摘要
研究背景和目的心血管疾病是严重危害人类健康的常见病。一氧化氮介导的内皮依赖性血管舒张反应降低是大多数心血管疾病的早期病理表现。大量研究表明,内源性一氧化氮合酶(NOS)抑制物—非对称性二甲基精氨酸(ADMA)是导致血管内皮功能不全的重要物质。二甲基精氨酸二甲胺水解酶(DDAH)是内源性ADMA的主要代谢酶。已知DDAH存在两种亚型——DDAH1和DDAH2;前者主要分布于表达神经型NOS的组织,后者则主要分布于表达内皮型NOS的心血管组织;因此,DDAH2是决定心血管系统ADMA含量的关键因素。但目前尚无直接增加DDAH2表达和活性的药物。近年来发现一种来源于人类免疫缺陷病毒HIV-1 Tat(Trans-activator transcription)蛋白的蛋白功能区,称之为PTD区段(Protein transduction domain),能有效地引导肽段或蛋白质进入细胞,具有蛋白传送功能。因此,本课题将Tat PTD区段融合于人DDAH2蛋白的N端,使DDAH2蛋白具备穿膜能力,定位于细胞内并发挥作用。在此基础上,用多种心血管的危险因子如高糖、同型半胱氨酸、游离脂肪酸如棕榈酸处理培养的人脐静脉内皮细胞系(HUVEC-12),观察上述因素对细胞内DDAH1和DDAH2表达及DDAH活性的影响;并进一步探讨外源性给予Tat-hDDAH2蛋白能否直接增加细胞中DDAH2蛋白含量,抵抗上述因素对DDAH活性的抑制,从而为大规模开发可应用的重组蛋白产品奠定技术基础,为心血管疾病防治拓开新途径。另一方面,用重组的DDAH2融合蛋白作为抗原,制备家兔来源的抗血清,用于DDAH2表达的检测。
方法①构建重组pGEX-6p-1-Tat-hDDAH2原核表达载体:以我室以前构建pcDNA3.1-hDDAH2质粒为模板,用Tat-hDDAH2上下游引物扩增Tat-hDDAH2基因,重组到T载体上,构建pGEM-Tat-hDDAH2质粒,然后再分别用SalⅠ和BamHⅠ双酶切pGEM-Tat-hDDAH2和原核表达载体pGEX-6p-1,连接重组成pGEX-6p-1-Tat-hDDAH2载体;再转化到宿主大肠杆菌BL21体内诱导表达,并通过优化诱导条件提高目的基因的原核表达水平和可溶性融合蛋白的含量。②重组Tat-hDDAH蛋白的纯化:将包涵体形式的重组蛋白溶于8mol/L的尿素溶液中,然后经过复性、透析得到复性蛋白GST-Tat-hDDAH;再将从细菌裂解液上清中的可溶性GST-Tat-hDDAH蛋白和从包涵体中得到的复性蛋白与还原型谷胱甘肽琼脂糖珠(Glutathione Sepharose 4B)充分混匀,经过Glutathione Sepharose4B的亲和吸附得到纯化的GST-Tat-hDDAH2融合蛋白,最后经过PreScission蛋白酶切,获得重组的Tat-hDDAH2蛋白。③抗hDDAH2抗血清的制备:重组pGEX-6p-1-hDDAH2载体;再转化到宿主细菌体内进行诱导表达,得到大量含GST-hDDAH2蛋白的包涵体;经2%的脱氧胆酸钠溶液洗涤后得到较纯化的包涵体,并将其作为抗原与佐剂充分乳化后经皮下注射免疫新西兰大白兔,制备抗DDAH2的多克隆抗体,并用ELISA、western blot检测DDAH2抗体的滴度和特异性,用于后续实验中DDAH2蛋白表达的检测。④重组Tat-hDDAH2蛋白的功能实验:培养人脐静脉内皮细胞系HUVEC-12,分别用心血管疾病的危险因子高糖、同型半胱氨酸、游离脂肪酸如棕榈酸(palmitic acid,PA)孵育HUVEC-12 16～48h,检测这些危险因子对内皮细胞DDAH1和DDAH2蛋白表达和DDAH活性的影响;并观察Tat-hDDAH2蛋白能否逆转上述因素对DDAH活性的损害。
结果①经PCR、酶切及测序鉴定,原核表达载体pGEX-6p-1-Tat-hDDAH2(Fig.1～3)和pGEX-6p-1-hDDAH2(Fig.4)构建成功;并转化BL21大肠杆菌诱导表达,通过筛选不同的诱导条件,发现用较低的IPTG浓度(0.05 mM)在低温(25℃)环境下和适当地延长诱导时间(8 h)有利于可溶性融合蛋白GST-Tat-hDDAH2(Fig.5～7)和GST-hDDAH2(Fig.9～11)在细菌体内的表达,但在原核细胞中仍以不溶性的包涵体为主;将包涵体变性、复性后,再经纯化和PreScission蛋白酶酶切,最后获得重组的Tat-hDDAH2(Fig.8)和hDDAH2蛋白(Fig.12)。②将1升含有pGEX-6p-1-Tat-hDDAH2质粒和pGEX-6p-1-hDDAH2质粒的细菌培养液于12000 rpm 4℃离心10 min,去上清,将细菌用100 mlPBS重悬;经超声裂解后于12000 rpm 4℃离心10 min,分离上清中的可溶性融合蛋白与包涵体;将包涵体变性、复性后,分别将包涵体中复性的融合蛋白和上清中可溶性的融合蛋白经Glutathione Sepharose 4B亲和吸附,得到纯化的GST-Tat-hDDAH2融合蛋白2.169 mg和纯化的GST-hDDAH2融合蛋白2.571 mg(Tab.1～2);再分别将GST-Tat-hDDAH2和GST-hDDAH2融合蛋白经PreScission蛋白酶酶切后,可得到总量为1.362 mg的Tat-hDDAH2重组蛋白和1.469 mg的DDAH2重组蛋白(Tab.1～2)。③用含GST-hDDAH2蛋白的包涵体免疫新西兰兔,10 w后用乌拉坦(0.8 g/kg,i.v.)麻醉,经颈动脉插管取血,3000 rpm 4℃离心10 min分离血清,加入终浓度为0.02%的叠氮钠,从而获得特异性抗hDDAH2、效价达1:10000以上的多克隆抗体(Fig.13～14),分装后于-80℃保存,用于本课题中重组DDAH2蛋白、重组Tat-hDDAH2蛋白的检测以及内皮细胞中的DDAH2蛋白表达测定。④分别用0.1～1μM的Tat-hDDAH2蛋白孵育内皮细胞60 min,检测内皮细胞中DDAH2的表达及DDAH活性,以了解Tat-hDDAH2蛋白作用的量-效关系,结果发现,Tat-hDDAH2蛋白呈剂量依赖性地上调DDAH2表达,增加DDAH活性(Fig.15);再分别用1μM的Tat-hDDAH2蛋白孵育内皮细胞5～60 min,以观测Tat-hDDAH2蛋白作用的时-效关系,同样发现,Tat-hDDAH2蛋白呈时间依赖性地上调DDAH2表达,增加DDAH活性(Fig.16)。此外,用10～30mM的葡萄糖孵育内皮细胞48 h,以及用1～3 mM的同型半胱氨酸和0.1～0.5 mM的棕榈酸孵育内皮细胞16 h,均能呈剂量依赖性地抑制DDAH2表达(Fig.17,19),降低DDAH活性(Fig.18,20);用0.1～0.5μM的Tat-hDDAH2蛋白预孵育内皮细胞1 h,能逆转上述损伤因素对DDAH2表达和DDAH活性的抑制(Fig.17～20)。
