重组纤维连接蛋白C端及N端肝素结合域两种多肽的制备及其对小鼠败血症治疗作用的研究
|
摘要
败血症是临床常见的危重症,尽管大量高效抗生素的不断问世和日臻完善的器官支持和重症监护技术,其病死率仍较高。所以,人们一直在研究开发有效的治疗药物。纤维连接蛋白是一种多功能的糖蛋白,能与肝素、胶原、纤维蛋白以及整合素家族的细胞表面受体结合,参与细胞的黏附、迁移、止血、抗感染以及维持血管的完整性等作用。大量研究表明纤维连接蛋白FN具有抗败血症的作用,但从血浆分离FN存在着血浆资源浪费和可能传播血源性传染病的问题,同时由于FN分子量大,全分子表达又存在技术上的难题。因此利用基因工程技术表达FN的功能区多肽并研究其功能,以代替FN的抗败血症作用不失为一种可行的方法。本课题组前期成功地用大肠杆菌表达系统表达纯化了FN的细胞结合域多肽(rhFN55多肽),用酵母表达系统表达纯化了FN N端肝素结合域多肽(FNNHBD),实验表明这两种多肽具有治疗小鼠败血症的作用;本课题前期也应用基因工程方法构建了FN C端的肝素结合域多肽的酵母表达载体,并筛选出了高表达的酵母表达菌株,但在酵母表达系统中纯化FN C端的肝素结合域多肽(FNCHBD)并研究其对鼠败血症的作用,目前国内外还未见报道。本实验在前期研究的基础上,表达纯化FNCHBD和FNNHBD多肽,研究FNCHBD多肽对鼠败血症的作用以及比较FNCHBD和FNNHBD两多肽对鼠败血症的治疗效果。
通过离心、超滤、过离子交换层析柱和分子筛层析柱对两多肽的酵母发酵上清进行纯化,通过Western-blot对纯化的多肽进行鉴定,并证实了两多肽都能够与FN的多克隆抗体结合,具有抗原性;将发酵上清通过过肝素亲和层析柱表明两多肽都能够挂柱,都具有肝素结合活性;经质谱分析测定FNCHBD的分子量为30.5KD,FNNHBD的分子量约为27.9KD。本研究按照A.-H. Kwon等用半乳糖胺(GalN)敏化LPS小鼠建立败血症模型,小鼠72小时的死亡率仅为60%,因此本研究在此基础上摸索了LPS的剂量,最后确定给每只小鼠腹腔注射LPS(100ug/kg)和GalN(400mg/kg)使小鼠的死亡率达到90%,建立了我们的败血症小鼠模型。通过观察72小时小鼠的存活率表明在LPS/GlaN注射半小时前给予FNCHBD和FNNHB两多肽都能提高模型小鼠的存活率,在生理盐水对照组中,小鼠的存活率为11.1%,而在FNCHBD和FNNHB两多肽的5mg/kg、10mg/kg、20mg/kg这三个剂量组的存活率分别为33.3%、55.5%、66.7%和27.8%、50%、61.1%,都表现出剂量依赖效应。通过测定注射LPS/GalN注射后9小时血清中AST、ALT、LDH和TBIL的值,表明FNCHBD和FNNHB两多肽都能够降低血清中AST、ALT、LDH和TBIL的值,改善小鼠的肝功;通过用ELISA方法检测通过测定注射LPS/GalN注射后9小时小鼠血浆中的炎症因子TNF-α、IL-1β、IL-6的水平,表明FNCHBD和FNNHB两多肽都能够抑制血浆中上述炎症因子的表达;通过病理组织学观察,FNCHBD和FNNHB两多肽都能够减轻肝脏的损伤;通过电镜和TUNEL方法表明FNCHBD和FNNHB两多肽都能够抑制肝细胞的凋亡;并且实验结果表明FNCHBD和FNNHB两多肽的上述作用都呈现出剂量依赖效应。同时本研究发现在20mg/kg剂量的基础上,在注射LPS/GlaN后增加FNCHBD和FNNHB两多肽的给药次数对提高小鼠的存活率都没有意义,预防性地给予FNCHBD和FNNHB两多肽的治疗效果优于LPS/GlaN注射后给药。最后通过比较FNCHBD和FNNHBD两多肽对败血症小鼠的治疗效果,发现FNCHBD和FNNHBD治疗效果相近,无统计学上的差异。
结论:本实验成功表达纯化出FNCHBD和FNNHBD多肽。成功建立了败血症小鼠模型,并表明了FNCHBD和FNNHBD肽都具有治疗鼠败血症的作用,且两者的治疗效果无统计学上的差异。治疗作用同样都具有剂量依赖性且作用机制相似,都是通过抑制TNF-α等炎症因子的产生,进而抑制肝细胞的凋亡,减轻肝脏的损伤。但在20mg/kg剂量的基础上,注射LPS/GlaN后增加给药次数都不能提高二者对鼠败血症的治疗效果,预防性地给予FNCHBD和FNNHB两多肽的治疗效果优于LPS/GlaN注射后给药。
Sepsis,an acute severe syndromes has still high mortality,although there are so many wonderful antibiotics , consummate organ support and surveille techniques. Fibronectin(FN)is known to be a large multifunction glycoprotein with binding sites for many substances including heparin, collagen, fibrinogen, fibrin and integrin cell surface receptors.It is involved in a variety of cellular processes, including tissue repair, blood clotting and cell migration or adhesion. Most studies suggested FN purified from plasma have the effect on sepsis, but it might transmit infectious diseases, such as HIV.Since it was difficult to express the whole molecular of FN by genetic engineering. Thus,it is necessary to express the related function polypeptides and study the function of recombinant polypeptides on sepsis.In our previous studies,we had expressed and purified the recombinant cell-binding domain polypeptide of FN ( rhFN55 polypeptide ) and N-terminal heparin-binding domain polypeptide (FNNHBD),and confirmed they both had the effect on mice with sepsis Our previous studies had constructed the recombinant yeast expressed vector with the C-terminal heparin-binding domain polypeptide(FNCHBD).But, the effect of FNCHBD on sepsis is unclear.In this study, we express and purify FNCHBD and FNNHBD, investigate the effect of FNCHBD and FNNHBD on sepsis in mice.
