【作者】 杨进；

【导师】 张寄南；

【作者基本信息】 南京医科大学 ， 内科学， 2008， 博士

【摘要】 背景:C-反应蛋白是一种急性时相反应的标志物,属于高度保守的正五聚体血浆蛋白家族,在先天免疫系统中作为模式识别受体发挥功能。急性心肌梗塞（AMI）时,由于心肌缺血、损伤、坏死等病变,可导致血清CRP水平发生变化,已有大量临床资料表明,CRP增高和动脉粥样硬化、急性心肌梗塞预后相关。CRP作为炎症标记物,在心血管事件形成过程中是否仅仅是一个“旁观者”,是否直接参与心血管疾病的发生与发展过程,仍存在争议。近年来,越来越多的体外研究证据表明CRP损伤血管平滑肌细胞,血管内皮细胞,在动脉粥样硬化的发生、发展过程中起着直接作用。有研究发现急性心肌梗塞患者心脏的梗塞区有CRP沉积,而且CRP的含量与梗塞的范围呈正相关。此外,动物实验已证明针对CRP进行治疗,可以防止大鼠心肌梗塞模型中梗塞面积的增加,并且能消除注射人CRP后引起心脏功能紊乱的现象。这些研究提示,CRP可能加重了心肌梗塞的发展,已证明线粒体细胞色素C在心肌梗塞区细胞凋亡过程中发生转移,细胞色素C释放到胞质后可引发caspase活化级联反应,导致细胞死亡,但这些重要蛋白是否参与了C-反应蛋白致心肌损伤过程仍不明确,因此,探讨CRP加重心肌梗塞,致心脏功能紊乱的分子机制,寻找一种针对CRP治疗的更为高效无毒的药物是一项重要的探索性研究领域,引起了研究工作者和临床医生的兴趣和关注。目的:为探讨CRP对心肌细胞的直接损伤作用和分子机制,寻找一种阻断CRP致病作用的生物学治疗方法,本研究用获得的高纯度的人CRP蛋白对体外培养的缺氧状态下的乳大鼠心肌细胞进行干预,确定信号通路中重要蛋白,作为治疗靶点进行干预研究;从稳定分泌CRP单克隆抗体的杂交瘤细胞中克隆出抗体可变区,VH、VL通过Linker连接形成单链抗体,通过大肠杆菌表达后进行纯化,初步探讨CRP单链抗体对CRP致心肌细胞损伤的保护作用。内容与方法:1.采用Immobilized p-Aminophenyl phosphoryl Cholnie Gel亲和层析柱从感染、炎症期及肿瘤患者胸水中分离、纯化CRP,用质谱鉴定其成分,并通过SDS-PAGE与western blot检测其纯度与特异性。2.从经TNF-alpha刺激的肝癌细胞系HepG2中,通过RT-PCR的方法,克隆人C-反应蛋白基因,并分别构建原核和真核表达载体,对原核表达载体进行了诱导表达和纯化,真核载体（pEGFP-N1-CRP）脂质体介导转染HEK293T细胞,荧光显微镜观察及western印迹法鉴定目的蛋白的表达。3.克隆大鼠Bcl-2 cDNA序列,构建真核表达载体（pEGFP-N1-Bcl2）（Bcl-2 cDNA中含终止密码子TGC）。分离乳大鼠心肌细胞,空载体pEGFP-N1和pEGFP-N1-Bcl2脂质体介导转染培养分离的心肌细胞,荧光显微镜观察及Western blot鉴定目的蛋白的表达。4.观察CRP干预对体外培养的乳大鼠心肌细胞的影响（1）实验分为三组:A.未转染组;B.pEGFP-N1转染组;C.pEGFP-N1-Bcl-2转染组。各组转染后48h,分别进行缺氧、100μg/ml CRP及缺氧+100μg/ml CRP干预;对照组为DMEM+10%FCS,正常氧含量、5%CO₂,37℃环境下培养。各组实验至少重复三次。（2）Hochest 33258检测各组细胞凋亡情况。（3）激光共聚焦免疫荧光定位和Western blot法分析细胞色素C在各组细胞中的分布,探讨细胞色素C的改变情况及其意义。（4）RT-PCR和western blot分析各组细胞中Bax,Bcl-2的表达以及Bax/Bci-2的比值。用Caspase活性测定试剂盒分析各组细胞中Caspase-3,Caspase-9的活性。5.从稳定分泌CRP单抗的杂交瘤中克隆出CRP-McAb可变区序列,通过Linker体外装配CRP单链抗体,构建原核、真核表达载体。对原核表达载体pET-28a-scFv诱导表达,包涵体蛋白经变性、复性,镍柱纯化,并通过SDS-PAGE与western blot进行鉴定。真核表达载体pEGFP-N1-scFv脂质体介导转染HEK293T细胞,荧光显微镜观察转染情况及western印迹法鉴定目的蛋白的表达。细胞免疫组化检测转染细胞表达的scFv对CRP的识别能力。6.观察CRP-scFv对CRP所致乳大鼠心肌细胞损伤的影响,初步探讨scFv对损伤乳大鼠心肌细胞保护作用。7.统计学方法:数据均测量数据以平均数加标准差（（?）±s）表示。组间差异采用独立样本t检验、单因素方差分析和x²检验（SPSS 13.0）,P＜0.05有统计学意义。结果1.采用亲和层析法从肿瘤患者腹水中成功纯化出大小为24KD蛋白,SDS-PAGE检测纯度超过95%,用anti-CRP抗体进行western blot检测未见杂带,质谱鉴定表明所得蛋白为人C-反应蛋白。2.成功克隆出人C-反应蛋白全长cDNA序列,测序证实与Genebank标准序列完全符合,并成功克隆至pET-28a和pEGFP-N1载体中。原核表达载体经1mM IPTG诱导表达后,SDS-PAGE检测在30KD处有一亮带,经western blot证实此条带为His-CRP融合蛋白。经镍柱亲和层析获得纯化的重组CRP蛋白。脂质体介导重组质粒pEGFP-N1-CRP转染HEK293T细胞,荧光显微镜观察及western印迹法均证实有目的蛋白表达。3.成功克隆出大鼠Bcl-2全长cDNA序列,测序证实与Genebank标准序列完全符合,并成功克隆至pEGFP-N1载体中。成功通过M-PEI介导成功将重组pEGFP-N1-Bcl-2转染至分离培养的乳大鼠心肌细胞中,并通过western印迹法证实有目的蛋白表达。4.CRP加重缺氧心肌细胞的凋亡。心肌细胞缺氧8h后凋亡率较对照组显著增加（P＜0.001）,缺氧同时用100μg/ml CRP进行干预,细胞凋亡率比单独进行缺氧干预有显著性增加（P＜0.05）。5.CRP加重缺氧心肌细胞线粒体中细胞色素C外流。细胞免疫荧光显示,对照组细胞中细胞色素C分布于线粒体中,缺氧8h细胞组中有少量细胞色素C发生移位,从线粒体转移至胞浆中,但当同时用缺氧和100μg/ml CRP进行干预时,细胞胞浆中弥散有大量细胞色素C。分别提取各组细胞线粒体和胞浆蛋白,用western blot分析进一步得到了证实。6.RT-PCR和western blot检测显示CRP显著升高缺氧心肌细胞线粒体膜蛋白Bax/Bcl-2的比值。7.CRP加重缺氧心肌细胞Caspase-9,Caspase-3的激活。单独缺氧干预8h,Caspase-9和Caspase-3活性与对照组相比均显著升高（P＜0.05）。缺氧和CRP同时干预组Caspase-9和Caspase-3活性与单独缺氧组相比显著升高（P＜0.05）,但单独CRP干预后,Caspase-9和Caspase-3活性与对照组相比无显著性差异（P＞0.05）。8.转染pEGFP-N1-Bcl-2后48h,DMEM培养基条件下,缺氧同时用100μg/ml CRP干预8h后心肌细胞比未转染细胞缺氧8h后凋亡率显著降低（P＜0.05）。在缺氧或同时用CRP干预条件下,过量表达Bcl-2蛋白不抑制细胞内Bax转录和表达水平,但Bax/Bcl-2比值比同样干预条件下的未转染组有显著下降。Bcl-2蛋白过表达抑制细胞色素C从线粒体向胞浆转移,进而抑制Caspase-9和Caspase-3活化。9.