结论①利用分子重组技术,成功地构建了pGEX-6p-1-Tat-hDDAH2和pGEX-6p-1-hDDAH2原核表达载体;②分别将pGEX-6p-1-Tat-hDDAH2和pGEX-6p-1-hDDAH2载体转化BL21大肠杆菌,经纯化后获得了较高纯度的Tat-hDDAH2和hDDAH2重组蛋白,实验证明,重组的Tat-hDDAH2具有强的穿膜能力,并呈剂量和时间依赖性地增加内皮细胞中DDAH2的蛋白含量,上调DDAH活性,具有良好的功能;③利用重组的GST-hDDAH2包涵体蛋白免疫新西兰大白兔,成功地制备了高效价的抗hDDAH2的多克隆抗体,并能特异性地检测重组的GST-hDDAH2、Tat-hDDAH2和hDDAH2以及人脐静脉内皮细胞表达的DDAH2蛋白;④本研究揭示,高糖、同型半胱氨酸和游离脂肪酸如棕榈酸等心血管危险因子抑制内皮细胞DDAH活性主要与下调DDAH2表达有关;重组的Tat-hDDAH2蛋白能呈剂量依赖性地逆转上述损伤因子对人脐静脉内皮细胞DDAH2表达和DDAH活性的抑制。
BACKGROUND Cardiovascular diseases,as the most common diseases of human being,have become one of the leading causes of death in developed countries as well as in many developing countries.Endothelial dysfunction, mainly characterized as reduction of nitric oxide(NO)-mediated endothelium-dependent vasodilatation,tends to precede diversed clinical manifestations of most cardiovascular diseases and to be an early pathological event.A substantial body of evidence suggests that elevated levels of endogenous nitric oxide synthase(NOS)inhibitor asymmetric dimethylarginine(ADMA)is involved in the mechanisms of endothelial dysfunction for cardiovascular diseases.Contribution of elevated endogenous ADMA to endothelial dysfunction predominantly lies in the decreased expression or activity of dimethylarginine dimethylaminohydrolase 2 (DDAH2),which is ADMA's main metabolic enzyme and extensively located in endothelial cells.DDAH is composed of 2 isoforms,DDAH1 and DDAH2. DDAH1 is typically found in tissues of expressing neuronal NOS,whereas DDAH2 predominates in cardiovascular tissues of expressing endothelial NOS. So DDAH2 play a pivotal role in modulating ADMA metabolism in cardiovascular system.However,no medicine has ever been found to directly increase expression and activity of DDAH2.Until recently,studies have identified a protein domain,namely protein transduction domain(PTD),from human immunodeficiency virus tans-activator transcription protein(HIV-1 Tat),which can efficiently conduct peptides or proteins into living cell,thus playing a role of protein transduction.The objectives of this study are firstly to fuse Tat PTD to the N-terminal of DDAH2 protein,so as to enable DDAH2 protein to possess the ability of go through cell membrane and produce effects inside cells.To go a step further,we will also study effects of cardiovascular risks such as high glucose,homocystein and palmitic acid on expressions of DDAH1 and DDAH2 protein and activities of DDAH in cultured human umbilical vein endothelial cells(HUVEC-12)pretreatmented with or without Tat-hDDAH2 protein.Our work will provide a potential therapeutic method of treating cardiovascular diseases with recombined protein products.Moreover, we will prepare the anti-serum from New Zealand rabbits against recombination of GST-hDDAH2 fusion protein for the detection of DDAH2 protein expression by western blotting in this study.
