古菌组蛋白（HphA）介导小发夹RNA抑制HCV 5'NCR和C区表达的研究
|
摘要
全世界目前己有1.7亿HCV感染者,我国普通人群抗-HCV阳性率为3.2%。急性HCV感染者中,大约80%可发展为慢性持续感染,其中20%的患者经历20-30年将发展为肝硬化,1%-5%的患者可进展为肝细胞癌。HCV所导致的终末期肝病是重要的死亡原因之一,也是肝移植的原因。目前慢性丙型肝炎的标准治疗是干扰素联合利巴韦林,但有效率仅仅40%-80%左右,人们试图开发新型、高效的治疗药物来解决这一问题。
自RNAi技术发现以来,已广泛应用于分子生物学领域的研究,并且已经证实其在细胞水平能有效抑制HIV、脊髓灰质炎病毒、HBV及HCV的复制和表达。
本文构建了三个靶向(targeting to)HCV 5'NCR和C区的小发夹RNA质粒表达载体,与含有HCV 5’NCR和C区部分基因及荧光素酶报告基因的质粒共转染人肝细胞HL-7702。通过检测荧光素酶报告基因和Western blot来检测目的蛋白表达。发现靶向HCV 5'NCR NCR和靶向C区的shRNA能明显抑制HCV 5'NCR和C区的表达。
目前小干涉RNA进行体内实验的主要障碍是缺乏安全有效的转基因载体。我们应用了一种非病毒类载体古菌组蛋白(HPhA)作为转基因载体。在实验中发现HPhA能与DNA结合,毒性很小,能在血清存在的条件下转染小干涉RNA表达质粒进入细胞抑制HCV 5’NCR和C区的表达。所以HPhA有潜力发展为适合体内实验的转基因载体。
本文首次应用HPhA作为转基因载体,为小干涉RNA抗病毒基因治疗的体内实验提供了重要的理论基础、技术路线和实验方法。
Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Effective therapies against viral infection are not available at present. There are some problems involved in the available techniques of HCV gene therapies, such as antioligonuclitide and ribozyme and deoxyribozyme. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.
RNA interference (RNAi) is a process during which double stranded RNA (dsRNA) induces the sequence-specific degradation of homologous single-stranded RNA. As a result, the expression of the target gene is suppressed. RNAi can therefore be used to develop specific dsRNA based gene therapeutics by knocking down specific genes such as the gene related to viral infection, carcinoma, and the others diseases.
In cultured mammalian cells, the sequence-specific gene silencing can be accomplished by introducing small interfering RNA (siRNA) that is cleaved from dsRNA. Only RNA molecules<30 bases in length can be used to exclusively induce RNAi in mammalian cells because longer molecules also activate the nonspecific double-stranded RNA-dependent IFN response. With the advance of producing siRNA, several laboratories made progress in using RNAi to control viral infection and showed that siRNA can inhibit infection by polio, HIV, HCV and influenza virus. Recently it was reported that RNAi could also reduced HCV replication and expression in the post-transcriptional level.
The likelihood of HCV developing escape mutations illustrates the importance of careful siRNA target sequence selection during the development of treatment strategies. Several conserved regions within the HCV5’noncoding region (5’-NCR) have been found to be viable targets for RNA interference and have potential in therapeutic RNA interference strategies. For example, the 5′UTR acts as an internal ribosome entry sequence, and its activity is determined by RNA structural characteristics. As such, the sequence does not tolerate nucleotide changes and is highly conserved between different HCV genotypes. Therefore, siRNA target sequences based on the 5′UTR region offer promise for siRNA-based treatments.
We construct HCV-specific shRNA expression plasmid targeted 5’NCR and C region (Psilencirle A, B and D), luciferase-specific siRNA expression plasmid (Psilencirle E) and scrambled siRNA expression plasmid(Psilencirle C) by SilenCircleTM RNAi Transcription Kit. Then we transfect above plasmids and plasmids pCMV/T7-NCRC?-luc to HL-7702 cells, pCMV/T7-NCRC?-luc containing HCV 5’NCR, partial core region and luciferase sequences. The luciferase gene is utilized as a reporter gene, its expression can reflect the synthesis of HCV 5’NCR and core protein. The results show that Psilencirle A,B and D can inhibit the expression of HCV 5’NCR and core protein in HL-7702 cells, the inhibiting rate are 38%, 49%, 55% respectedly.
