【作者】 周生余；

【导师】 肖伟；

【作者基本信息】 山东大学 ， 内科学， 2011， 博士

【摘要】 研究背景各种急慢性呼吸系统疾病常存在病原体感染、内毒素产生、炎性介质和炎性因子释放、缺氧、缺血、酸中毒等诸多因素中的一种或多种,这些因素可导致血管内皮受损和组织因子释放,激活各种凝血因子,使血液呈高凝状态或(和)血栓形成。凝血酶是参与此过程中的重要凝血因子之一。已有研究资料证实,除发挥凝血功能外,凝血酶还具有重要的非凝血功能,即通过与细胞膜表面的蛋白酶激活受体-1(PAR1)结合,激活一系列下游信号通路,在细胞的生长、增殖、分泌,粘附等方面发挥重要作用。在急性肺损伤和各种慢性肺部疾病如慢性阻塞性肺病(COPD)、肺间质性疾病中,肺泡灌洗液中的凝血酶浓度显著升高,其与PAR1结合,产生各种病理生理作用,如促进各类炎症因子IL-6、IL-8、PGE2、GM-CSF的释放,调控肺部的炎症反应及修复增殖。另有研究表明凝血酶可通过激活NADPH氧化酶亚单位p47(phox)和p67 (phox)诱导多种细胞如血管平滑肌细胞、血管内皮细胞、海马神经元细胞等产生以活性氧(ROS)为主的自由基。ROS对细胞的作用具有两面性,一方面,高浓度下具有损伤作用,参与促进细胞的凋亡、坏死过程；另一方面,低浓度可作为重要的信号分子,参与细胞信号转导,主要激活MAPK通路,通过激活转录因子,影响基因的表达,促进细胞增殖、分化等。核因子NF-κB是重要的转录因子之一,在氧化应激、感染、炎症反应、细胞凋亡及异常增殖等病理过程中,因调节多种靶基因的表达而发挥重要作用。肺成纤维细胞是肺组织重要结构细胞,占肺间质全部常驻细胞的95%以上,也是参与肺损伤修复的主要细胞。在损伤后的修复过程中,成纤维细胞表现出极高增殖反应。各种损害因子导致肺部毛细血管通透性增加,血浆成分渗漏到毛细血管外并与肺成纤维细胞接触,刺激该细胞的增殖修复反应。推测凝血酶可能在其中发挥一定作用。研究目的凝血酶作用于肺成纤维细胞,检测细胞产生ROS情况及产生途径,观察ROS是否可以活化MAPK信号系统,进而影响核转录因子NF-κB,调控生长基因的表达。研究方法选择人肺成纤维细胞(HLF)作为体外研究对象,一定浓度的凝血酶作用于该细胞。MTT法和流式细胞Brdu掺入法检测凝血酶促人肺成纤维细胞增殖现象；利用化学荧光法、流式细胞检测技术和GSH检测试剂盒检测活性氧的产生；Western blot法检测PAR1, NADPH氧化酶、ERK1/2及p38MAPK信号蛋白的表达；激光共聚焦显微扫描技术观察NF-κB中亚单位p65向核内转移情况。研究结果1.在0.1U/ml-20U/ml浓度范围内,凝血酶对细胞的增殖能力随浓度递增而增强。在10 U/ml及以上时,与对照组比较,可显著促进HLF细胞的增殖(P<0.05),但当浓度超过20U/ml,增殖作用不再增强；20 U/ml的凝血酶分别作用HLF 24 h、36 h、48 h、60h、72h,从36h时间点开始,与对照组比较,凝血酶组细胞增殖能力显著增强(P<0.05)；BrdU掺入增殖实验结果显示,凝血酶浓度在10 U/ml及以上时,较对照组可显著促进HLF细胞增殖(P<0.05),并随浓度增加而增强,但当凝血酶浓度超过20U/ml,增殖作用不再更强。2.5U/ml及以上浓度的凝血酶可显著增加HLF内的活性氧数量(P<0.05),并呈剂量依赖关系,但当浓度大于20U/ml,活性氧的量不再增加；20U/ml凝血酶作用HLF细胞30min,流式检测显示细胞内ROS产生量较对照组增加64.7%,作用1h时细胞内ROS量较对照组增加138.8%,而加入凝血酶拮抗剂水蛭素组细胞内ROS量与对照组比较,差异无统计学意义(P>0.05)；10U/ml和20 U/ml的凝血酶作用HLF细胞,细胞内GSH/GSSG比值较对照组显著降低(P<0.05),而加入水蛭素或氧自由基拮抗剂NAC组,GSH/GSSG之比与对照组无统计学差异(P>0.05)。3.20 U/ml凝血酶作用于HLF细胞10min和30min,胞浆内NADPH氧化酶亚单位p47phox和Rac2表达显著降低(P<0.05),1h后几乎无蛋白表达。加入水蛭素组与对照组比较,p47phox和Rac2表达无统计学意义(P>0.05)。4.凝血酶作用于HLF细胞10min到2h,细胞内磷酸化ERK1/2表达水平较对照组显著升高(P<0.05),而提前加入NAC或水蛭素组则无表达；细胞内p38MAPK磷酸化不受凝血酶影响,即凝血酶作用HLF细胞10min到2h,蛋白磷酸化水平较对照组无统计学差异(P>0.05)。5.凝血酶分别作用HLF细胞5min、10min、30min、1h和2h,各时间点细胞核内代表p65的荧光含量与对照组比较,差异无统计学意义(P>0.05),而佛波酯组荧光含量明显增加,差异有统计学意义(P<0.05)。6.支气管上皮细胞和肺成纤维细胞均有PAR1蛋白表达,肺成纤维细胞受体表达更明显(P<0.05)。结论1.凝血酶具有促进人肺成纤维细胞HLF增殖的能力。2.凝血酶促进HLF细胞增殖机制包括如下。2.1凝血酶受体PAR1在HLF中有较高表达。2.2凝血酶通过与PAR1受体结合,激活NADPH氧化酶,诱导细胞产生活性氧。2.3凝血酶通过磷酸化ERK1/2而不是p38MAPK,激活生长信号转导通路。2.4凝血酶促进HLF增殖过程中,无核因子NF-κB参与。意义凝血酶通过与肺成纤维细胞上的受体PAR1结合,激活NADPH氧化酶,诱导细胞产生活性氧,活性氧激活MAPK信号通路中的ERK1/2信号途径,实现促人肺成纤维细胞增殖过程。该研究初步探讨了凝血酶对人肺成纤维细胞的促增殖作用机理,为寻找凝血功能紊乱相关肺部疾病的新的防治靶点提供依据。更多还原

【Abstract】 BackgroundVarious acute or chronic lung diseases commonly exist diversity infections, endotoxin, inflammatory media and inflammatory factors, ischemia, acidosis. The factors can result in the vascular endothelium damage and tissue factor releasing, activating various coagulation factors, making blood in the condition of high coagulation or thrombogenesis. Thrombin is one of the key coagulation factors. In addition, it is confirmed that thrombin has important non-coagulation functions, including promoting cells growth, proliferation, secretion, adhere, and so on through binding to protease activated receptor (PAR) in cell membrane and activating a series of downstream signal pathways. The concentration of thrombin