METHODS①Construction of recombination prokaryotic expression vector of pGEX-6p-1-Tat-hDDAH2:Taken the plasmid pcDNA3.1-hDDAH2 as template,we amplified Tat-hDDAH2 gene using the forward and reverse primers of Tat-hDDAH2,and ligated it with T-vector to construct pGEM-Tat-hDDAH2 plasmid.The plasmid and prokaryotic expression vector pGEX-6p-1 were digested with SalⅠand BamHⅠfor over night,respectively. We ligated the incised plasmid and vector to recombinate the vector of pGEX-6p-1-Tat-hDDAH2,which were then transformed into host bacteria E coli BL21 to induce maximal expression of the targeted gene and produce soluble fusion proteins in optimized conditions.②Purification of Tat-hDDAH recombination protein:First,recombinant proteins in form of inclusion bodies were dissolved in urea solution(8 M)and then after renaturation and dialysis, the renaturated protein GST-Tat-hDDAH was achieved.Nextly,the soluble GST-Tat-hDDAH protein in the supernatant of the bacterial lysate and the renaturated protein collected from the inclusion bodies were thoroughly mixed with Glutathione Sepharose 4B and then get purified by the affinity adsorption. Finally,the purified GST-Tat-hDDAH2 protein was incised by PreScission protease,and recombinant protein Tat-hDDAH2 was obtained.③Preparation of anti-hDDAH2 anti-serum:After recombination and transformation of pGEX-6p-1-hDDAH2 vector,the host bacteria were induced to express and produce a great quantity of inclusion bodies containing GST-hDDAH2 protein.The inclusion bodies washed by 2%sodium deoxycholate solution was thoroughly emulsified with the adjuvant and used to immunize New Zealand rabbits by subcutaneous injections.After the anti-DDAH2 polyclonal antibody was prepared,its titer and specificity were determined by ELISA and western blotting,respectively.We used it to detect expression of DDAH2 protein in the present study.④Functional study of Tat-hDDAH2 recombination protein:The human umbilical venous endothelial cell line HUVEC-12 were cultured and incubated with different risk factors of cardiovascular diseases,including high glucose,homocysteic acid and free fatty acid such as palmitic acid(PA)in the absence or presence of Tat-hDDAH2 protein for 16～48 h.Expressions of DDAH1 and DDAH2 proteins,as well as activities of DDAH,were detected to review the important role of DDAH2 in endothelial function and to explore the potential protective effects of Tat-hDDAH2 protein against these risk factors.
RESULTS①As identified by PCR,digestion and sequencing,prokaryotic expression vectors of both pGEX-6p-1-Tat-hDDAH2(Fig.1～3)and pGEX-6p-1-hDDAH2(Fig.4)were successfully constructed and then transformed to E.coli BL21,in which were efficiently amplified to express soluble fusion proteins of GST-Tat-hDDAH2(Fig.5～7)and GST-hDDAH2 (Fig.9～11)by using a low concentration of IPTG(0.05 mM),at low temperature(25℃)and for a extended cultural time(8 h).However,in prokaryotic cells,the amplified fusion proteins occur in the form of insoluble inclusion bodies predominantly.So the inclusion bodies were denaturated, renaturated,purified and incised by PreScission protease to obtain the Tat-hDDAH2(Fig.8)and DDAH2(Fig.12)recombination proteins finally.