An obvious challenge in the use of siRNA as a therapeutic agent is the development of suitable delivery methods. There have been recent advances in delivery of siRNA using peptide and polymeric vehicles and in vivo application through injection of a large volume of liquid siRNA solutions into the tail veins of mice. The latter treatment could induce RNA interference in the mouse liver and prevent Fas-mediated apoptosis. In our study we introduced the new transgene carrier HPhA (gene engineering production of jilin university) to transfect siRNA expression plasmid into HL-7702 cells.
Gel electrophoresis mobility shift assays demonstrate that the purified HphA has high affinity to DNA. The HphA can increase plasmid electrophoresis mobilities with proper HphA/DNA mass ratios.
We determined the toxicty of HphA to cells by MTT methods, and compared with LipofectamineTM 2000. The results shows that HphA almost don’t affect the growth of cells in relative low concentration(20ug/ml), but LipofectamineTM 2000 cause relative cell viability obviously come down. Cells will die only when the concentration of HphA is higher than 100ug/ml . The toxicty of 1000ug/ml HphA to HL-7702 cells is similar with that of 40ug/ml LipofectamineTM 2000. Considering of the maximum amount of HphA per well is only 9ug when transfection, we can conclude that HphA is nontoxic to cells.
HCV-specific siRNA expression plasmid mediated by HphA can inhibit the expression of HCV 5’NCR and core protein, its efficiency is similar with LipofectamineTM2000. The most important is that when medium containe10% fetal calf serum (FCS), the efficiency of HphA mediated transfection is similar with that without fetal calf serum, and LipofectamineTM2000 can’t transfec plasmids into HL-7702 cells to express HCV 5’NCR and core protein ,when medium containe10% fetal calf serum (FCS). So we concluded that HphA is suitable to transfection in vivo, and it is a safe and effective transgene carrier and will play an import role in RNAi-based genetherapy.
自RNAi技术发现以来,已广泛应用于分子生物学领域的研究,并且已经证实其在细胞水平能有效抑制HIV、脊髓灰质炎病毒、HBV及HCV的复制和表达。
本文构建了三个靶向(targeting to)HCV 5'NCR和C区的小发夹RNA质粒表达载体,与含有HCV 5’NCR和C区部分基因及荧光素酶报告基因的质粒共转染人肝细胞HL-7702。通过检测荧光素酶报告基因和Western blot来检测目的蛋白表达。发现靶向HCV 5'NCR NCR和靶向C区的shRNA能明显抑制HCV 5'NCR和C区的表达。
目前小干涉RNA进行体内实验的主要障碍是缺乏安全有效的转基因载体。我们应用了一种非病毒类载体古菌组蛋白(HPhA)作为转基因载体。在实验中发现HPhA能与DNA结合,毒性很小,能在血清存在的条件下转染小干涉RNA表达质粒进入细胞抑制HCV 5’NCR和C区的表达。所以HPhA有潜力发展为适合体内实验的转基因载体。
本文首次应用HPhA作为转基因载体,为小干涉RNA抗病毒基因治疗的体内实验提供了重要的理论基础、技术路线和实验方法。
Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Effective therapies against viral infection are not available at present. There are some problems involved in the available techniques of HCV gene therapies, such as antioligonuclitide and ribozyme and deoxyribozyme. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.
RNA interference (RNAi) is a process during which double stranded RNA (dsRNA) induces the sequence-specific degradation of homologous single-stranded RNA. As a result, the expression of the target gene is suppressed. RNAi can therefore be used to develop specific dsRNA based gene therapeutics by knocking down specific genes such as the gene related to viral infection, carcinoma, and the others diseases.
In cultured mammalian cells, the sequence-specific gene silencing can be accomplished by introducing small interfering RNA (siRNA) that is cleaved from dsRNA. Only RNA molecules<30 bases in length can be used to exclusively induce RNAi in mammalian cells because longer molecules also activate the nonspecific double-stranded RNA-dependent IFN response. With the advance of producing siRNA, several laboratories made progress in using RNAi to control viral infection and showed that siRNA can inhibit infection by polio, HIV, HCV and influenza virus. Recently it was reported that RNAi could also reduced HCV replication and expression in the post-transcriptional level.
The likelihood of HCV developing escape mutations illustrates the importance of careful siRNA target sequence selection during the development of treatment strategies. Several conserved regions within the HCV5’noncoding region (5’-NCR) have been found to be viable targets for RNA interference and have potential in therapeutic RNA interference strategies. For example, the 5′UTR acts as an internal ribosome entry sequence, and its activity is determined by RNA structural characteristics. As such, the sequence does not tolerate nucleotide changes and is highly conserved between different HCV genotypes. Therefore, siRNA target sequences based on the 5′UTR region offer promise for siRNA-based treatments.
