HCV核心蛋白长期稳定表达小鼠模型的建立
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摘要
丙型病毒性肝炎是一种严重危害人类健康的疾病。丙型肝炎病毒感染是造成慢性肝炎,肝硬化及肝癌的主要原因之一,全球大约有1.7亿人感染HCV。目前尚无有效的防治方法,IFN-α和利巴韦林联合治疗是临床上标准的治疗方法,但只对不到50%的患者有效。
HCV是一单股正链RNA病毒,病毒基因组全长9600bp,含有一个大的开放读码框(open reading region,ORF)。它编码一个多聚蛋白前体,经宿主信号肽酶和病毒基因编码的蛋白酶切割产生4个结构蛋白(核心蛋白、包膜蛋白E1、E2和P7)和6个非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A和NS5B)。核心蛋白是HCV编码的一个重要结构蛋白,位于整个多聚蛋白的N-末端,是病毒核衣壳的组成部分,在不同型的HCV中具有高度的保守性。越来越多的研究结果表明核心蛋白与HCV感染所致肝细胞癌的发生有密切关系。但由于HCV具有极强的宿主特异性,目前对HCV及核心蛋白的机制和功能的研究多在细胞模型,缺乏合适的小动物模型阻碍了在动物整体水平上的研究。
我国HCV流行株主要为基因1b型,且感染1b型HCV患者运用IFN治疗效果较差,与肝硬化及肝癌的关系较为密切。因此研究1b型HCV核心蛋白在HCV持续感染及肝癌发病机制中的作用,对防治我国丙型肝炎具有重要的实际意义。因此本课题基于成年小鼠,联合水动力转染技术与噬菌体整合酶系统,建立1b型HCV核心蛋白长期稳定表达小鼠模型,作为经典转基因技术的补充,并初步将其用于HCV核心蛋白抑制剂-shRNAs在体内抗HCV的作用效果的评价。具体研究内容及结果如下:
1.构建HCV核心蛋白表达载体pGL3-attB-Core-Fluc。该载体含有核心蛋白基因、报告基因Fluc和?C31整合酶识别位点attB。将pGL3-attB-Core-Fluc与对照载体pGL3-attB-Core、pGL3-attB-Fluc转染Huh7细胞,pGL3-attB-Core-Fluc核心蛋白表达量与pGL3-attB-Core核心蛋白表达量是一致的,而pGL3-attB-Core-Fluc的Fluc活性却低于pGL3-attB-Fluc。在C57BL/6小鼠体内也证实了上述结果。
2.利用?C31整合酶介导位点特异性整合的特点,建立HCV核心蛋白稳定表达小鼠模型。通过水动力转染技术将核心蛋白表达载体pGL3-attB-Core-Fluc与编码?C31整合酶的表达载体pCMV-int共转染至小鼠体内。实验证实,在?C31整合酶作用下,HCV核心蛋白及报告基因Fluc整合到小鼠肝脏第2号染色体的mpsL1位点,在小鼠肝脏获得稳定表达,长达420d。
3.在上述动物模型基础上,对靶向HCV核心蛋白不同位点的shRNAs进行了体内作用效果的评价。本实验在pSilencerTM2.1-U6 neo载体基础上,构建发夹型siRNA-shRNA452、shRNA479、shRNA523表达质粒。结果显示:在细胞水平, shRNA-452、shRNA-479和shRNA-523对核心蛋白和报告基因Fluc均有抑制作用,抑制率可达到40-55%;在核心蛋白瞬时表达小鼠模型中, shRNA-Fluc和shRNA-523的抑制作用在转染后24 hr已被检测到,并且随时间的延长抑制作用更加明显。而shRNA-452在转染后48 hr还未出现抑制作用;在核心蛋白稳定表达小鼠模型中,在第6hr、12hr,shRNA523抑制Fluc的活性分别达到了76.4±26.0%和91.8±8.0%,一次注射该抑制作用持续24hr。
总之,本研究将水动力转染技术及噬菌体整合酶系统联合应用,建立了HCV核心蛋白长期稳定表达小鼠模型。利用该模型可以在小鼠体内实时监测靶向HCV核心蛋白shRNA的作用。这种新颖而简单的方法可用于评价抗HCV核酸药物。
Viral hepatitis C is a major public health concern worldwide. HCV causes chronic hepatitis, cirrhosis and liver cancer. Approximately 170 million people suffer from this infectious disease around the world. Today no therapy is proved to be enough valuable to hepatitis C treatment. IFN-αand ribavirin combination therapy is the standard clinical treatment, but this treatment is only less than 50% of patients.