②One liter of bacterial culture solution containing pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-DDAH2 plasmids was centrifuged at 12000 rpm,4℃,for 10 min.The supernatant was discarded and the pellet was resuspended in 100 ml PBS.After spallation by ultrasound,the resuspension solution was centrifuged at 12000 rpm,4℃for 10 min to separate the soluble protein in supernatant and insoluble inclusion bodies.After the inclusion bodies were denaturated, renaturated,the renaturated protein collected from the inclusion bodies and the soluble fusion protein in the supernatant were purified by affinity adsorption with Glutathione Sepharose 4B to get 1.545 mg of purified GST-Tat-hDDAH2 protein and 1.735 mg of GST-hDDAH2 fusion protein(Tab.1～2).These proteins were incised by PreScission protease to obtain 1.085 mg of recombinant Tat-hDDAH2 protein and 1.154 mg of recombinant hDDAH2 protein(Tab.1～2).③After immunizating New Zealand rabbits with inclusion bodies containing GST-hDDAH2 for 10 weeks,we obtained the polyclonal antibody against human DDAH2 protein in a titer of 1:10000 by centrifuging the blood from the rabbits.In addtion,we used this anti-serum for measurement of recombinant DDAH2 protein,recombinant Tat-hDDAH2 proteins and expression of DDAH2 protein in HUVEC-12.④Incubation of HUVEC-12 with 0.1～1μM Tat-hDDAH2 for 60 min,the expression of DDAH2 and activity of DDAH in the HUVEC-12 were examined to see the dose-response of Tat-hDDAH2.The results showed that Tat-hDDAH2 upregulated DDAH2 expression and enhanced DDAH activity in HUVEC-12 in a dose-dependent manner(Fig.15).Moreover,the time course for Tat-hDDAH2 action was checked by treatment of endothelial cells with 1μM Tat-hDDAH2 for 5～60 min.Similiarly,Tat-hDDAH2 upregulated DDAH2 expression and enhanced DDAH activity in HUVEC-12 in a time-dependent manner.However,incubation of endothelial cells with 10～30 mM glucose for 48 h or with 1～3 mM Hcy and 0.1～0.5 mM PA for 16 h,not only DDAH2 expression was suppressed(Fig.17,19)but also DDAH activity was inhibited (Fig.18,20)in a dose-dependent manner.Pretreatment with 0.1～0.5μM Tat-hDDAH2 for 1 h could protect endothelial cells from the deleterious effect of these risk factors for cardiovascular deseases on DDAH2 expression and DDAH activity.
CONCLUSIONS①The construction of prokaryotic expression vectors pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-Tat-hDDAH2 was achieved.②The prokaryotic expression vector of pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-Tat-hDDAH2 was transformed into Escherichia coli BL21.After purification,GST-hDDAH2 and GST-Tat-hDDAH2 recombination proteins in high purity were obtained.Further experiments confirmed that Tat-hDDAH2 protein can not only cross biological membranes efficiently but also prossess favourable function of up-regulating the DDAH2 expression and DDAH activity in endothelial cell in time- and dose-dependent manner.③Polyclonal antibody agaist hDDAH2 with a high potency was prepared successfully by immunizating New Zealand rabbits with inclusion bodies containing GST-hDDAH2.This antibody can be used to detect recombination GST-hDDAH2,Tat-hDDAH2 hDDAH2 protein and DDAH2 expressing in human umbilical vein endothelial cells.④This study showed that reductions of DDAH activity induced by cardiovascular risk factors such as high glucose, homocystein and free fatty acids attributed to down-regulation of expression of DDAH2.Tat-hDDAH2 can protect endothelial cells from the deleterious effect of these risk factors for cardiovascular deseases on DDAH2 expression and DDAH activity in in time- and dose-dependent manner.
引文
1. Suwaidi JA, Hamasaki S, Higano ST, Nishimura RA, Holmes DR Jr, Lerman A. Long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction. Circulation 2000;101(9):948-54.
2. Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 2001;104(22):2673-8.
3. Vallance P, Leone A, Calver A, Collier J, Moncada S. Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. J Cardiovasc Pharmacol 1992;20 Suppl 12.S60-2.
4. Yu XJ, Li YJ, Xiong Y. Increase of an endogenous inhibitor of nitric oxide synthesis in serum of high cholesterol fed rabbits. Life Sci 1994;54(12):753-8.
5. Fu YF, Xiong Y, Fu SH. Captopril restores endothelium-dependent relaxation of rat aortic rings after exposure to homocysteine. J Cardiovasc Pharmacol 2003;42(4):566-72.
6. Xiong Y, Lu R, Li YJ, Deng HW. Elevation of an endogenous inhibitor of nitric oxide synthase in diabetic rat serum. Zhongguo Yao Li Xue Bao 1997; 18(6):511-4.
7. Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M, et al. Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation 2002;106(8):987-92.
8. Tarnow L, Hovind P, Teerlink T, Stehouwer CD, Parving HH. Elevated plasma asymmetric dimethylarginine as a marker of cardiovascular morbidity in early diabetic nephropathy in type 1 diabetes. Diabetes Care 2004;27(3):765-9.