We construct HCV-specific shRNA expression plasmid targeted 5’NCR and C region (Psilencirle A, B and D), luciferase-specific siRNA expression plasmid (Psilencirle E) and scrambled siRNA expression plasmid(Psilencirle C) by SilenCircleTM RNAi Transcription Kit. Then we transfect above plasmids and plasmids pCMV/T7-NCRC?-luc to HL-7702 cells, pCMV/T7-NCRC?-luc containing HCV 5’NCR, partial core region and luciferase sequences. The luciferase gene is utilized as a reporter gene, its expression can reflect the synthesis of HCV 5’NCR and core protein. The results show that Psilencirle A,B and D can inhibit the expression of HCV 5’NCR and core protein in HL-7702 cells, the inhibiting rate are 38%, 49%, 55% respectedly.
An obvious challenge in the use of siRNA as a therapeutic agent is the development of suitable delivery methods. There have been recent advances in delivery of siRNA using peptide and polymeric vehicles and in vivo application through injection of a large volume of liquid siRNA solutions into the tail veins of mice. The latter treatment could induce RNA interference in the mouse liver and prevent Fas-mediated apoptosis. In our study we introduced the new transgene carrier HPhA (gene engineering production of jilin university) to transfect siRNA expression plasmid into HL-7702 cells.
Gel electrophoresis mobility shift assays demonstrate that the purified HphA has high affinity to DNA. The HphA can increase plasmid electrophoresis mobilities with proper HphA/DNA mass ratios.
We determined the toxicty of HphA to cells by MTT methods, and compared with LipofectamineTM 2000. The results shows that HphA almost don’t affect the growth of cells in relative low concentration(20ug/ml), but LipofectamineTM 2000 cause relative cell viability obviously come down. Cells will die only when the concentration of HphA is higher than 100ug/ml . The toxicty of 1000ug/ml HphA to HL-7702 cells is similar with that of 40ug/ml LipofectamineTM 2000. Considering of the maximum amount of HphA per well is only 9ug when transfection, we can conclude that HphA is nontoxic to cells.
HCV-specific siRNA expression plasmid mediated by HphA can inhibit the expression of HCV 5’NCR and core protein, its efficiency is similar with LipofectamineTM2000. The most important is that when medium containe10% fetal calf serum (FCS), the efficiency of HphA mediated transfection is similar with that without fetal calf serum, and LipofectamineTM2000 can’t transfec plasmids into HL-7702 cells to express HCV 5’NCR and core protein ,when medium containe10% fetal calf serum (FCS). So we concluded that HphA is suitable to transfection in vivo, and it is a safe and effective transgene carrier and will play an import role in RNAi-based genetherapy.
引文
1. 人类病毒性疾病, 人民卫生出版社. 第十三节丙型病毒性肝炎, P611.
2. Handbook of Liver Diaease, Second Edition, CHURCHILL LIVINGSTONE. Lawrence S. Friedman, Chronic viral Hepatitis, P51.
3. Wagner M Leevy Lurie Y, Pakula R, Malnick S, et al. Efficacy and safty of the combination of histamine dihydrochloride and interferon alfa-2b in phase Ⅱtrail in naive patients with chronic hepatitis C [Abstract]. HEPATOLOGY, 2001;34(Suppl.):350A.
4. Lin C-C, Luu K, Lourenco D, Corritori S, Hong Z, Lau JY. Viramidine, a prodrug of ribavirin, demonstrated superior liver-targeting properties and much improved toxicity profile in cynomolgus monkey studies [Abstract]. Hepatology, 2001;34(Suppl.):453A.
5. Lau JY, Lin C-C. Assessment of liver delivery of ribavirin and viramidine, a liver-targeting ribavirin prodrug, using whole body autoradiography [Abstract]. Hepatology, 2001;34(Suppl.)427A.
6. Eldrup AB, Allerson CR, Bennett CF, Bera S,Bhat B, Bhat N, Bosserman MR, Brooks J, Burlein C, Carroll SS, Cook PD. (2004) Structure-activity relationship of purineribonucleosides for inhibition of hepatitis Cvirus RNA-dependent RNA polymerase. Med Chem;47:2283-95.
7. Carroll SS, Tomassini JE, Bosserman M, GettyK, Stahlhut MW, Eldrup AB, Bhat B, Hall D,Simcoe AL, La Femina R, Rutkowski CA,Wolanski B, Yang Z, Migliaccio G, DeFrancesco R, Kuo LC, MacCoss M, Olsen DB. (2003) Inhibition of hepatitis C virus RNA replication by 2_-modified nucleoside analogs. J Biol Chem;278:11979-84.
