
When the time is 2h,the concentration of TFPI in each groups(normal group,sham-operative group,model group,C5a antisence peptide intervention group) are:(14.60±0.76)ng/ml,(16.67±2.19)ng/ml,(26.2±2.21)ng/ml,(26.18±3.3)ng/ml.Whe-n the time is 4h,the concentration of each groups are:(15.11±0.37)ng/ml, (18.94±0.39)ng/ml,(28.08±2.46)ng/ml,(28.72±1.46)ng/ml,respectively. When the time is 8h,the concentration of each groups are:(15.72±0.92)ng/ml,(18.01±2.92) ng/ml, (27.45±2.52) ng/ml,(29.20±1.61)ng/ml,respectively. When the time is 2h,the concentration of each groups are:(15.00±0.42)ng/ml,(19.1±0.96)ng/ml,(29.2±1.41) ng/ml,(29.4±1.32)ng/ml. The oncentration of TFPI is not increased after 2h.There is no significant difference between model groups and C5a antisence peptide groups, p>0.05.
It suggests that coagulation disorders emerged in the early stage of sepsis. Mainly expressed in the imbalance of TF and TFPI. C5a antisence peptide can not change the concentration of TFPI. So the primary mechanism of antisence peptide effects coagulation disorder maybe reducing the production of TF then striking the balance of TF and TFPI.
We have discussed the relationship of C5a and TF in cells and in animals.From cell experiments,we found that TF increased dose-dependently and time-dependently after C5a stimulation.The potential mechanism is C5a binds to its receptor then activiates NF-κB through specific signaling pathways and finally regulates TF expression and the newly synthetized reagent has the antagonism in vitro.
From animal experiments,we found that there is coagulation disorders in sepsis.Exhibited with persistent hyper-coagulation activation but relative insufficient anti-coagulation.After the treatment of C5a antisense peptide,the coagulation disorders have a certain degree of alleviate.It evidence that the newly synthetized reagent has the antagonism in vivo.If we can improve the experiment conditions and design the experiment more reasonable,potent evidences will support our hypothesis.
引文
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[21]Vander Poll T.Tissue factor as an initiator of coagulation and inflammation in the lung.Crit Care.2008;12 Suppl 6:S3.
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[23]董春霞,胡豫.内皮细胞表达组织因子的信号转导.医学综述.2005,11(5)：407-410
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[25]Anne Karin Lindahl.Tissue factor pathway inhibitor:from unknown coagulation inhibitor to major antithrombotic principle.Cardiovascular Research.1997,33:286-291.
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[30]Palomba L,Persichini T,Mazzone V.Inhibition of nitric-oxide synthase-I (NOS-I)-dependent nitric oxide production by lipopolysaccharide plus interferon-gamma is mediated by arachidonic acid. Effects on NFkappaB activation and late inducible NOS expression.J Biol Chem.2004,279 (29):29895-901.
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[32]Ikeda,K.C5a induces tissue factor activity on endothelial cells. [J].Thromb Haemost,1997,77:394-398.
[33]吕凤林,巫振洪,李元朝.人C5a B细胞表位的设计与其单克隆抗体的结合.中华微生物学和免疫学杂志.2003,23(10)：256-257.
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[7]Jonathan Cohen.The immunopathogenesis of sepsis.NATURE.2002,420:19-26.
[8]Dierk H.Endemann,Ernestol.Schiffrin.Endothelial Dysfunction.J Am Soc Nephrol.2004,15:1983-1992.
[9]WilliamC.Aird.The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome.BLOOD,2003101(10):3765-3777.
[10]李磊,汤耀卿.内皮细胞与脓毒症.生命科学.2005,17(3)：236-239.
[11]Marcel Schouten,Willem Joost Wiersinga,Marcel Levi.Inflammation, endothelium,and coagulation in sepsis.Journal of Leukocyte Biology.2008,83: 536-545.