9. Jiang DJ, Jia SJ, Dai Z, Li YJ. Asymmetric dimethylarginine induces apoptosis via p38 MAPK/caspase-3-dependent signaling pathway in endothelial cells. J Mol Cell Cardiol 2006.
10. Wells SM, Holian A. Asymmetric dimethylarginine induces oxidative and nitrosative stress in murine lung epithelial cells. Am J Respir Cell Mol Biol 2007;36(5):520-8.
11. Antoniades C, Tousoulis D, Marinou K, Vasiliadou C, Tentolouris C, Bouras G, et al. Asymmetrical dimethylarginine regulates endothelial function in methionine-induced but not in chronic homocystinemia in humans: effect of oxidative stress and proinflammatory cytokines. Am J Clin Nutr 2006;84(4):781-8.
12. Smirnova IV, Kajstura M, Sawamura T, Goligorsky MS. Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation. Am J Physiol Heart Circ Physiol 2004;287(2):H782-90.
13. Tang J, Frankel A, Cook RJ, Kim S, Paik WK, Williams KR, et al. PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells. J Biol Chem 2000;275(11):7723-30.
14. Ogawa T, Kimoto M, Sasaoka K. Occurrence of a new enzyme catalyzing the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline in rats. Biochem Biophys Res Commun 1987;148(2):671-7.
15. Xiao S, Wagner L, Schmidt RJ, Baylis C. Circulating endothelial nitric oxide synthase inhibitory factor in some patients with chronic renal disease. Kidney Int 2001 ;59(4): 1466-72.
16. Chen X, Niroomand F, Liu Z, Zankl A, Katus HA, Jahn L, et al. Expression of nitric oxide related enzymes in coronary heart disease. Basic Res Cardiol 2006.
17. Okubo K, Hayashi K, Wakino S, Matsuda H, Kubota E, Honda M, et al. Role of asymmetrical dimethylarginine in renal microvascular endothelial dysfunction in chronic renal failure with hypertension. Hypertens Res 2005;28(2): 181-9.
18. Dayoub H, Achan V, Adimoolam S, Jacobi J, Stuehlinger MC, Wang BY, et al. Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence. Circulation 2003; 108(24): 3042-7.
19. Leiper J, Nandi M, Torondel B, Murray-Rust J, Malaki M, O'Hara B, et al. Disruption of methylarginine metabolism impairs vascular homeostasis. Nat Med 2007;13(2):198-203.
20. Konishi H, Sydow K, Cooke JP. Dimethylarginine dimethylaminohydrolase promotes endothelial repair after vascular injury. J Am Coll Cardiol 2007;49(10): 1099-105.
21. Smith CL, Birdsey GM, Anthony S, Arrigoni FI, Leiper JM, Vallance P. Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype. Biochem Biophys Res Commun 2003;308(4):984-9.
22. Hasegawa K, Wakino S, Tanaka T, Kimoto M, Tatematsu S, Kanda T, et al. Dimethylarginine Dimethylaminohydrolase 2 Increases Vascular Endothelial Growth Factor Expression Through Sp1 Transcription Factor in Endothelial Cells. Arterioscler Thromb Vasc Biol 2006.
23. Valkonen VP, Tuomainen TP, Laaksonen R. DDAH gene and cardiovascular risk. Vasc Med 2005;10 Suppl 1:S45-8.
24. Tojo A, Welch WJ, Bremer V, Kimoto M, Kimura K, Omata M, et al. Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. Kidney Int 1997;52(6):1593-601.
25. Pullamsetti S, Kiss L, Ghofrani HA, Voswinckel R, Haredza P, Klepetko W, et al. Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension. FASEB J 2005; 19(9): 1175-7.
26. Chen Y, Li Y, Zhang P, Traverse JH, Hou M, Xu X, et al. Dimethylarginine dimethylaminohydrolase and endothelial dysfunction in failing hearts. Am J Physiol Heart Circ Physiol 2005;289(5):H2212-9.
27. Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 1997;272(25): 16010-7.
28. Wadia JS, Dowdy SF. Protein transduction technology. Curr Opin Biotechnol 2002;13(1):52-6.
29. Park J, Ryu J, Kim KA, Lee HJ, Bahn JH, Han K, et al. Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells. J Gen Virol 2002;83(Pt 5): 1173-81.
30. Eum WS, Jang SH, Kim DW, Choi HS, Choi SH, Kim SY, et al. Enhanced transduction of Cu,Zn-superoxide dismutase with HIV-1 Tat protein transduction domains at both termini. Mol Cells 2005; 19(2): 191-7.
31. Asoh S, Mori T, Nagai S, Yamagata K, Nishimaki K, Miyato Y, et al. Zonal necrosis prevented by transduction of the artificial anti-death FNK protein. Cell Death Differ 2005;12(4):384-94.
32. Mendoza V, Klein D, Ichii H, Ribeiro MM, Ricordi C, Hankeln T, et al. Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction. Transplant Proc 2005;37(1):237-40.