8. Ni Z-J, Wagman AA. (2004) Progress and development of small molecule HCV antivirals. Curr Opin Drug Discovery Dev;7:446-59.
9. Stuyver LJ, Whitaker T, McBrayer TR, Hernandez-Santiago BI, Lostia S, Tharnish PM, Ramesh M, Chu CK, Jordan R, Shi J, Rachakonda S, Watanabe KA, Otto MJ, Schinazi RF. (2003) Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture. Antimicrob Agents Chemother;47:244-54.
10. Maag D, Castro C, Hong Z, Cameron CE. (2001) Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. J Biol Chem;276:46094-8.
11. Standring D. (2003) NM283 has potent antiviral activity against chronic hepatitis C virus genotype-1 in the chimpanzee. HEP DART2003;Frontiers in Drug Development for Viral Hepatitis, Kauai, Hawaii, HI USA, Abs.28.
12. Altamura A, Tomei L, Koch U, Neuner PJS, Summa V. (2000) Diketoacid- derivatives as Inhibitors of Polymerases. WO 00/06529.
13. De Francesco R. (2000) Diketobutanoic acids as HCV polymerase inhibitors. 7th International Meeting on hepatitis and related viruses, Gold Coast, Queensland, Australia, 3-7December.
14. Love RA, Parge HE, Wickersham JA, Hostomsky Z, Habuka N, Moomaw EW, Adachi T, Hostomska Z. (1996) The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell;87:331-42.
15. McCoy MA, Senior MM, Gesell JJ, Ramanathan L, Wyss DF. (2001) Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex. Mol Biol;305:1099-110.
16. Llinas-Brunet M, Bailey M, Fazal G, Ghiro E, Gorys V, Goulet S, Halmos T, Maurice R, Poirier M, Poupart MA, Rancourt J, Thibeault D, Wernic D, Lamarre D. (2000) Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease:towards smaller inhibitors Bioorg Med ChemLett;10:2267-70.
17. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, Boes M, Cameron DR, Cartier M, Cordingley MG, Faucher AM. (2003) An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature;426:186-9.
18. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, Boes M, Cameron DR, Cartier M, Cordingley MG, Faucher AM. (2003) An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature;426:186-9.
19. Benhamou Y, Hinrichsen H, Sentijens R, Reiser M, Manns MP, Forns X, Avendano C, Cronlein J, Nehmiz G, Steinmann G. (2002) Safety, tolerability and antiviral effect of BILN 2061, a novel HCV serine protease inhibitor, after oral treatment over 2 days in patients with chronic hepatitis C genotype 1, with advanced liver fibrosis. Hepatology;36:304A.
20. Zhang X, Schmitt AC, Decicco CP. (2002) Design and synthesis of 6-amino-5-oxo-1,2,3,5-tetrahydro-tetrahydro-3-indolizinecarboxylic acids as- sheet peptidomimetics. Tetrahedron Lett;43:9663–6.
21. Glunz PW, Douty BD, Decicco CP. (2003) Design and synthesis of bicyclic pyrimidinone-based HCV NS3 protease inhibitors. Bioorg Med Chem Lett; 13:785-8.
22. Yao N, Hesson T, Cable M, Hong Z, Kwong AD,Le HV, Weber PC. (1997) Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol.; 4: 463-7.
23. Gallinari P, Brennan D, Nardi C, Brunetti M, Tomei L, Steinkühler C, De Francesco R. (1998) Multiple enzymatic activities associated with recombinant NS3 of hepatitis C virus. J Virol; 72:6758–69.
24. Colman RF. (1983) Affinity labeling of purine nucleotide sites in proteins.Annu Rev Biochem;52:67-91.
25. Bretner M, Schalinski S, Haag A, Lang M, Schmitz H, Baier A, Behrens SE, Kulikowski T, Borowski P. (2004a) Synthesis and evaluation of ATP-binding site directed potential inhibitors of nucleoside triphosphatases helicases and polymerases of hepatitis C and other selected Flaviviridae viruses. Antivir Chem Chemother;15:35-42.
26. Sun LQ, Cairns MJ, Saravolac EG, et al.Catalytic nucleic acid:Form Lab to Applications. Pharmacol Rev 2000 sep;52(3):325-47.
27. Sriram B, Banerjea AC. IN ritro-selected RNA cleaving DNA enzymes from a combinatorial library are potent ingibitors of HIV-1 gene expression Biochem J 2000 Dec 15;352Pt5:667-73.