The FNCHBD and FNNHBD were expressed and purified and t were identified by western-blot and by passing the heparin affinity column. The molecular weights of FNCHBD is 30.5KD, and that of FNNHBD is 27.9KD by the mass spectrogram.
According to A.-H. Kwon,s and our test condiction,the septic mouse model was established by the injection of 100ug/kg lipopolysaccharide(LPS ) and 400mg/kg D-Galactosamine ( GalN ) into the mice from abdomen.The result show that both FNCHBD and FNNHBD can arise the survival of the experimental mice by administrating intravenously 30 min before LPS/GalN injection. In the control group, the survival rate of the mice was 11.1%,and the survival rate was 33.3% , 55.5% ,66.7% respectively at 5mg/kg ,10mg/kg , 20mg/kg of FNCHBD in the FNCHBD-treated group and that was 27.8%,50% 61.1% in FNNHBD-treated group respectively. The levels of serum AST、ALT、LDH and TBIL increased in the control group, and decreased in the FNCHBD-treated group and FNCHBD-treated group with the increasing dosage of FNCHBD and FNNHBD. Histopathology of liver showed that mass denaturation, necrosis, congestion, haemorrhage and hyperemia in the control group, both FNCHBD and FNNHBD can inhibited the injury of liver in a dose-dependent manner. The level of TNF-α、IL-6、IL-1βin plasma determined by ELISA in the control group were significantly higher than in the normal control, while both FNCHBD and FNNHBD can significantly inhibited the expression of these apoptosis of hepatocyte in a dose-dependent manner.A lot of apoptosis of hepatocyte were showed in the control group by electron microscope and TUNEL. However,both FNCHBD and FNNHBD both can significantly inhibited hepatocyte apoptosis in a dose-dependent manner. The results also showed that increasing the times of administration of FNCHBD and FNNHBD both can not contribute to the survival rate of mice with sepsis after LPS/GalN injecting at the base of 20mg/kg of FNCHBD and FNNHBD and administration with FNCHBD and FNNHBD respctivly before LPS/GalN injecting had the better effect on sepsis in mice than after LPS/GalN injecting.
Conclusion:In this study, FNCHBD and FNNHBD were expressed and purified and the model of sepsis in mice was constructed successfully. FNCHBD and FNNHBD were both demonstrated to have the effect on mice with sepsis in a dose-dependent manner The effect on mice with sepsis of two polypeptides have no significant deviationin statistics. Therapeutic mechanism is similar, both through inhibiting expression of TNF-αand other inflammatory cytokines and suppressing subsequent apoptotic processes in the liver. Increasing the times of administration of FNCHBD and FNNHBD after LPS/GalN injecting do not contribute to their effect on mice with sepsis at the base of 20mg/kg of FNCHBD and FNNHBD. Administration with FNCHBD and FNNHBD respctivly beforeLPS/GalN injecting had the better effect on sepsis in mice than after LPS/GalN injecting.