33．金伯泉．细胞和分子免疫学实验技术．西安：第四军医大学出版社,2004．45．
34. Krzyzanowska K, Mittermayer F, Krugluger W, Schnack C, Hofer M, Wolzt M, et al. Asymmetric dimethylarginine is associated with macrovascular disease and total homocysteine in patients with type 2 diabetes. Atherosclerosis 2006.
35. Leiper JM, Santa Maria J, Chubb A, MacAllister RJ, Charles IG, Whitley GS, et al. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases. Biochem J 1999;343 Pt 1:209-14.
36．周宇荀,魏东芝,王二力．融合蛋白表达载体pGEX及其应用．生命科学 1998；10(3)：122-124．
37. Yu L, Deng K, Yu CA. Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase. J Biol Chem 1995;270(43):25634-8.
38. Kimoto M, Miyatake S, Sasagawa T, Yamashita H, Okita M, Oka T, et al. Purification, cDNA cloning and expression of human NG,NG-dimethylarginine dimethylaminohydrolase. Eur J Biochem 1998;258(2):863-8.
39. McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 1990;348(6301):552-4.
40. Mann DA, Frankel AD. Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J 1991;10(7): 1733-9.
41. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285(5433):1569-72.
42. Stuhlinger MC, Stanger O. Asymmetric dimethyl-L-arginine (ADMA): a possible link between homocyst(e)ine and endothelial dysfunction. Curr Drug Metab 2005;6(1):3-14.
43. Tyagi N, Collins K, Steed MM, Ovechkin AV, Moshal KS, Tyagi SC. Mechanisms of Homocysteine-Induced Oxidative Stress. Am J Physiol Heart Circ Physiol 2005.
44. Stuhlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF, Cooke JP. Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine. Circulation 2001;104(21):2569-75.
45. Mikael LG, Genest J Jr, Rozen R. Elevated homocysteine reduces apolipoprotein A-I expression in hyperhomocysteinemic mice and in males with coronary artery disease. Circ Res 2006;98(4): 564-71.
46. Jones LC, Tran CT, Leiper JM, Hingorani AD, Vallance P. Common genetic variation in a basal promoter element alters DDAH2 expression in endothelial cells. Biochem Biophys Res Commun 2003;310(3):836-43.
47. Yu YR, Li HL, Yu HL, Wang C, Pu S. [The relationship between insulin resistance and endothelium-dependent vasodilatation in obese subjects]. Zhonghua Yi Xue Za Zhi2003;83(17): 1467-70.
48. Steinberg HO, Tarshoby M, Monestel R, Hook G, Cronin J, Johnson A, et al. Elevated circulating free fatty acid levels impair endothelium-dependent vasodilation. J Clin Invest 1997;100(5): 1230-9.
49. Mittermayer F, Schaller G, Pleiner J, Krzyzanowska K, Kapiotis S, Roden M, et al. Rosiglitazone prevents free fatty acid-induced vascular endothelial dysfunction. J Clin Endocrinol Metab 2007.
50. Namiranian K, Mittermayer F, Artwohl M, Pleiner J, Schaller G, Mayer BX, et al. Free fatty acids do not acutely increase asymmetrical dimethylarginine concentrations. Horm Metab Res 2005;37(12):768-72.
51. Murray-Rust J, Leiper J, McAlister M, Phelan J, Tilley S, Santa Maria J, et al. Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase. Nat Struct Biol 2001;8(8):679-83.
52. Leiper J, Murray-Rust J, McDonald N, Vallance P. S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase. Proc Natl Acad Sci U S A 2002;99(21):13527-32.
53. Jiang JL, Zhu HQ, Chen Z, Xu HY, Li YJ. Angiotensin-converting enzyme inhibitors prevent LDL-induced endothelial dysfunction by reduction of asymmetric dimethylarginine level. Int J Cardiol 2005;101(1): 153-5.
54. Holden DP, Cartwright JE, Nussey SS, Whitley GS. Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine. Circulation 2003;108(13): 1575-80.
55. Selley ML. Homocysteine increases the production of asymmetric dimethylarginine in cultured neurons. J Neurosci Res 2004;77(1):90-3.
56. Wakino S, Hayashi K, Tatematsu S, Hasegawa K, Takamatsu I, Kanda T, et al. Pioglitazone lowers systemic asymmetric dimethylarginine by inducing dimethylarginine dimethylaminohydrolase in rats. Hypertens Res 2005;28(3):255-62.
57. Achan V, Tran CT, Arrigoni F, Whitley GS, Leiper JM, Vallance P. all-trans-Retinoic acid increases nitric oxide synthesis by endothelial cells: a role for the induction of dimethylarginine dimethylaminohydrolase. Circ Res 2002;90(7):764-9.
1. Park J, Ryu J, Kim KA, Lee HJ, Bahn JH, Han K, et al. Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells. J Gen Virol 2002;83(Pt 5):1173-81.
2. Ziegler A, Nervi P, Durrenberger M, Seelig J. The cationic cell-penetrating peptide CPP(TAT) derived from the HTV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence. Biochemistry 2005;44(1): 138-48.