28. Dianne S. Schwarz, Benjamin Haley, Phillip D.Zamore, et al. Evidence that siRNA funtion as guides, not primers,in the Drosophila and human RNAi pathways. Molecular Cell September, 2002(10):537-548.
29. Joyce a. Wilson, Sumedha Jayasena, Anastasia Khvorova, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. PNAS2003;100(5):2783-88.
30. Lipardi C, Wei Q, and Paterson BM. (2001) RNAi as random degradative PCR. siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs. Cell 107:297-307.
31. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, and Mello CC. (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34.
32. Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics? Cancer Cell 2002;2:17-23
33. Elbashir SM, et al. Functional anatomy of siRNAs for mediating efficientRNAi in Drosophila melanogaster embryo lysate. EMBO 2001;20:6877-6888
34. 张 明 , 邵 宁 生 . RNA 阻 抑 和 基 因 沉 默 . 军 事 医 学 科 学 院 院 刊 2002;26:61-65.
35. Tuschl T, et al. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 1999;13:3191-3197.
36. Tuschl T, et al. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 1999;13:3191-3197.
37. Dykxhoorn DM, Norina CD, sharp PA. Killing the messenger: short RNAs that silence gene expression. Nature Cell Biology 2003;4:457-466.
38. Chiu YL, Rana TM. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol Cell 2002;10:549-561.
39. Stein P, Svobada P, Anger M, et al. RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 2003;9:187-192.
40. Kapadia SB , Brideou2Andersen A , Chisari FV1 Interference of hepatitis C virus RNA replication by shore interfering RNA[J ]1 Proc Natl Acad Sci USA , 2003,100:20141.
41. 吴峰, 陈惠鹏. 通过 RNA 干涉沉默 SARS 病毒的研究前景生物技术通讯, 2003,3:252.
42. Susan L. Uprichard, Bryan Boyd, Alana Althage, and Francis V. Chisari , Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs ,MEDICAL SCIENCES, January 18, 2005 vol. 102 773-778
43. Cheng TL, Chang WW, Su IJ, Lai MD, Huang W, Lei HY, Chang WT, Therapeutic inhibition of hepatitiss B virus surfacee antigen expression by RNA interference, Biochem Biophys Res Commun. 2005 Oct 28;336(3): 820-30.
44. Morrissey DV, Lockridge JA, Shaw L, Blanchard K, Jensen K, Breen W,Hartsough K, Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.Nat Biotechnol. 2005 Aug;23(8):1002-7.
45. Soutschek, J. et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432,173-178.
46. Sorensen, D.R. et al. (2003) Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J. Mol. Biol. 327,761–766.
47. Chien, P.Y. et al. (2005) Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo. Cancer Gene Ther. 12, 321–328
48. Leng, Q. et al. (2005) Highly branched HK peptides are effective carriers of siRNA.J. Gene Med. 7, 977–986
49. Leng, Q. et al. (2005) Small interfering RNA targeting Raf-1 inhibits tumor
50. growth in vitro and in vivo. Cancer Gene Ther. 12, 682–690
51. Filleur, S. et al. (2003) SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res. 63, 3919–3922
52. Ge, Q. et al. (2004) Inhibition of influenza virus production in virus-infected
53. mice by RNA interference. Proc. Natl. Acad. Sci. U. S. A. 101, 8676–8681
54. Kariko, K. et al. (2004) Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3. J. Immunol. 172, 6545–6549.
55. Sledz, C.A. et al. (2003) Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839.
56. 56 Chi, J.T. et al. (2003) Genomewide view of gene silencing by small interfering RNAs. Proc. Natl. Acad. Sci. U. S. A. 100, 6343–6346.
57. Semizarov, D. et al. (2003) Specificity of short interfering RNA determinedthrough gene expression signatures. Proc. Natl. Acad. Sci. U. S. A. 100, 6347–6352.
58. Hornung, V. et al. (2005) Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270.
59. Judge, A.D. et al. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457–462
60. Liang weng, Dan liu, Yuanyuan Li, Shugui Cao, Yan Feng, An archaeal histone-like protein as an efficient DNA carrier in gene transfer. Biochimica et Biophysica Acta 1702(2004)209-216
61. Lewis, D.L. et al. (2002) Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat. Genet. 32,107–108.
62. McCaffrey, A.P. et al. (2002) RNA interference in adult mice. Nature 418, 38–39.
63. Song, E. et al. (2003) RNA interference targeting Fas protects mice from fulminant hepatitism. Nat. Med. 9,347-351.
64. Zender, L. et al. (2003) Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc. Natl. Acad. Sci. U. S. A. 100,7797-7802.