成功从稳定分泌CRP单抗的杂交瘤中克隆出CRP-McAb可变区序列,测序后与V-Base数据库比对,确定阅读框,重叠延伸（overlap extend PCR）PCR将VH和VL通过Linker连接,并成功克隆入pET-28a和pEGFP-N1载体中。原核表达载体经1mM IPTG诱导表达后,SDS-PAGE检测在30KD处有一亮带,经western blot证实此条带为His-scFv融合蛋白。经镍柱亲和层析获得纯化的重组scFv蛋白。脂质体介导重组pEGFP-N1-scFv转染HEK293T细胞,荧光显微镜观察,western印迹法均证实有目的蛋白表达。细胞免疫组化结果证明表达的scFv可识别人CRP蛋白。10.凋亡检测初步结果表明anti-CRP单克隆抗体（4E8C6H6F12E7D11）和重组scFv蛋白可抑制CRP诱导的缺氧心肌细胞的凋亡。结论与讨论:1.本研究中采用Immobilized p-Aminophenyl phosphoryl CholnieGel亲和层析柱从感染、炎症期及肿瘤患者胸水中分离、纯化CRP,此法得到的CRP纯度高,无NaN3污染,符合体外干预实验要求。2.CRP加重缺氧诱导的乳大鼠心肌细胞的损伤,而对体外正常培养心肌细胞无影响。该结果与Pepys MB等在大鼠心肌梗塞模型中注射人类CRP后,出现梗死面积增加的现象相一致。表明体外培养心肌细胞进行缺氧+CRP处理是可用的细胞水平实验模型。3.本研究发现心肌细胞线粒体膜蛋白Bax/Bcl-2的比值降低,导致线粒体膜通透性发生改变,细胞色素C从线粒体向胞浆转移,活化了Caspase-9和Caspaes-3可能是CRP加重缺氧诱导的乳大鼠心肌细胞凋亡的重要途径。过量表达Bcl-2蛋白后,CRP促凋亡作用被抑制,提示Bcl-2是该信号通路中重要蛋白,可作为靶点进行干预治疗。但在该通路上游,CRP是如何作用于损伤心肌细胞,还有待进一步深入研究。4.本研究中从稳定分泌CRP单抗的杂交瘤中克隆出CRP-McAb可变区序列,经过表达、纯化获得了针对CRP的单链抗体,初步研究结果表明该重组单链抗体可抑制CRP诱导的缺氧心肌细胞的凋亡,为运用基因工程抗体作为生物技术药物针对CRP进行治疗提供了理论基础。更多还原

【Abstract】 Background:C-reactive protein（CRP）is an acute-phase reactant,which belongs to the highly conserved pentraxin family of plasma proteins and serves as a pattern-recognition molecule in the innate immune system. Myocardial necrosis following acute myocardial infarction is a potent acute-phase stimulus;there is a major CRP response.Furthermore,there is considerable clinical considence supporting a strong association between the peak CRP values in circulating blood after the onset and the outcome of myocardial infarction.What remains less clear is whether CRP acts simply as a marker of vascular disease burden and activity or indeed participates in the development,progression,and complications of cardiovascular disease.In past few years,an increasing number of in vitro studies have indicated that CRP had exerting adverse and ultimately harmful effects on vascular smooth muscle cells（VSMCs）,aortic endothelial cells,and therefore acts as a potential initiator and mediator of atherosclerosis.CRP was also found in the area of acute myocardial infarction,and showed positive correlation between the level of CRP and the infarct size.Furthermore,recently,therapeutic inhibition of CRP has been showed a promising new approach to cardio-protection in acute myocardial infarction in rat models.These results indicate that CRP may play an important role in the progression of cardiac dysfunction.Previous studies have confirmed that cytochrome C releasing from mitochondria then activates Caspase signal pathway may played important role in the cardiac apoptosis,but whether the key proteins mentioned above take part in the progress of CRP induced cardiac damage is little known.So,it is important to illustrate the molecular mechanism involved in the effects of CRP on the myocardial infarction and the cardiac-disorder,and it is a perspective work to explore a new atoxic drug with higher efficiency. Objective:To investigate the direct effects of CRP on the cardiac myocyte and the molecular mechanism involved,and to explore a new efficient biological therapy against CRP,in this study,we used the hypoxia stimulated primary neonatal rat cardiac myocytes in vitro to simulate the cardiac myocyte in myocardial infarction in vivo.Within this system,we detected the effects