[12]MarcelLevi,TymenT.Keller,EricvanGorp.Infection and inflammation and the coagulation system.Cardiovascular Research.2003,60:26-39.
[13]Hotchkiss Richard S,Tinsley Kevin W, Swanson Paul E. Endothelial cell apoptosis in sepsis.Crit Care Med.2002,30[Suppl]:S225-S228.
[14]PN Monk,A-M Scola, P Madala et al.Function,structure and therapeutic potential of complement C5a receptors. British Journal of Pharmacology.2007, 152,429-448.
[15]Ward PA.The dark side of C5a in sepsis.Nat Rev Immunol,2004,4(2):133-142.
[16]Konstantinos Kambas, Maciej M. Markiewski et al.C5a and TNF-Up-Regulate the Expression of Tissue Factor in Intra-Alveolar Neutrophils of Patients with the Acute Respiratory Distress Syndrome J Immunol.2008 Jun 1;180(11): 7368-75.
[17]Sacha Zeerleder,C. Erik Hack,Walter A. Wuillemin.Disseminated Intravascular Coagulation in Sepsis.CHEST.2005,128(4):2864-2875.
[18]Maciej M.Markiewski,Bo Nilsson,Kristina Nilsson Ekdahl,et al.Complement and coagulation:strangers or partners in crime?TRENDS in Immunology, 2007,28(4):184-192.
[19]Amara U, Rittirsch D, Flierl M,et al.Interaction between the coagulation and complement system. Adv Exp Med Biol,2008,632:71-79.
[20]姜巧,陈蕊,郭振辉.C5a与凝血系统的相互作用.国际病理科学与临床杂志.2009,29(6)：503-506.
[21]Vander Poll T.Tissue factor as an initiator of coagulation and inflammation in the lung.Crit Care.2008;12 Suppl 6:S3.
[22]郑贵军,李银平.组织因子和组织因子途径抑制物与脓毒症.中国中西医结合急救杂志.2008,15(2)：126-128.
[23]董春霞,胡豫.内皮细胞表达组织因子的信号转导.医学综述.2005,11(5)：407-410
[24]Bajaj MS,Birktoft JJ,Steer SA.Structure and biology of tissue factor pathway inhibitor.Thromb Haemost.2001,86(4):959-972.
[25]Anne Karin Lindahl.Tissue factor pathway inhibitor:from unknown coagulation inhibitor to major antithrombotic principle.Cardiovascular Research.1997,33:286-291.
[26]Gando S,Kameue T,Matsuda N.Imbalances between the levels of tissue factor and tissue factor pathway inhibitor in ARDS patients.Thromb Res.2003,109(2-3):119-124.
[27]May,Ghosh.Rel/NF-KB and IKB proteins:an overview.seminars in Cancer Biology.1997,8:63-73.
[28]May,Ghosh.Signal transducion through NF-KB.Immun.1998,19(2):80-88.
[29]Rahman A,Kefer J,Bando M.E-selectin expression in human endothelial cells by TNF-alpha-induced oxidant generation and NF-kappaB activation.Am J Physiol.1998,275(3 Pt 1):L533-544.
[30]Palomba L,Persichini T,Mazzone V.Inhibition of nitric-oxide synthase-I (NOS-I)-dependent nitric oxide production by lipopolysaccharide plus interferon-gamma is mediated by arachidonic acid. Effects on NFkappaB activation and late inducible NOS expression.J Biol Chem.2004,279 (29):29895-901.
[31]Kenneth J,Livak,Thomas D.Schmittgen. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-D D CT Method[J].METHODS 2001,25:402-408
[32]Ikeda,K.C5a induces tissue factor activity on endothelial cells. [J].Thromb Haemost,1997,77:394-398.
[33]吕凤林,巫振洪,李元朝.人C5a B细胞表位的设计与其单克隆抗体的结合.中华微生物学和免疫学杂志.2003,23(10)：256-257.