HCV is an enveloped, positive-sense RNA virus of the family Flaviviridae. The 9.6-kb genome encodes one large polyprotein that is processed by viral and cellular proteinases to produce the virion structural proteins (core protein、glycoproteins E1、E2 and P7) as well as nonstructural proteins (NS2 through NS5B). The core protein which is derived from the N-terminus of the viral polyprotein forms the viral nucleocapsid. The amino acid sequence of this protein is well conserved among different HCV strains in comparison with other HCV proteins. More and more reports indicate that core protein is related with carcinogenesis of HCV. However, HCV has host specificity. The mechanism and function of HCV and core protein were mostly studied in cell models. Lacking of appropriate small animal models that can infect HCV hampers the study at the whole level in animal.
In china, the HCV strain is mainly genotype 1b. And the patients infected with HCV-1b is less effective using IFN-α. So study of HCV-1b core protein in HCV persistent infection and HCC pathopoiesis mechanism has important significance for HCV prevention and cure. We established a mouse model which stably express HCV 1b core protein. And the model could be used for evaluating the effect of shRNAs which target HCV core gene. The main research content and results are as follows:
1. We constructed an eukaryotic expression vector pGL3-attB-Core-Fluc. This vector contains HCV core protein, report gene Fluc and attB site that can be recognized by phage integrase. To investigate whether these constructs could express core protein and Fluc, pGL3-attB-Core, pGL3-attB-Fluc and pGL3-attB-Core-Fluc were transfected into Huh7 cells respectively. The expression level of core protein in cells transfected with pGL3-attB-Core-Fluc was equivalent of that in cells transfected with pGL3-attB-Core. But the expression level of Fluc was lower than that in cells transfected with pGL3-attB-Fluc. The same result was also disovered in mice.
2. By site-special recombination of the phage ?C31 integrase,we established a mouse model with stable expression of HCV core protein. pGL3-attB-Core-Fluc was injected with the integrase expression vector pCMV-Int into the liver of mice by hydrodynamics-based procedure. The experiment confirmed that when pCMV-Int was included, core protein and Fluc integrated to mpsL1 site located at mice chromosomal 2 and got a stable expression.
3. Based on the stable mouse model, the shRNAs targeted the highly conserved core protein region of the HCV genome were evaluated in vivo.Based on pSilencerTM2.1-U6 neo vector, we constructed shRNAs expressing plasmids: shRNA452, shRNA479 and shRNA523. In cultured cells, the results revealed that the luciferase activities and core protein expression were reduced about 40-55% in the cells co-transfected with shRNA452, shRNA479 and shRNA523. The inhibitory effects of these three shRNAs had no statistical difference. In the transient mouse model, the effect of shRNA-523 was detectable as early as 24 hr and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48hr post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6hr and 12 hr after injection respectively, and the inhibitory effect persisted for 1 day after a single injection.
In conclusion, this study combined hydrodynamic transfection and phage integrase system to establish a mouse model which can stably express HCV core protein. Moreover, shRNA targeting HCV core protein can effectively downregulate core gene and reporter gene expression in the liver of mouse model. This luminescence-based method allows continuous monitoring of the kinetics of HCV core protein inhibitors in live animal. This novel and simple method can be used for screening anti-HCV compounds.