of CRP on cardiac myocyte and look for the key point protein in the signal transduction pathway for intervention as the therapeutic target;the sequence of variable region was cloned from mouse hybridoma cells stably secreting anti-CRP antibody,the VH and VL were conjuncted with linker.The protein of scFv was obtained in the E.Coli,and the effects of scFv on CRP induced cardiac damage were observed in vitro.Materials and methods:1.Human CRP was isolated from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel.The malignant ascites fluid was obtained from cancer patients and the investigation conforms to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects.Purified human CRP was assayed by SDS-PAGE,mass-spectrum and western-blot analysis.2.The full length of human C-reactive protein gene was cloned from TNF-alpha induced hepatoma cell line（HepG2）by RT-PCR and subcloned into prokaryotic and eukaryotic expression vector plasmid, named as pET-28a-CRP and pEGFP-N1-CRP,respectively.His-CRP was induced to express and purified by Ni²⁺affinity chromatography. After transfection pEGFP-N1-CRP into HEK293T cells,the expression of CRP was observed under fluorescence microscope,and confirmed by western blotting.3.The full length of rat Bcl-2 gene was cloned into pEGFP-N1 plasmid and named as pEGFP-N1-Bcl-2（Stop codon TGC was contained in the cDNA of Bcl-2）.After transfected into cultured neonatal cardiac myocytes using M-PEI,the expression of Bcl-2 was observed under fluorescence microscope,and confirmed by western blotting.4.The effects of CRP on cultured neonatal cardiac myocytes in virto were observed.（1）Myocytes were divided into three groups:A.None transfected myocytes;B.pEGFP-N1 transfected myocytes;C.pEGFP-N1-Bcl-2 transfected myocytes.48 hours after transfection,myocytes were treated with 100μg/ml CRP,hypoxia and cotreatment with them respectively. Control myocytes were incubated in DMEM containing 10%FBS under normoxia.A hypoxic condition was created by incubating the cells with serum-free DMEM in an airtight Plexiglas chamber with an atmosphere of 5%CO₂/95%N₂ at 37℃for 8h in experiment.（2）Determination of myocyte apoptosis was performed using cell apoptosis assay Kit（Hoechst 33258）.（3）The localization of Cyt c was detected with immunofluorescent under confocal microscope and the quantity of translocation of cytochrome c from mitochondria to cytolist was analyzed by western blot.（4）The expressions of Bax and Bcl-2 and the ratio of Bax/Bcl-2 were analyzed by RT-PCR and western blot.Caspase-9 and Caspase-3 activities were determined with a Caspase assay kit.5.CRP-McAb variable region（VH and VL）was cloned from mouse hybridoma cells stably secreting anti-CRP antibody,the VH and VL were conjuncted with Linker.The prokaryotic and eukaryotic expression vector were constructed,His-scFv was induced to expression existed majorly in the inclusion body,after denaturation and renaturation,the protein was purified by Ni²⁺affinity chromatography and detected by SDS-PAGE and western blot.The plasmid pEGFP-N1-scFv was transfected into HEK293T cells using M-PEI,the expression of scFv was observed under fluorescence microscope and confirmed by western blotting.Cellular