65. Layzer, J.M. et al. (2004) In vivo activity of nuclease-resistant siRNAs. RNA 10,766-771.
66. Giladi, H. et al. (2003) Small interfering RNA inhibits hepatitis B virus replication in mice. Mol. Ther. 8,769-776.
67. Sen, A. et al. (2003) Inhibition of hepatitis C virus protein expression by RNA interference. Virus Res. 96,27-35.
68. Lorenz, C. et al. (2004) Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells. Bioorg. Med. Chem. Lett. 14, 4975–4977.
69. Woodle, M.C. and Lu, P.Y. (2005). Nanoparticles deliver RNAi therapy. Nanotoday, 34–41.
70. Li, B.J. et al. (2005) Using siRNA in prophylactic and therapeutic regimens against SARS Coronavirus in Rhesus Macaque. Nat. Med. 11,944–951.
71. Morrissey, D. et al. (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23,1002–1007.
72. Lu, P.Y. et al. (2005) Modulation of angiogenesis with siRNA inhibitors for novel therapeutics. Trends Mol. Med. 11, 104–113.
73. osher JM, Labouesse M. RNA interference: genetic wand and genetic watchdog. Nature Cell Biology 2000;2:E31-36.
74. Pasquinelli AE. MicroRNAs: deviants no longer. Trends Genet 2002;18:171-173.
75. Chiu YL, Rana TM. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol Cell 2002;10:549-561.
76. Stein P, Svobada P, Anger M, et al. RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 2003;9:187-192.
77. Svoboda P, stoin P, Schultz RM. RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA. Biochem Biophys Res Commun 2001;287:1099-1104.
78. Tavernarakis N, Wang SL, Dorovkov M, et al. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genet 2000;24:180-183.
79. Brummelkamp TR, Bernard R, Agami R, A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-553.
80. McManus MT, Petersen CP, Haines BB, et al. Gene silencing using micro-RNA designed hairpins. RNA 2002;8:842-850.
81. Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expressionby RNAi in mammalian cells. PNAS USA 2002;99:1443-1448.
82. Paddison PJ, Caudy AA, Bernstein E, etal. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 2002;16:948-958.
83. Paul CP, Good PD, Winer I, et al. Effective expression of small interfering RNA in human cells. Nature Biotechnol 2002;20(5):505-508.
84. Sui G.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. PNAs. USA 2002;99(8):5515-5526.
85. Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnol 2002;20(5):500-505.
86. Yokota T, et al. Inhibition of intracellular hepatitis V virus replication by synthetic and vector-derived small interfering RNAs. EMBO J 2003;4(6):1-7
87. Hutvagner G, Zamore PD, A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002;297:2056-2060.
88. Kavasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNAVa promoter significantly induce RNAi mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 2003;31:700-707.
89. Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics? Cancer Cell 2002;2:17-23.
90. ammalian cells. PNAs. USA 2002;99(8):5515-5526.
91. 马鑫, 鲁晓春, 彭建强, 等. AAV 载体质粒介导的荧光素酶 shRNA 抑制其在哺乳动物细胞中的表达. 中华实验和临床病毒学杂志 2002;16: 253-255.
92. Hijikata M, Kato N, Ootsuyama Y, et al. Gene mapping of the putative structural region of the hepatitis C genome by in vitro processing analysis. Proc Natl Acad Sci USA. 1991;88:5547-51.
93. Sharookh B. Kapadia, Amy Brideau-Andersen, and Francis V. Chisari* Interference of hepatitis C virus RNA replication by short interfering RNAs Proc Natl Acad Sci U S A. 2003 February 18; 100 (4): 2014–2018.
94. Joyce A. Wilson,* Sumedha Jayasena, RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells Proc Natl Acad Sci U S A. 2003 March 4; 100 (5): 2783–2788
95. Joyce A. Wilson1 and Christopher D. Richardson1,2* Hepatitis C Virus Replicons Escape RNA Interference Induced by a Short Interfering RNA Directed against the NS5b Coding Region, J Virol. 2005 June; 79(11): 7050–7058.
96. Das, A. T., T. R. Brummelkamp, E. M. Westerhout, M. Vink, M. Madiredjo, R. Bernards, and B. Berkhout. 2004. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J. Virol. 78:2601-2605.
97. Bertolini L., S. Lacovacci., A. Ponzetto. et al. (1993): The human bone-marrow-derived B-cell line CE. susceptible to hepatitis C virus infection. Res. Virol., 144: 281-285.