通过离心、超滤、过离子交换层析柱和分子筛层析柱对两多肽的酵母发酵上清进行纯化,通过Western-blot对纯化的多肽进行鉴定,并证实了两多肽都能够与FN的多克隆抗体结合,具有抗原性;将发酵上清通过过肝素亲和层析柱表明两多肽都能够挂柱,都具有肝素结合活性;经质谱分析测定FNCHBD的分子量为30.5KD,FNNHBD的分子量约为27.9KD。本研究按照A.-H. Kwon等用半乳糖胺(GalN)敏化LPS小鼠建立败血症模型,小鼠72小时的死亡率仅为60%,因此本研究在此基础上摸索了LPS的剂量,最后确定给每只小鼠腹腔注射LPS(100ug/kg)和GalN(400mg/kg)使小鼠的死亡率达到90%,建立了我们的败血症小鼠模型。通过观察72小时小鼠的存活率表明在LPS/GlaN注射半小时前给予FNCHBD和FNNHB两多肽都能提高模型小鼠的存活率,在生理盐水对照组中,小鼠的存活率为11.1%,而在FNCHBD和FNNHB两多肽的5mg/kg、10mg/kg、20mg/kg这三个剂量组的存活率分别为33.3%、55.5%、66.7%和27.8%、50%、61.1%,都表现出剂量依赖效应。通过测定注射LPS/GalN注射后9小时血清中AST、ALT、LDH和TBIL的值,表明FNCHBD和FNNHB两多肽都能够降低血清中AST、ALT、LDH和TBIL的值,改善小鼠的肝功;通过用ELISA方法检测通过测定注射LPS/GalN注射后9小时小鼠血浆中的炎症因子TNF-α、IL-1β、IL-6的水平,表明FNCHBD和FNNHB两多肽都能够抑制血浆中上述炎症因子的表达;通过病理组织学观察,FNCHBD和FNNHB两多肽都能够减轻肝脏的损伤;通过电镜和TUNEL方法表明FNCHBD和FNNHB两多肽都能够抑制肝细胞的凋亡;并且实验结果表明FNCHBD和FNNHB两多肽的上述作用都呈现出剂量依赖效应。同时本研究发现在20mg/kg剂量的基础上,在注射LPS/GlaN后增加FNCHBD和FNNHB两多肽的给药次数对提高小鼠的存活率都没有意义,预防性地给予FNCHBD和FNNHB两多肽的治疗效果优于LPS/GlaN注射后给药。最后通过比较FNCHBD和FNNHBD两多肽对败血症小鼠的治疗效果,发现FNCHBD和FNNHBD治疗效果相近,无统计学上的差异。
结论:本实验成功表达纯化出FNCHBD和FNNHBD多肽。成功建立了败血症小鼠模型,并表明了FNCHBD和FNNHBD肽都具有治疗鼠败血症的作用,且两者的治疗效果无统计学上的差异。治疗作用同样都具有剂量依赖性且作用机制相似,都是通过抑制TNF-α等炎症因子的产生,进而抑制肝细胞的凋亡,减轻肝脏的损伤。但在20mg/kg剂量的基础上,注射LPS/GlaN后增加给药次数都不能提高二者对鼠败血症的治疗效果,预防性地给予FNCHBD和FNNHB两多肽的治疗效果优于LPS/GlaN注射后给药。
Sepsis,an acute severe syndromes has still high mortality,although there are so many wonderful antibiotics , consummate organ support and surveille techniques. Fibronectin(FN)is known to be a large multifunction glycoprotein with binding sites for many substances including heparin, collagen, fibrinogen, fibrin and integrin cell surface receptors.It is involved in a variety of cellular processes, including tissue repair, blood clotting and cell migration or adhesion. Most studies suggested FN purified from plasma have the effect on sepsis, but it might transmit infectious diseases, such as HIV.Since it was difficult to express the whole molecular of FN by genetic engineering. Thus,it is necessary to express the related function polypeptides and study the function of recombinant polypeptides on sepsis.In our previous studies,we had expressed and purified the recombinant cell-binding domain polypeptide of FN ( rhFN55 polypeptide ) and N-terminal heparin-binding domain polypeptide (FNNHBD),and confirmed they both had the effect on mice with sepsis Our previous studies had constructed the recombinant yeast expressed vector with the C-terminal heparin-binding domain polypeptide(FNCHBD).But, the effect of FNCHBD on sepsis is unclear.In this study, we express and purify FNCHBD and FNNHBD, investigate the effect of FNCHBD and FNNHBD on sepsis in mice.
The FNCHBD and FNNHBD were expressed and purified and t were identified by western-blot and by passing the heparin affinity column. The molecular weights of FNCHBD is 30.5KD, and that of FNNHBD is 27.9KD by the mass spectrogram.