immunostaining was used to detect the ability of recognization between expressed scFv and human CRP.6.The anti-apoptosis effect of CRP-scFv was analyzed with in situ apoptosis detection kit—TUNEL.7.Statistics:The data mentioned above at least derived from three independent experiments.All data are presented as mean±SD.The independent samples T test,one way ANOVA and chi-square test were used to analyze differences and statistical tests were performed with the use of SPSS 13.0 statistical software.Statistical significance was accepted at P＜0.05.Results:1.CRP purified from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel was in the monomeric form （24KD）with no detection of other proteins by silver staining on SDS-PAGE（purity up to 95%）,western-blot or mass-spectrum.2.The full length cDNA of human CRP was successfully cloned,DNA sequencing and BLAST algorithm revealed that the cDNA was exactly identical to the reported sequence in the Genebank.The CRP gene was then successfully subcloned into pET-28a and pEGFP-N1. After induction with 1mM IPTG at 37℃for 4h,E.coli BL21（DE3） transformed with pET28a-CRP produced a fusion protein of approximately 30 kDa,which matched well with the theoretical molecular weight of His-CRP.The expressed recombinant CRP was purified by Ni²⁺affinity chromatography and detected by anti-CRP antibody.The plasmid pEGFP-N1-Bcl-2 was successfully transfected into HEK293T cells using M-PEI which resulted in significant increased CRP protein expression.3.The full length cDNA of rat Bcl-2 was successfully cloned,DNA sequencing and BLAST algorithm revealed that the cDNA was exactly identical to the reported sequence in the Genebank.The Bcl-2 gene was then successfully subcloned into pEGFP-N1.Cultured neonatal cardiac myocytes transfected using M-PEI which resulted in 70%to 80%transfection efficiency and significant increase in Bcl-2 protein expression.4.CRP augmented hypoxia-induced cell apoptosis.The percentage of apoptotic myocytes increased significantly after 8 hours hypoxia,as compared with control.Cotreatment of hypoxia with CRP（100μg/mL） significantly increased the percentage of apoptotic cells.However, CRP did not induce myocytes apoptosis significantly under normoxia.5.CRP induced more cytochrome c release than hypoxia alone.Control cardiac myocyte demonstrated organized speckled patterns of cytochrome c that colocalized with mitochondria.In contrast,after hypoxia for 8 hours,there was little cytochrome c in cytolist but after cotreatment with hypoxia and 100μg/mL CRP,cytochrome c staining diffused throughout the cells,no longer colocalized to the mitochondria.The phenomenon was further conformed by western blot which detected the quantity of translocation of cytochrome c from mitochondria to cytolist.6.CRP increased the ratio of Bax/Bcl-2 in the hypoxia-induced cardiac myocyte.7.CRP further increased the hypoxia-induced activation of Caspase-9 and Caspase-3 significantly.However CRP showed no action on the activity Caspase-9 and Caspase-3 during normoxia.8.Bcl-2 over-expression in cultured neonatal cardiomyocytes resulted in significant inhibition of cotreatment of hypoxia with CRP-induced apoptosis,cytochrome c release,Bax/Bcl-2 