3. Chauhan A, Turchan J, Pocernich C, Bruce-Keller A, Roth S, Butterfield DA, et al. Intracellular human immunodeficiency virus Tat expression in astrocytes promotes astrocyte survival but induces potent neurotoxicity at distant sites via axonal transport. J Biol Chem 2003;278(15):13512-9.
4. Tasciotti E, Zoppe M, Giacca M. Transcellular transfer of active HSV-1 thymidine kinase mediated by an 11-amino-acid peptide from HIV-1 Tat. Cancer Gene Ther 2003;10(1):64-74.
5. Weinberger LS, Burnett JC, Toettcher JE, Arkin AP, Schaffer DV. Stochastic gene expression in a lentiviral positive-feedback loop: HIV-1 Tat fluctuations drive phenotypic diversity. Cell 2005;122(2):169-82.
6. Michienzi A, Li S, Zaia JA, Rossi JJ. A nucleolar TAR decoy inhibitor of HIV-1 replication. Proc Natl Acad Sci U S A 2002;99(22): 14047-52.
7. Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 1997;272(25): 16010-7.
8. Soane L, Fiskum G. TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death. J Neurochem 2005;95(1):230-43.
9. Mann DA, Frankel AD. Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J 1991;10(7): 1733-9.
10. Barka T, Gresik EW, van Der Noen H. Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo. J Histochem Cytochem 2000;48(11): 1453-60.
11. Hidema S, Maruyama T, Kato S, Nishimori K. Highly induced DNA recombination mediated by membrane permeabilized recombinant cre protein in mouse primary cells. Biosci Biotechnol Biochem 2007;71(3):817-20.
12. Toro A, Grunebaum E. TAT-mediated Intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice. J Clin Invest 2006;116(10):2717-26.
13. Jalota-Badhwar A, Kaul-Ghanekar R, Mogare D, Boppana R, Paknikar KM, Chattopadhyay S. Smar1 derived p44 peptide retains its tumor suppressor function through modulation of p53. J Biol Chem 2007.
14. Abu-Amer Y, Dowdy SF, Ross FP, Clohisy JC, Teitelbaum SL. TAT fusion proteins containing tyrosine 42-deleted IkappaBalpha arrest osteoclastogenesis. J Biol Chem 2001;276(32):30499-503.
15. Astriab-Fisher A, Sergueev D, Fisher M, Shaw BR, Juliano RL. Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions. Pharm Res 2002; 19(6):744-54.
16. Polyakov V, Sharma V, Dahlheimer JL, Pica CM, Luker GD, Piwnica-Worms D. Novel Tat-peptide chelates for direct transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy. Bioconjug Chem 2000;11(6):762-71.
17. Wunderbaldinger P, Josephson L, Weissleder R. Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles. Bioconjug Chem 2002;13(2):264-8.
18. Dodd CH, Hsu HC, Chu WJ, Yang P, Zhang HG, Mountz JD Jr, et al. Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticles. J Immunol Methods 2001;256(1-2):89-105.
19. Marty C, Meylan C, Schott H, Ballmer-Hofer K, Schwendener RA. Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes. Cell Mol Life Sci 2004;61(14): 1785-94.
20. Hyndman L, Lemoine JL, Huang L, Porteous DJ, Boyd AC, Nan X. HIV-1 Tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes. J Control Release 2004;99(3):435-44.
21. Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci U S A 2000;97(24):13003-8.
22. Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB. Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 2000;56(5):318-25.
23. Rusnati M, Taraboletti G, Urbinati C, Tulipano G, Giuliani R, Molinari-Tosatti MP, et al. Thrombospondin-1/HIV-1 tat protein interaction: modulation of the biological activity of extracellular Tat. FASEB J 2000;14(13): 1917-30.
24. Tyagi M, Rusnati M, Presta M, Giacca M. Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J Biol Chem 2001;276(5):3254-61.
25. Silhol M, Tyagi M, Giacca M, Lebleu B, Vives E. Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat. Eur J Biochem 2002;269(2):494-501.
26. Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, et al. Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J Biol Chem 2001;276(28):26204-10.
27. Ferrari A, Pellegrini V, Arcangeli C, Fittipaldi A, Giacca M, Beltram F. Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time. Mol Ther 2003;8(2):284-94.
28. Console S, Marty C, Garcia-Echeverria C, Schwendener R, Ballmer-Hofer K. Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J Biol Chem 2003;278(37):35109-14.
29. Lundberg M, Wikstrom S, Johansson M. Cell surface adherence and endocytosis of protein transduction domains. Mol Ther 2003;8(1): 143-50.
30. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003;422(6927):37-44.
31. Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 2004;10(3):310-5.
32. Nichols BJ, Lippincott-Schwartz J. Endocytosis without clathrin coats. Trends Cell Biol 2001; 11(10)406-12.
33. Liu NQ, Lossinsky AS, Popik W, Li X, Gujuluva C, Kriederman B, et al. Human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway. J Virol 2002;76(13):6689-700.