According to A.-H. Kwon,s and our test condiction,the septic mouse model was established by the injection of 100ug/kg lipopolysaccharide(LPS ) and 400mg/kg D-Galactosamine ( GalN ) into the mice from abdomen.The result show that both FNCHBD and FNNHBD can arise the survival of the experimental mice by administrating intravenously 30 min before LPS/GalN injection. In the control group, the survival rate of the mice was 11.1%,and the survival rate was 33.3% , 55.5% ,66.7% respectively at 5mg/kg ,10mg/kg , 20mg/kg of FNCHBD in the FNCHBD-treated group and that was 27.8%,50% 61.1% in FNNHBD-treated group respectively. The levels of serum AST、ALT、LDH and TBIL increased in the control group, and decreased in the FNCHBD-treated group and FNCHBD-treated group with the increasing dosage of FNCHBD and FNNHBD. Histopathology of liver showed that mass denaturation, necrosis, congestion, haemorrhage and hyperemia in the control group, both FNCHBD and FNNHBD can inhibited the injury of liver in a dose-dependent manner. The level of TNF-α、IL-6、IL-1βin plasma determined by ELISA in the control group were significantly higher than in the normal control, while both FNCHBD and FNNHBD can significantly inhibited the expression of these apoptosis of hepatocyte in a dose-dependent manner.A lot of apoptosis of hepatocyte were showed in the control group by electron microscope and TUNEL. However,both FNCHBD and FNNHBD both can significantly inhibited hepatocyte apoptosis in a dose-dependent manner. The results also showed that increasing the times of administration of FNCHBD and FNNHBD both can not contribute to the survival rate of mice with sepsis after LPS/GalN injecting at the base of 20mg/kg of FNCHBD and FNNHBD and administration with FNCHBD and FNNHBD respctivly before LPS/GalN injecting had the better effect on sepsis in mice than after LPS/GalN injecting.
Conclusion:In this study, FNCHBD and FNNHBD were expressed and purified and the model of sepsis in mice was constructed successfully. FNCHBD and FNNHBD were both demonstrated to have the effect on mice with sepsis in a dose-dependent manner The effect on mice with sepsis of two polypeptides have no significant deviationin statistics. Therapeutic mechanism is similar, both through inhibiting expression of TNF-αand other inflammatory cytokines and suppressing subsequent apoptotic processes in the liver. Increasing the times of administration of FNCHBD and FNNHBD after LPS/GalN injecting do not contribute to their effect on mice with sepsis at the base of 20mg/kg of FNCHBD and FNNHBD. Administration with FNCHBD and FNNHBD respctivly beforeLPS/GalN injecting had the better effect on sepsis in mice than after LPS/GalN injecting.
引文
[1]姚咏明。MODS 抗炎治疗研究的反思[J]。中国危重医学, 1999,1 1(8):85O-854。
[2]Yamada KM. Fibronectin peptides in cell migration and wound repair[J]. Clin Invest ,2000; 105: 1507-9.
[3] Ruiz Martín G, Prieto Prieto J, et al.Plasma fibronectin as a marker of sepsis[J].Int J Infect Dis, 2004 Jul;8(4):236-43.
[4]陈元仲,吕联煌,张学敏等。血液疾病血浆纤维结合蛋白水平的研究[J]。中国实验临床免疫学杂志,1991,3(4):30-32。
[5]SabaTM,F.A.Blumenstock,WA Scovill,et al. Cryoprecipitate reversal of opsonic α2-surface binding glycoprotein deficiency in septic surgical and trauma patients[J].Science,1978,201:622-624.
[6]Saba TM, F.A.Blumenstock, DM Shah, et al. Reversal of fibronectin and opsonic deficiency in patients: a controlled study[J]. Ann.surg,1984, 199:87-96.
[7]Barkaga ZS,Shoikhetla N,Elykomov VA,et al。Comparative data on the use of a cryosupernatant and fresh-frozen plasma in the therapy of a protracted infectious-septic syndrome of disseminated intravascular coagulation[J].Ter Arkh,1998,70(7):70-72.
[8]吴勇, 陈元仲, 吕联煌。纤维连接蛋白对内毒素血症小鼠保护作用的实验研究[J]。中国病理生理杂志,2003 ,19(6):799–802。
[9]Takamichi Saito, A-HON Kwon,Zwyu Qiu et al。Protective Effect of Fibronectin for Endotoxin Induced Liver Injury after Partial Hepatectomy in Rats[J].Journal of Surgical Research,2005,124:79–84.
[10]A.-H. Kwon, Z. Qiu, H. Nagahama, M. Kaibori, et al. Fibronectin Suppresses Apoptosis and Protects Mice From Endotoxic Shock[J].Transplantation Proceedings,2004,36:2432–2435.
[11]Qiu Z, Kwon AH, Tsuji K et al.Fibronectin prevents D-galactosamine /lipopolysaccharide-induced lethalhepatic failure in mice[J].SHOCK, 2006,25(1):80-87.
[12]Wu Y, Chen YZ, Huang HF et al. Recombinant fibronectin polypeptide antagonizes hepaticfailure induced by endotoxin in mice[J]. Acta Pharmacol Sin,2004,25(6): 783-788.