was high significantly in lung alveoli in acute lung injury and chronic lung diseases such as COPD, lung interstitial diseases. Thrombin binding to PAR1 results in many kinds of effects including promoting various kinds inflammation factors such as IL-6、IL-8、PGE2、GM-CSF releasing, regulating the lung inflammation reaction and recovery. Another reseach indicates that thrombin stimulates various cells such as vascular smooth muscle, vascular endothelia cells, hippocampus neuron to produce ROS through activating NADPH oxidase subunit p47(phox) and p67(phox). ROS have two sides effects, one is injury effect via participating cell apoptosis and necrosis at the middle to high concentrations of ROS. Another side is participating cells signal transduction, activating MAPK pathway mainly at the lower concentration of ROS, finally to activate transcription factor and impact gene expression, resulting in cells proliferation and differentiation. NF kappa B is an important transcription factor and has the extensive biological effects and has the important roles in the pathological process such as oxidative stress, infection, inflammation reaction, cell apoptosis and abnormal proliferation.As we know lung fibroblast is the important structure cell in lung tissue and more than 95 percent of all lung interstitial cells in quantity. It is the main cell which participates the injury recovery process and manifestes higher proliferation activation. A great deal of damage factors make the capillary permeability increase and the plasm fractions leak into the outer of blood capillary. The leakage substances can touch fibroblast and then make cell proliferate. We presume that thrombin as one of the leakage substances could play a certain role in the recovery process.ObjectiveThrombin stimulated lung fibroblast and cell were detected to generate reactive oxygen series(ROS) and the produce pathway. ROS were investigated to activate MAPK signal pathway as the second messager, even more it has the effect on NF kappa B, regulating growth gene expression.MethodsHLF cells were chosen as the research object in vitro, a certain concentration of thrombin was added to cells. MTT method and Brdu incorporation assay were used to observe thrombin promoting HLF proliferation. Chemiluminescence method, flow cytometry and GSH detection kit were used to detect ROS in HLF. Western blot method was used to detect PAR1, NADPH oxidase, ERK1/2 and P38MAPK protein expression. Laser copolymerization technology was used to detect the p65 translation from kytoplasm to nucleus.Results1. Thrombin had reproductive activity with concentration dependence during the concentration of 0.1U/ml-20U/ml.Compared with control group, thrombin could significantly facilitate HLF to proliferate at the concentration of 10 U/ml or above (P<0.05), but there was no more proliferation effect when thrombin concentration was more than 20U/ml. Thrombin at the concentration of 20U/ml for 24h,36h,48h,60h,72h,respectively, compared with the control group, had significant enhance in proliferation from 36h to 60h (P<0.05).Brdu incorporation assay showed that thrombin enhanced cells to