34. Nath A, Psooy K, Martin C, Knudsen B, Magnuson DS, Haughey N, et al. Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic. J Virol 1996;70(3): 1475-80.
35. Hallbrink M, Floren A, Elmquist A, Pooga M, Bartfai T, Langel U. Cargo delivery kinetics of cell-penetrating peptides. Biochim Biophys Acta 2001 ;1515(2): 101-9.
36. Jones SW, Christison R, Bundell K, Voyce CJ, Brockbank SM, Newham P, et al. Characterisation of cell-penetrating peptide-mediated peptide delivery. Br J Pharmacol 2005;145(8): 1093-102.
37. Cashman SM, Morris DJ, Kumar-Singh R. Evidence of protein transduction but not intercellular transport by proteins fused to HIV tat in retinal cell culture and in vivo. Mol Ther2003;8(1): 130-42.
38. Hu M, Chen P, Wang J, Scollard DA, Vallis KA, Reilly RM. (123)I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1). Eur J Nucl Med Mol Imaging 2007;34(3):368-77.
39. Brask J, Chauhan A, Hill RH, Ljunggren HG, Kristensson K. Effects on synaptic activity in cultured hippocampal neurons by influenza A viral proteins. J Neurovirol 2005;11(4):395-402.
40. Theisen DM, Pongratz C, Wiegmann K, Rivero F, Krut O, Kronke M. Targeting of HIV-1 Tat traffic and function by transduction-competent single chain antibodies. Vaccine 2006;24(16):3127-36.
41. Mi MY, Zhang J, He Y. Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein. Retrovirology 2005;2:71.
42. Falnes PO, Wesche J, Olsnes S. Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment. Biochemistry 2001;40(14):4349-58.
43. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285(5433): 1569-72.
44. Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for Intracellular protein delivery. J Biol Chem 2001;276(8):5836-40.
45. Leifert JA, Harkins S, Whitton JL. Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity. Gene Ther 2002;9(21): 1422-8.
46. Yang Y, Ma J, Song Z, Wu M. HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors. FEBS Lett 2002;532(1-2):36-44.
47. Ford KG, Souberbielle BE, Darling D, Farzaneh F. Protein transduction: an alternative to genetic intervention?. Gene Ther 2001;8(1):1-4.
48. Sacchetti A, Cappetti V, Marra P, Dell'Arciprete R, El Sewedy T, Crescenzi C, et al. Green fluorescent protein variants fold differentially in prokaryotic and eukaryotic cells. J Cell Biochem Suppl 2001;Suppl 36:117-28.
49. Liu J, Zhang Q, Remsen EE, Wooley KL. Nanostructured materials designed for cell binding and transduction. Biomacromolecules 2001;2(2):362-8.
50. Lindsay MA. Peptide-mediated cell delivery: application in protein target validation. Curr Opin Pharmacol 2002;2(5):587-94.
51. Sengoku T, Bondada V, Hassane D, Dubai S, Geddes JW. Tat-calpastatin fusion proteins transduce primary rat cortical neurons but do not inhibit cellular calpain activity. Exp Neurol 2004;188(1): 161-70.
52. Caron NJ, Torrente Y, Camirand G, Bujold M, Chapdelaine P, Leriche K, et al. Intracellular delivery of a Tat-eGFP fusion protein into muscle cells. Mol Ther 2001;3(3):310-8.
53. Violini S, Sharma V, Prior JL, Dyszlewski M, Piwnica-Worms D. Evidence for a plasma membrane-mediated permeability barrier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate. Biochemistry 2002;41(42):12652-61.
54. Melan MA, Sluder G. Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy. J Cell Sci 1992;101(Pt 4):731-43.
55. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, et al. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 2003 ;278(1):585-90.
56. Vives E. Present and future of cell-penetrating peptide mediated delivery systems: "is the Trojan horse too wild to go only to Troy?". J Control Release 2005;109(1-3):77-85.
57. Sandgren S, Cheng F, Belting M. Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans. J Biol Chem 2002;277(41):38877-83.
58. Kim D, Jeon C, Kim JH, Kim MS, Yoon CH, Choi IS, et al. Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo. Exp Cell Res 2006.
59. Goun EA, Pillow TH, Jones LR, Rothbard JB, Wender PA. Molecular transporters: synthesis of oligoguanidinium transporters and their application to drug delivery and real-time imaging. Chembiochem 2006;7(10): 1497-515.
60. Cai SR, Xu G, Becker-Hapak M, Ma M, Dowdy SF, McLeod HL. The kinetics and tissue distribution of protein transduction in mice. Eur J Pharm Sci 2006;27(4):311-9.
61. Chen J, Li G, Lu J, Chen L, Huang Y, Wu H, et al. A novel type of PTD, common helix-loop-helix motif, could efficiently mediate protein transduction into mammalian cells. Biochem Biophys Res Commun 2006.