[13]Lin Cereghino GP, Sunga AJ, Lin Cereghino J, et al. Expression of foreigngenes in the yeast Pichia pastoris[J]. Genet Eng (N Y),2001, 23:157-169.
[14]Cereghino GP, Cereghino JL, Ilgen C, et al. Production of recombinantproteins in fermenter cultures of the yeast Pichia pastoris[J].Curr Opin Biotechnol, 2002, 13(4): 329-332.
[15]Jin F, Xu X, Wang L, et al. Expression of recombinant hybridpeptide cecropinA(1-8)-magainin2(1-12)in Pichia pastoris: purification and characterization[J]. Protein Expr Purif, 2006,50(2):147-156.
[16]Cereghino J L,Cregg J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMS Micro.bial Rev,2000,24(1):45-66.
[17]Wijelath ES, Rahman S, Namekata M,et al.Heparin-II domain of fibronectin is a vascular endothelial growth fact- or-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism[J].Circ Res,2006,99(8):853-60.
[18]Viji RI,Kumar VB,Kiran MS,et al.Angiogenic response of endothelial cells to heparin-binding domain of fibronectin[J].Int J Biochem Cell Biol, 2008,40(2):215-26.
[19]Kim JH,Park SO,Jang HJ,et al.Importance of the heaprin-binding domain of fibronectin for enhancing cell adh- esion activity of the recombinant fibronectin. Biotechnology Letters[J].2006,28 (17):1409 -13.
[20]Kang W,Park S,Jang JH.Kinetic and functional analysis of the heparin-binding domain of fibronectin[J].Biotechnol Lett, 2008, 30(1):55-9.
[21]Liu Y, Huang B, Yuan Y, et al.Inhibition of hepatocarcinoma and tumor metastasis to liver by gene therapy with recombinant CBD- HepII polypeptide of fibronectin[J].Int J Cancer. 2007, 121(1):184- 92.
[22]Kapila YL, Wang S, Dazin P,et al.The heparin-binding domain and V region of fibronectin regulate apoptosis by suppression of p53 and c-myc in human primary cells[J].Biol Chem. 2002,277(10):8482-91.
[23]Arciola CR,Bustanji Y,Conti M,et al.Staphylococcus epidermidis- fibronectin binding andits inhibition by heparin.Biomaterials[J]. 2003,24(18):3013-9.
[24]Dabo SM, Confer AW, Anderson BE,et al Bartonella henselae Pap31, an extracellular matrix adhesin, binds the fibr- onectin repeat III13 moduleInfect[J].Immun,2006,74 (5): 2513-21.
[25]Naito M.Fukuda T.Sekiguchi K,et al.The domains of human fibronectin mediating the binding of alpha antigen,the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterialinfection[J].Bio chemical Journal, 2000,347(3):725-31.
[26]Emma Andersson,Victoria Rydengard,Andreas Sonesson,et al. Antimicrobial activities of heparin-binding peptides[J].Eur, Biochem,2004,271:1219–1226.
[27]Verfaillie CM, Benis A,Iida J,et al.Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain offironectin between the integrin alpha 4 beta and the CD44 adhesion receptor[J].BLOOD,1994,15180-2.
[28]Stack MS,Pizzo SV.Modulation of tissue plasminogen activator catalyzed plasminogen activation by synthetic peptides derived from the amino-terminal heparin binding domain of fibronectin[J].BiolChem.1993,268(25): 18924-8.
[29]Jalkanen S, Jalkanen M.Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin[J]. Cell Biol.1992, 116(3): 817-25.
[30]Yasuda T,Kakinuma T,Julovi Sm,et al.COOH-terminal heparin-binding fibronectin fragment induces nitric- oxide production in rheumatoid cartilage through CD44[J].Rheumatology(Oxford), 2004,43(9): 1116-20.
[31]Sakamoto S,Okanoue T,Itoh Y,et al.Involvement of Kupffer cells in the interaction between neutrophils and sinusoidal endothelial cells in rats[J].Shock,2002,18:152-157.
[32]Ozmen L,Pericin M,Hakimi J,et al.Interleukin 12,interf- eron,and tumor necrosis factor are the key cytokines of the generated Shwarzman reaction[J].Exp Med.1994,180: 907-915.
[33]Satoshi GD,Takashi KE,Statoshi NK et al.Dissemianate intravascular coagulation is a frequent complication of systemic inflammatory response syndrome[J].Throm and Hemost. 1996,75(2):224-228.
[34]Mignon A, Rouquet N, Fabre M,et al.LPS challenge in D-galactosamine -sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock. Am J Respir Crit Care Med.1999,159:1308-1315.
[35]Bohlinger I, Leist M, Gantner F,et al.DNA fragmentation in mouse organs during endotoxic shock[J].Am Pathol,1996, 149:1381-1393.