proliferate significantly above the concentration of lOU/ml, compared with control group (P<0.05), and it was a dose-response relationship. But there was no more proliferation ability when thrombin’s concentration was above 20U/ml.2. Thrombin significantly increased the amount of ROS at the concentration of 5U/ml compared with control group (P<0.05), showing a dose-response relationship. ROS did not increase more when the concentration was more than 40U/ml. Thrombin at the concentration of 20 U/ml for 30 minutes increased intracellular ROS (mean value 298.0) by 64.7% compared with the control (mean value 180.6). The same concentration of thrombin for 1 hour caused the increase ROS (mean value 431.2) by 138.8% compared with the control. ROS generation in HLF pretreated with hirudin (20 U/ml) for 30 minutes had no significant change (mean value 171.2) compared with the control.Ratio of GSH/GSSG had a significant decrease in HLF treated with thrombin at the concentration of 10U/ml,20U/ml(2.1、1.7 respectively) compared with the control group(5.2) (P<0.05). The ratio was no significant change (5.0,4.9) compared with the control group (5.2) when pretreated with hirudin or NAC (P> 0.05)3.Subunits p47phox and Rac2 specific bands markedly decreased in cytosol following treatment with thrombin(20 U/ml) at 10 minutes and 30 minutes time points(P<0.05). There was little protein expression at 1h time point (P<0.05). But there was no significant change between the control group and hirudin+thrombin group (P> 0.05)4.p-ERK1/2 protein expressing increased significantly from 10 minutes to 2 hours time points with thrombin treatment compared with control (P<0.05). There was little protein expression in the cells pretreated with NAC and hirudin. Thrombin did not affect the change of p-p38 expression at all from 10 minutes to 2 hours time point (P >0.05)5. Thrombin stimulated HLF at 5min,10min,30min,1h and 2h respectively. The fluorescence contents representing p65 at different time points in nucleus had no statistical difference compared with control group (P>0.05). The fluorescence contents in cells stimulated with TPA had the significantly increase and there was a statistical difference (P<0.05)6. The protein expression of PAR1 was found in both bronchial epithelial cell and human lung fibroblast and there was more in the later (P<0.05)Conclusion1. Thrombin promotes human lung fibroblast to proliferation.2. The mechanism of promoting proliferation is as follows.2.1 PAR1 is expressed higher in HLF than that in bronchial epithelial cell.2.2 Thrombin induced HLF generate ROS via activating NADPH oxidase.2.3 Thrombin activated growth signal pathway via phosphorylating ERK1/2 but not p38MAPK.2.4 NF kappa B was not activated in thrombin promoting proliferation process.SignificanceThrombin activates protease activated receptor-PAR1, inducing NADPH oxidase generate ROS and ROS activate ERK1/2 signal pathway, resulting to HLF proliferation. Our study explore primarily the mechanism of thrombin on human lung fibroblast, which provides the theory evidence for blood coagulation disorder relative respiratory diseases revention and therapy.更多还原