HCV是一单股正链RNA病毒,病毒基因组全长9600bp,含有一个大的开放读码框(open reading region,ORF)。它编码一个多聚蛋白前体,经宿主信号肽酶和病毒基因编码的蛋白酶切割产生4个结构蛋白(核心蛋白、包膜蛋白E1、E2和P7)和6个非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A和NS5B)。核心蛋白是HCV编码的一个重要结构蛋白,位于整个多聚蛋白的N-末端,是病毒核衣壳的组成部分,在不同型的HCV中具有高度的保守性。越来越多的研究结果表明核心蛋白与HCV感染所致肝细胞癌的发生有密切关系。但由于HCV具有极强的宿主特异性,目前对HCV及核心蛋白的机制和功能的研究多在细胞模型,缺乏合适的小动物模型阻碍了在动物整体水平上的研究。
我国HCV流行株主要为基因1b型,且感染1b型HCV患者运用IFN治疗效果较差,与肝硬化及肝癌的关系较为密切。因此研究1b型HCV核心蛋白在HCV持续感染及肝癌发病机制中的作用,对防治我国丙型肝炎具有重要的实际意义。因此本课题基于成年小鼠,联合水动力转染技术与噬菌体整合酶系统,建立1b型HCV核心蛋白长期稳定表达小鼠模型,作为经典转基因技术的补充,并初步将其用于HCV核心蛋白抑制剂-shRNAs在体内抗HCV的作用效果的评价。具体研究内容及结果如下:
1.构建HCV核心蛋白表达载体pGL3-attB-Core-Fluc。该载体含有核心蛋白基因、报告基因Fluc和?C31整合酶识别位点attB。将pGL3-attB-Core-Fluc与对照载体pGL3-attB-Core、pGL3-attB-Fluc转染Huh7细胞,pGL3-attB-Core-Fluc核心蛋白表达量与pGL3-attB-Core核心蛋白表达量是一致的,而pGL3-attB-Core-Fluc的Fluc活性却低于pGL3-attB-Fluc。在C57BL/6小鼠体内也证实了上述结果。
2.利用?C31整合酶介导位点特异性整合的特点,建立HCV核心蛋白稳定表达小鼠模型。通过水动力转染技术将核心蛋白表达载体pGL3-attB-Core-Fluc与编码?C31整合酶的表达载体pCMV-int共转染至小鼠体内。实验证实,在?C31整合酶作用下,HCV核心蛋白及报告基因Fluc整合到小鼠肝脏第2号染色体的mpsL1位点,在小鼠肝脏获得稳定表达,长达420d。
3.在上述动物模型基础上,对靶向HCV核心蛋白不同位点的shRNAs进行了体内作用效果的评价。本实验在pSilencerTM2.1-U6 neo载体基础上,构建发夹型siRNA-shRNA452、shRNA479、shRNA523表达质粒。结果显示:在细胞水平, shRNA-452、shRNA-479和shRNA-523对核心蛋白和报告基因Fluc均有抑制作用,抑制率可达到40-55%;在核心蛋白瞬时表达小鼠模型中, shRNA-Fluc和shRNA-523的抑制作用在转染后24 hr已被检测到,并且随时间的延长抑制作用更加明显。而shRNA-452在转染后48 hr还未出现抑制作用;在核心蛋白稳定表达小鼠模型中,在第6hr、12hr,shRNA523抑制Fluc的活性分别达到了76.4±26.0%和91.8±8.0%,一次注射该抑制作用持续24hr。
总之,本研究将水动力转染技术及噬菌体整合酶系统联合应用,建立了HCV核心蛋白长期稳定表达小鼠模型。利用该模型可以在小鼠体内实时监测靶向HCV核心蛋白shRNA的作用。这种新颖而简单的方法可用于评价抗HCV核酸药物。
Viral hepatitis C is a major public health concern worldwide. HCV causes chronic hepatitis, cirrhosis and liver cancer. Approximately 170 million people suffer from this infectious disease around the world. Today no therapy is proved to be enough valuable to hepatitis C treatment. IFN-αand ribavirin combination therapy is the standard clinical treatment, but this treatment is only less than 50% of patients.
HCV is an enveloped, positive-sense RNA virus of the family Flaviviridae. The 9.6-kb genome encodes one large polyprotein that is processed by viral and cellular proteinases to produce the virion structural proteins (core protein、glycoproteins E1、E2 and P7) as well as nonstructural proteins (NS2 through NS5B). The core protein which is derived from the N-terminus of the viral polyprotein forms the viral nucleocapsid. The amino acid sequence of this protein is well conserved among different HCV strains in comparison with other HCV proteins. More and more reports indicate that core protein is related with carcinogenesis of HCV. However, HCV has host specificity. The mechanism and function of HCV and core protein were mostly studied in cell models. Lacking of appropriate small animal models that can infect HCV hampers the study at the whole level in animal.
In china, the HCV strain is mainly genotype 1b. And the patients infected with HCV-1b is less effective using IFN-α. So study of HCV-1b core protein in HCV persistent infection and HCC pathopoiesis mechanism has important significance for HCV prevention and cure. We established a mouse model which stably express HCV 1b core protein. And the model could be used for evaluating the effect of shRNAs which target HCV core gene. The main research content and results are as follows:
1. We constructed an eukaryotic expression vector pGL3-attB-Core-Fluc. This vector contains HCV core protein, report gene Fluc and attB site that can be recognized by phage integrase. To investigate whether these constructs could express core protein and Fluc, pGL3-attB-Core, pGL3-attB-Fluc and pGL3-attB-Core-Fluc were transfected into Huh7 cells respectively. The expression level of core protein in cells transfected with pGL3-attB-Core-Fluc was equivalent of that in cells transfected with pGL3-attB-Core. But the expression level of Fluc was lower than that in cells transfected with pGL3-attB-Fluc. The same result was also disovered in mice.