[36]Leist M,Gantner F,Bohlinger I,et al.Tumor necrosis factor-[alpha] induced hepatocyte apoptosis precedes liver failure in experimental murine shock models[J].Am Pathol,1995,146:1220-1234.
1. Ruiz G, Veiga J, Luisa GLM,et al.Plasmatic concentrations of fibronectin as marker of clinical course among septic patients[J].Enferm Infecc Microbiol Clin, 2001, 19:93-98.
1. Saba TM ,Jaffe E. Plasma fibronectin(opsonic glycoprotein ):its synthesis by vascular endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function[J].AM J Med,1980,68(4):577-594.
2. Bortolussi R, Rajaraman k, Qing GF,et al .Fibronectin enhances in vitro lipopolysaccharide peiming of polymorphonuclear leukocytes[J].Blood ,1997,89(11):4182-4189.
3. Everitt EA,Malik AB,Hendey B, et al. Fibronectin enhances the migration rate of human neutrophils in vitro[J].Leukoc Biol,1996,60:199-206.
4. Oritiz A, Alonso J,Gomez-chiarri M,et al.Fibronectin(FN)decreases glomerular lesions and synthesis of tumour necrosis factor–alpha(TNF-alpha)platelet activating factor(PAF)and FN in proliferative glomerulonephritis[J].Clin Exp Immunol,1995,101(2)334-340.
5. Resnikoff M,Brien T ,Vincent PA,et al.Lung matrix incorporation of pasma fibronectin rdduces vascular permeability in postsurgical bacteremia[J].Am J Plysiol,1999,277:749-759.
7. Nooteboom A,Hendriks T,Otteholler I,et al .Permeability characteristics of human ehdothelial monolayers seeded on different extracellular mateix proteins[J].Mediators Inflamm,2000,9(5)235-241.
8.Takamichi Saito, A-HON Kwon,Zwyu Qiu et al。Protective Effect of Fibronectin for Endotoxin Induced Liver Injury after Partial Hepatectomy in Rats [J].Journal of Surgical Research,2005,124, 79–84.
9. Ruiz Martín G, Prieto Prieto J, et al.Plasma fibronectin as a marker of sepsis[J].Int J Infect Dis. 2004 ,8(4):236-43.
10. 陈元仲,吕联煌,张学敏等。血液疾病血浆纤维结合蛋白水平的研究[J]。中国实验临床免疫学杂志,1991,3(4):30-32。
11.王光,李立红,王玉兰等。严重感染患儿血浆纤维连接蛋白的变化及替代疗法观察[J]。中国实用儿科杂志,2000,15(11)。
12.Barkaga ZS,Shoikhetla N,Elykomov VA,et al。Comparative data on the use of a cryosupernatant and fresh-frozen plasma in the therapy of a protracted infectious-septic syndrome of disseminated intravascular coagulation[J].Ter Arkh,1998,70(7):70-72。
13.吴勇, 陈元仲, 吕联煌。纤维连接蛋白对内毒素血症小鼠保护作用的实验研究中国病理生理杂志[J]。2003 ,19(6): 799–802。
14.A.-H. Kwon, Z. Qiu, H. Nagahama, M. Kaibori, and Y. Kamiyama Fibronectin Suppresses Apoptosis and Protects Mice From Endotoxic Shock[J]. Transplantation Proceedings, 2004,36:2432–2435.
15.Sakai T, Johnson KJ, Murozono M, Fassler R, et al. Plasma fibronectin supports neuronal survival andreduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis[J]. Nat. Med. 2001,7: 324.
[2]Yamada KM. Fibronectin peptides in cell migration and wound repair[J]. Clin Invest ,2000; 105: 1507-9.
[3] Ruiz Martín G, Prieto Prieto J, et al.Plasma fibronectin as a marker of sepsis[J].Int J Infect Dis, 2004 Jul;8(4):236-43.
[4]陈元仲,吕联煌,张学敏等。血液疾病血浆纤维结合蛋白水平的研究[J]。中国实验临床免疫学杂志,1991,3(4):30-32。
[5]SabaTM,F.A.Blumenstock,WA Scovill,et al. Cryoprecipitate reversal of opsonic α2-surface binding glycoprotein deficiency in septic surgical and trauma patients[J].Science,1978,201:622-624.
[6]Saba TM, F.A.Blumenstock, DM Shah, et al. Reversal of fibronectin and opsonic deficiency in patients: a controlled study[J]. Ann.surg,1984, 199:87-96.
[7]Barkaga ZS,Shoikhetla N,Elykomov VA,et al。Comparative data on the use of a cryosupernatant and fresh-frozen plasma in the therapy of a protracted infectious-septic syndrome of disseminated intravascular coagulation[J].Ter Arkh,1998,70(7):70-72.