98. Carloni G., S. Lacovacci, M. Sargiacomo, et al. (1993): Susceptibility of human liver cell cultures to hepatitis C virus infection. Arch. Virol. Suppl., 8: 31-39.
99. Lanford R.E., C. Sureau, J.R. Jacob, et al. (1994): Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand- specific RT/PCR. Virology, 202: 606-614.
100. V. Lohmann, F. K?rner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager , Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line science.vol285. 110-113.
101. Thomas Pietschmann,Volker Lohmann, Gabriel Rutter, KatharinaKurpanek,and Ralf Bartenschlager Characterization of Cell Lines Carrying Self-Replicating Hepatitis C Virus RNAs Journal of Virology, February 2001, Vol75, 1252-1264.
102. JL. Workman, and RE Kingston. Alteration of Nucleosome Structure as a Mechanism of Transcriptional Regulation. Ann Rev Biochem. 1998;67: 545-579.
103. AP Wolffe, and JJ Hayes. Chromation Disruption and Modification. Nucl Acids Res. 1999;27:711-720.
104. G Orphanides, and D Reinberg. RNA polymerase II elongation through chromation. Nature. 2000; 407:471-475.
105. Aiuti A, Slavin S, et al. Science. 2002; 296: 2410-2413.
106. Audouy S, Molema G, deleij L, Hoekstra D. Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability. Journal of Gene Med. 2002;2:465-476.
107. Jan Kr?nke,1 Ralf Kittler,2 Frank Buchholz,2 Marc P. Windisch,1 Thomas Pietschmann,1 Ralf Bartenschlager,1* and Michael Frese, Alternative Approaches for Efficient Inhibition of Hepatitis C Virus RNA Replication by Small Interfering RNAs. J Virol. 2004 April; 78(7): 3436–3446.
2. Handbook of Liver Diaease, Second Edition, CHURCHILL LIVINGSTONE. Lawrence S. Friedman, Chronic viral Hepatitis, P51.
3. Wagner M Leevy Lurie Y, Pakula R, Malnick S, et al. Efficacy and safty of the combination of histamine dihydrochloride and interferon alfa-2b in phase Ⅱtrail in naive patients with chronic hepatitis C [Abstract]. HEPATOLOGY, 2001;34(Suppl.):350A.
4. Lin C-C, Luu K, Lourenco D, Corritori S, Hong Z, Lau JY. Viramidine, a prodrug of ribavirin, demonstrated superior liver-targeting properties and much improved toxicity profile in cynomolgus monkey studies [Abstract]. Hepatology, 2001;34(Suppl.):453A.
5. Lau JY, Lin C-C. Assessment of liver delivery of ribavirin and viramidine, a liver-targeting ribavirin prodrug, using whole body autoradiography [Abstract]. Hepatology, 2001;34(Suppl.)427A.
6. Eldrup AB, Allerson CR, Bennett CF, Bera S,Bhat B, Bhat N, Bosserman MR, Brooks J, Burlein C, Carroll SS, Cook PD. (2004) Structure-activity relationship of purineribonucleosides for inhibition of hepatitis Cvirus RNA-dependent RNA polymerase. Med Chem;47:2283-95.
7. Carroll SS, Tomassini JE, Bosserman M, GettyK, Stahlhut MW, Eldrup AB, Bhat B, Hall D,Simcoe AL, La Femina R, Rutkowski CA,Wolanski B, Yang Z, Migliaccio G, DeFrancesco R, Kuo LC, MacCoss M, Olsen DB. (2003) Inhibition of hepatitis C virus RNA replication by 2_-modified nucleoside analogs. J Biol Chem;278:11979-84.
8. Ni Z-J, Wagman AA. (2004) Progress and development of small molecule HCV antivirals. Curr Opin Drug Discovery Dev;7:446-59.
9. Stuyver LJ, Whitaker T, McBrayer TR, Hernandez-Santiago BI, Lostia S, Tharnish PM, Ramesh M, Chu CK, Jordan R, Shi J, Rachakonda S, Watanabe KA, Otto MJ, Schinazi RF. (2003) Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in culture. Antimicrob Agents Chemother;47:244-54.
10. Maag D, Castro C, Hong Z, Cameron CE. (2001) Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. J Biol Chem;276:46094-8.
11. Standring D. (2003) NM283 has potent antiviral activity against chronic hepatitis C virus genotype-1 in the chimpanzee. HEP DART2003;Frontiers in Drug Development for Viral Hepatitis, Kauai, Hawaii, HI USA, Abs.28.