ratio increase and Caspases activation.9.The cDNA of CRP-McAb variable region（VH and VL）was successfully cloned from mouse hybridoma cells stably secreting anti-CRP antibody.After DNA sequencing and read frame definition according to the V-base,the VH and VL were conjuncted with Linker using overlapping extension PCR.The scFv gene was successfully subcloned into pET-28a and pEGFP-N1.After induction with 1mM IPTG,a fusion protein of approximately 30 kDa was produced,which matched well with the theoretical molecular weight of His-scFv.The expressed recombinant scFv was purified by Ni²⁺affinity chromatography and detected by anti-His antibody.The plasmid pEGFP-N1-scFv was successfully transfected into HEK293T cells using M-PEI which resulted in significant increasing of scFv expression.Cellular immunostaining showed that the expressed scFv could be recognized by human CRP.10.Our preliminary data of myocyte apoptosis analysis indicated that anti-CRP antibody（4E8C6H6F12E7D11）and recombinant scFv protein could inhibit cardiac myocyte apoptosis induced by hypoxia cotreated with CRP.Discussion and conclusion:1.A number of recent studies have used commercial CRP preparations that remain poorly characterized and indeed contain known quantities of biologically active contaminants such as sodium azide.So,in this study,we used affinity chromatography（Immobilized p-Aminophenyl Phosphoryl Choline Gel）to purify human C-reactive protein from malignant ascites fluid of the patients.Our purified CRP is of high purity,without Na₃N contamination,and is suitable for the in vitro intervention.2.The data of this study suggest a potential mechanism that CRP could enhance apoptosis in hypoxia-stimulated neonatal rat cardiac myocytes through the mitochondrion-dependent pathway but CRP alone has no effects on neonatal rat cardiac myocytes under normoxia, which is consist with the results of Pepys MB’s group that injection of human CRP to rats undergoing acute myocardial infarction increased infarct size and aggravated cardiac dysfunction.3.The present study demonstrates for the first time CRP can regulate the Bax and Bcl-2 proteins in mitochondria and decrease the ratio of Bax/Bcl-2,augment mitochondrial death pathway through provoking the mitochondrial permeability transition,therefore cytochrome c released from the mitochondria to the cytosol,activate Caspase-9 and Caspase-3 in hypoxic cardiac myocytes,a fact which indicate that therapeutic inhibition of CRP may have significant implications in the development of future therapies to combat the effects of myocardial infarction.But,further studies are needed to clarify the upstream signal pathway of the pro-apoptotic action of CRP and the receptor of CRP in the cell membrane.4.In this study,we cloned cDNA of CRP-McAb variable region（VH and VL）from mouse hybridoma cells stably secreting anti-CRP antibody and obtained scFv antibody against CRP after expression and purification.Our preliminary study suggested that recombinant scFv protein could inhibit inhibit cardiac myocyte apoptosis induced by hypoxia cotreated with CRP.These results provide exciting future for using genetically engineered antibody to biological therapy against CRP.更多还原