[8]吴勇, 陈元仲, 吕联煌。纤维连接蛋白对内毒素血症小鼠保护作用的实验研究[J]。中国病理生理杂志,2003 ,19(6):799–802。
[9]Takamichi Saito, A-HON Kwon,Zwyu Qiu et al。Protective Effect of Fibronectin for Endotoxin Induced Liver Injury after Partial Hepatectomy in Rats[J].Journal of Surgical Research,2005,124:79–84.
[10]A.-H. Kwon, Z. Qiu, H. Nagahama, M. Kaibori, et al. Fibronectin Suppresses Apoptosis and Protects Mice From Endotoxic Shock[J].Transplantation Proceedings,2004,36:2432–2435.
[11]Qiu Z, Kwon AH, Tsuji K et al.Fibronectin prevents D-galactosamine /lipopolysaccharide-induced lethalhepatic failure in mice[J].SHOCK, 2006,25(1):80-87.
[12]Wu Y, Chen YZ, Huang HF et al. Recombinant fibronectin polypeptide antagonizes hepaticfailure induced by endotoxin in mice[J]. Acta Pharmacol Sin,2004,25(6): 783-788.
[13]Lin Cereghino GP, Sunga AJ, Lin Cereghino J, et al. Expression of foreigngenes in the yeast Pichia pastoris[J]. Genet Eng (N Y),2001, 23:157-169.
[14]Cereghino GP, Cereghino JL, Ilgen C, et al. Production of recombinantproteins in fermenter cultures of the yeast Pichia pastoris[J].Curr Opin Biotechnol, 2002, 13(4): 329-332.
[15]Jin F, Xu X, Wang L, et al. Expression of recombinant hybridpeptide cecropinA(1-8)-magainin2(1-12)in Pichia pastoris: purification and characterization[J]. Protein Expr Purif, 2006,50(2):147-156.
[16]Cereghino J L,Cregg J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMS Micro.bial Rev,2000,24(1):45-66.
[17]Wijelath ES, Rahman S, Namekata M,et al.Heparin-II domain of fibronectin is a vascular endothelial growth fact- or-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism[J].Circ Res,2006,99(8):853-60.
[18]Viji RI,Kumar VB,Kiran MS,et al.Angiogenic response of endothelial cells to heparin-binding domain of fibronectin[J].Int J Biochem Cell Biol, 2008,40(2):215-26.
[19]Kim JH,Park SO,Jang HJ,et al.Importance of the heaprin-binding domain of fibronectin for enhancing cell adh- esion activity of the recombinant fibronectin. Biotechnology Letters[J].2006,28 (17):1409 -13.
[20]Kang W,Park S,Jang JH.Kinetic and functional analysis of the heparin-binding domain of fibronectin[J].Biotechnol Lett, 2008, 30(1):55-9.
[21]Liu Y, Huang B, Yuan Y, et al.Inhibition of hepatocarcinoma and tumor metastasis to liver by gene therapy with recombinant CBD- HepII polypeptide of fibronectin[J].Int J Cancer. 2007, 121(1):184- 92.
[22]Kapila YL, Wang S, Dazin P,et al.The heparin-binding domain and V region of fibronectin regulate apoptosis by suppression of p53 and c-myc in human primary cells[J].Biol Chem. 2002,277(10):8482-91.
[23]Arciola CR,Bustanji Y,Conti M,et al.Staphylococcus epidermidis- fibronectin binding andits inhibition by heparin.Biomaterials[J]. 2003,24(18):3013-9.
[24]Dabo SM, Confer AW, Anderson BE,et al Bartonella henselae Pap31, an extracellular matrix adhesin, binds the fibr- onectin repeat III13 moduleInfect[J].Immun,2006,74 (5): 2513-21.
[25]Naito M.Fukuda T.Sekiguchi K,et al.The domains of human fibronectin mediating the binding of alpha antigen,the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterialinfection[J].Bio chemical Journal, 2000,347(3):725-31.
[26]Emma Andersson,Victoria Rydengard,Andreas Sonesson,et al. Antimicrobial activities of heparin-binding peptides[J].Eur, Biochem,2004,271:1219–1226.
[27]Verfaillie CM, Benis A,Iida J,et al.Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain offironectin between the integrin alpha 4 beta and the CD44 adhesion receptor[J].BLOOD,1994,15180-2.
[28]Stack MS,Pizzo SV.Modulation of tissue plasminogen activator catalyzed plasminogen activation by synthetic peptides derived from the amino-terminal heparin binding domain of fibronectin[J].BiolChem.1993,268(25): 18924-8.
[29]Jalkanen S, Jalkanen M.Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin[J]. Cell Biol.1992, 116(3): 817-25.