2. By site-special recombination of the phage ?C31 integrase,we established a mouse model with stable expression of HCV core protein. pGL3-attB-Core-Fluc was injected with the integrase expression vector pCMV-Int into the liver of mice by hydrodynamics-based procedure. The experiment confirmed that when pCMV-Int was included, core protein and Fluc integrated to mpsL1 site located at mice chromosomal 2 and got a stable expression.
3. Based on the stable mouse model, the shRNAs targeted the highly conserved core protein region of the HCV genome were evaluated in vivo.Based on pSilencerTM2.1-U6 neo vector, we constructed shRNAs expressing plasmids: shRNA452, shRNA479 and shRNA523. In cultured cells, the results revealed that the luciferase activities and core protein expression were reduced about 40-55% in the cells co-transfected with shRNA452, shRNA479 and shRNA523. The inhibitory effects of these three shRNAs had no statistical difference. In the transient mouse model, the effect of shRNA-523 was detectable as early as 24 hr and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48hr post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6hr and 12 hr after injection respectively, and the inhibitory effect persisted for 1 day after a single injection.
In conclusion, this study combined hydrodynamic transfection and phage integrase system to establish a mouse model which can stably express HCV core protein. Moreover, shRNA targeting HCV core protein can effectively downregulate core gene and reporter gene expression in the liver of mouse model. This luminescence-based method allows continuous monitoring of the kinetics of HCV core protein inhibitors in live animal. This novel and simple method can be used for screening anti-HCV compounds.
引文
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53. Keravala A, Lee S, Thyagarajan B, et al. Mutational derivatives of phiC31 integrase with increased efficiency and specificity[J]. Mol Ther. 2009,17:112-120.
54. Aneja MK, Imker R, Rudolph C. Phage phiC31 integrase-mediated genomicintegration and long-term gene expression in the lung after nonviral gene delivery [J]. J Gene Med. 2007, 9:967-975.
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62. Kronke J, Kittler R, Buchholz F, et al. Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs[J]. J virol. 2004,78:3436-3446.
63. Yokota T, Sakamoto N, Enomoto N, et al. Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs[J]. EMBO Rep. 2003,4:602-608.
64. Wilson JA, Jayasena S, Khvorova A, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells[J]. Proc Natl Acad Sci. 2003,100:2783-2788.
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67. Suzuki H, Kaneko H, Tamai N, et al. Suppression of hepatitis C virus core protein by short hairpin RNA expression vectors in the core protein expression Huh-7 cells[J]. Nucleosides Nucleotides and Nucleic Acids. 2007,26:815-820.
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56. Hannon G J. RNA interference[J]. Nature. 2002, 418:244-251.
57. Moss EG. RNA interference: it's a small RNA world[J]. Curr Biol. 2001, 11: R772 -R775.
58. Cullen BR.RNA interference: antiviral defense and genetic tool[J]. Nat Immunol. 2002, 3:597-599.
59. Lindenbach BD, Rice CM. RNAi targeting an animal virus: news from the front[J]. Mol Cell . 2002, 9:925-927.
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64. Wilson JA, Jayasena S, Khvorova A, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells[J]. Proc Natl Acad Sci. 2003,100:2783-2788.
65. Carmichael GG. Silencing viruses with RNA[J].Nature.2002,418 (6896) :379-380.
66. Suzuki H, Tamai N, Habu Y, et al. Suppression of hepatitis C virus replication by baculovirus vector-mediated short-hairpin RNA expression[J]. FEBS Letters. 2008,582:3085-3089.
67. Suzuki H, Kaneko H, Tamai N, et al. Suppression of hepatitis C virus core protein by short hairpin RNA expression vectors in the core protein expression Huh-7 cells[J]. Nucleosides Nucleotides and Nucleic Acids. 2007,26:815-820.
1. Seeff LB. Natural history of chronic hepatitis C. Hepatology 2002;36(Suppl 1): S35-46.
2. Ray RB , Meyer K, Ray R. Hepatitis C virus core protein promotes immortalization of primary human hepatocytes[J ] . Virology. 2000; 271(1) :197-204.
3. Marusawa H , Hijikata M ,Chiba T, Shimotohno K. Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation. J Virol. 1999;73 (6) :4713-4720.
4. Sacco R, Tsutsumi T, Suzuki R,Otsuka M, Aizaki H, Sakamoto S, Matsuda M, Seki N,Matsuura Y, Miyamura T, Suzuki T. Antiapoptotic regulation by hepatitis C virus core protein through up-regulation of inhibitor of caspase-activated DNase . Virology. 2003 ; 317 (1) :24-35.