[30]Yasuda T,Kakinuma T,Julovi Sm,et al.COOH-terminal heparin-binding fibronectin fragment induces nitric- oxide production in rheumatoid cartilage through CD44[J].Rheumatology(Oxford), 2004,43(9): 1116-20.
[31]Sakamoto S,Okanoue T,Itoh Y,et al.Involvement of Kupffer cells in the interaction between neutrophils and sinusoidal endothelial cells in rats[J].Shock,2002,18:152-157.
[32]Ozmen L,Pericin M,Hakimi J,et al.Interleukin 12,interf- eron,and tumor necrosis factor are the key cytokines of the generated Shwarzman reaction[J].Exp Med.1994,180: 907-915.
[33]Satoshi GD,Takashi KE,Statoshi NK et al.Dissemianate intravascular coagulation is a frequent complication of systemic inflammatory response syndrome[J].Throm and Hemost. 1996,75(2):224-228.
[34]Mignon A, Rouquet N, Fabre M,et al.LPS challenge in D-galactosamine -sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock. Am J Respir Crit Care Med.1999,159:1308-1315.
[35]Bohlinger I, Leist M, Gantner F,et al.DNA fragmentation in mouse organs during endotoxic shock[J].Am Pathol,1996, 149:1381-1393.
[36]Leist M,Gantner F,Bohlinger I,et al.Tumor necrosis factor-[alpha] induced hepatocyte apoptosis precedes liver failure in experimental murine shock models[J].Am Pathol,1995,146:1220-1234.
1. Ruiz G, Veiga J, Luisa GLM,et al.Plasmatic concentrations of fibronectin as marker of clinical course among septic patients[J].Enferm Infecc Microbiol Clin, 2001, 19:93-98.
1. Saba TM ,Jaffe E. Plasma fibronectin(opsonic glycoprotein ):its synthesis by vascular endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function[J].AM J Med,1980,68(4):577-594.
2. Bortolussi R, Rajaraman k, Qing GF,et al .Fibronectin enhances in vitro lipopolysaccharide peiming of polymorphonuclear leukocytes[J].Blood ,1997,89(11):4182-4189.
3. Everitt EA,Malik AB,Hendey B, et al. Fibronectin enhances the migration rate of human neutrophils in vitro[J].Leukoc Biol,1996,60:199-206.
4. Oritiz A, Alonso J,Gomez-chiarri M,et al.Fibronectin(FN)decreases glomerular lesions and synthesis of tumour necrosis factor–alpha(TNF-alpha)platelet activating factor(PAF)and FN in proliferative glomerulonephritis[J].Clin Exp Immunol,1995,101(2)334-340.
5. Resnikoff M,Brien T ,Vincent PA,et al.Lung matrix incorporation of pasma fibronectin rdduces vascular permeability in postsurgical bacteremia[J].Am J Plysiol,1999,277:749-759.
7. Nooteboom A,Hendriks T,Otteholler I,et al .Permeability characteristics of human ehdothelial monolayers seeded on different extracellular mateix proteins[J].Mediators Inflamm,2000,9(5)235-241.
8.Takamichi Saito, A-HON Kwon,Zwyu Qiu et al。Protective Effect of Fibronectin for Endotoxin Induced Liver Injury after Partial Hepatectomy in Rats [J].Journal of Surgical Research,2005,124, 79–84.
9. Ruiz Martín G, Prieto Prieto J, et al.Plasma fibronectin as a marker of sepsis[J].Int J Infect Dis. 2004 ,8(4):236-43.
10. 陈元仲,吕联煌,张学敏等。血液疾病血浆纤维结合蛋白水平的研究[J]。中国实验临床免疫学杂志,1991,3(4):30-32。
11.王光,李立红,王玉兰等。严重感染患儿血浆纤维连接蛋白的变化及替代疗法观察[J]。中国实用儿科杂志,2000,15(11)。
12.Barkaga ZS,Shoikhetla N,Elykomov VA,et al。Comparative data on the use of a cryosupernatant and fresh-frozen plasma in the therapy of a protracted infectious-septic syndrome of disseminated intravascular coagulation[J].Ter Arkh,1998,70(7):70-72。
13.吴勇, 陈元仲, 吕联煌。纤维连接蛋白对内毒素血症小鼠保护作用的实验研究中国病理生理杂志[J]。2003 ,19(6): 799–802。
14.A.-H. Kwon, Z. Qiu, H. Nagahama, M. Kaibori, and Y. Kamiyama Fibronectin Suppresses Apoptosis and Protects Mice From Endotoxic Shock[J]. Transplantation Proceedings, 2004,36:2432–2435.
15.Sakai T, Johnson KJ, Murozono M, Fassler R, et al. Plasma fibronectin supports neuronal survival andreduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis[J]. Nat. Med. 2001,7: 324.