5. Ray RB , Meyer K, Ray R. Hepatitis C virus core protein promotes immortalization of primary human hepatocytes[J ] . Virology. 2000; 271(1) :197-204.
6. Yao ZQ, Nguyen DT, Hiotellis AI, Hahn YS: Hepatitis C virus core protein inhibits human T lymphocyte responses by a complement dependent regulatory pathway. J Immunol. 2001; 167: 5264-5272.
7. Bartenschlager, R., and V. Lohmann. 2000. Replication of hepatitis C virus. J. Gen. Virol. 81(Pt. 7):1631-1648.
8. A. Grakoui, C. Wychowski, C. Lin, S.M. Feinstone, C.M. Rice, Expression and identification of hepatitis C virus polyprotein cleavage products, J. Virol. 67 (1993) 1385-1395.
9. M. Hijikata, N. Kato, Y. Ootsuyama, M. Nakagawa, K. Shimotohno, Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis, Proc. Natl. Acad. Sci. USA 88 (1991) 5547-5551.
10. Steeve Boulant, Christophe Vanbelle, Christine Ebel, Francois Penin and Jean-Pierre Lavergne. Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features. Journal of Virology, Sept. 2005, p. 11353-11365
11. Matsuura Y, Harada T, Makimura M, Sato M, Aizaki H, Suzuki T, Miyamura T. Characterization of HCV structural proteins expressed in various animal cells. [ J ].Intervirology. 1994;37(2):114-8.
12. Dubuisson J, Hsu HH, Cheung RC, Greenberg HB, Russell DG, Rice CM. Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses[ J ].J Virol. 1994 Oct;68(10):6147-60.
13. Tokushige K, Moradpour D, Wakita T, Geissler M, Hayashi N, Wands JR. Comparison between cytomegalovirus promoter and elongation factor-1 alpha promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein[ J ].J Virol Methods. 1997 Feb;64(1):73-80.
14. Chen CM, You LR, Hwang LH, Lee YH. Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor[ J ].J Virol. 1997 Dec;71(12):9417-26.
15.全俊,胡国玲,范学工,刘双虎,谭德明.丙型肝炎病毒核心区蛋白在HepG2细胞中的表达.中国现代医学杂志2002年10月第十二卷第20期。
16.单于,陈系古,黄冰,胡安斌,郭忠敏,赵宁.表达丙肝病毒核心蛋白基因的人肝细胞模型的建立.中华实验外科杂志2003年11月第20卷第11期.
17. Moriya K, Yotsuyanagi H, Shintani Y, et al. Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol,1997, 78 ( Pt 7) : 1527-1531.
18.Honda A,Arai Y,Hirota N,et al. Hepatitis C virus structural proteins induce liver cell injury in transgenic mice[ J ].J Med Virol,1999,59(3):281-289.
19. LERAT H, HONDA M, BEARD M R , et al. Steatosis and liver cancer intransgenic mice exp ressing the structural and nonstructural proteins of hepatitis C virus [ J ] 1 Gastroenterology, 2002, 122(2) : 3521.
20. Soguero C,Joo M,Chianese-Bullock KA,et al. Hepatitis C virus core protein leads to immune suppression and liver damage in a transgenic murine model [ J ]. J Virol, 2002,76:9345-9354.
21. Kato T, Miyamoto M, DateT, et al. Repeated hepatocyte injury promotes hepatic tumorigenesis in hepatitis C virus transgenic mice [ J ].Cancer Sci, 2003, 94: 679-685.
22. Omura T,Yoshiyama M,Hayashi T,et al.Core protein of hepatitis C virus induces cardiomyopathy[ J ].Circ Res,2005,96(2):148-150.
23. Moriya K,Fujie H,Shintani Y,et al.The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice.Nat Med,1998,4:1065-1067.
24. Moriya K,Nakagawa K,Santa T,et al.Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Res,2001,61:4356-4370.
25. Perlemuter G,Letteron P,Carnot F et al.Alcohol and hepatitis C virus core protein additively increase lipid peroxidation and synergistically trigger hepatic cytokine expression in a transgenic mouse model.J Hepatol,2003,39(6):1020
26. Tsutsumi T,Suzuki T,Moriya K et al. Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic mice. Hepatology, 2003, 38(4):820
27.杨国柱,傅思莹,黄冰,等.肝脏特异性可调控丙型肝炎病毒核心蛋白表达的转基因小鼠模型的建立[ J ]中山大学学报(医学科学版) ,2007, 28 (4) : 373 - 377.
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