12. Altamura A, Tomei L, Koch U, Neuner PJS, Summa V. (2000) Diketoacid- derivatives as Inhibitors of Polymerases. WO 00/06529.
13. De Francesco R. (2000) Diketobutanoic acids as HCV polymerase inhibitors. 7th International Meeting on hepatitis and related viruses, Gold Coast, Queensland, Australia, 3-7December.
14. Love RA, Parge HE, Wickersham JA, Hostomsky Z, Habuka N, Moomaw EW, Adachi T, Hostomska Z. (1996) The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell;87:331-42.
15. McCoy MA, Senior MM, Gesell JJ, Ramanathan L, Wyss DF. (2001) Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex. Mol Biol;305:1099-110.
16. Llinas-Brunet M, Bailey M, Fazal G, Ghiro E, Gorys V, Goulet S, Halmos T, Maurice R, Poirier M, Poupart MA, Rancourt J, Thibeault D, Wernic D, Lamarre D. (2000) Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease:towards smaller inhibitors Bioorg Med ChemLett;10:2267-70.
17. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, Boes M, Cameron DR, Cartier M, Cordingley MG, Faucher AM. (2003) An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature;426:186-9.
18. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, Boes M, Cameron DR, Cartier M, Cordingley MG, Faucher AM. (2003) An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature;426:186-9.
19. Benhamou Y, Hinrichsen H, Sentijens R, Reiser M, Manns MP, Forns X, Avendano C, Cronlein J, Nehmiz G, Steinmann G. (2002) Safety, tolerability and antiviral effect of BILN 2061, a novel HCV serine protease inhibitor, after oral treatment over 2 days in patients with chronic hepatitis C genotype 1, with advanced liver fibrosis. Hepatology;36:304A.
20. Zhang X, Schmitt AC, Decicco CP. (2002) Design and synthesis of 6-amino-5-oxo-1,2,3,5-tetrahydro-tetrahydro-3-indolizinecarboxylic acids as- sheet peptidomimetics. Tetrahedron Lett;43:9663–6.
21. Glunz PW, Douty BD, Decicco CP. (2003) Design and synthesis of bicyclic pyrimidinone-based HCV NS3 protease inhibitors. Bioorg Med Chem Lett; 13:785-8.
22. Yao N, Hesson T, Cable M, Hong Z, Kwong AD,Le HV, Weber PC. (1997) Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol.; 4: 463-7.
23. Gallinari P, Brennan D, Nardi C, Brunetti M, Tomei L, Steinkühler C, De Francesco R. (1998) Multiple enzymatic activities associated with recombinant NS3 of hepatitis C virus. J Virol; 72:6758–69.
24. Colman RF. (1983) Affinity labeling of purine nucleotide sites in proteins.Annu Rev Biochem;52:67-91.
25. Bretner M, Schalinski S, Haag A, Lang M, Schmitz H, Baier A, Behrens SE, Kulikowski T, Borowski P. (2004a) Synthesis and evaluation of ATP-binding site directed potential inhibitors of nucleoside triphosphatases helicases and polymerases of hepatitis C and other selected Flaviviridae viruses. Antivir Chem Chemother;15:35-42.
26. Sun LQ, Cairns MJ, Saravolac EG, et al.Catalytic nucleic acid:Form Lab to Applications. Pharmacol Rev 2000 sep;52(3):325-47.
27. Sriram B, Banerjea AC. IN ritro-selected RNA cleaving DNA enzymes from a combinatorial library are potent ingibitors of HIV-1 gene expression Biochem J 2000 Dec 15;352Pt5:667-73.
28. Dianne S. Schwarz, Benjamin Haley, Phillip D.Zamore, et al. Evidence that siRNA funtion as guides, not primers,in the Drosophila and human RNAi pathways. Molecular Cell September, 2002(10):537-548.
29. Joyce a. Wilson, Sumedha Jayasena, Anastasia Khvorova, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. PNAS2003;100(5):2783-88.
30. Lipardi C, Wei Q, and Paterson BM. (2001) RNAi as random degradative PCR. siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs. Cell 107:297-307.
31. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, and Mello CC. (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34.
32. Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics? Cancer Cell 2002;2:17-23
33. Elbashir SM, et al. Functional anatomy of siRNAs for mediating efficientRNAi in Drosophila melanogaster embryo lysate. EMBO 2001;20:6877-6888
34. 张 明 , 邵 宁 生 . RNA 阻 抑 和 基 因 沉 默 . 军 事 医 学 科 学 院 院 刊 2002;26:61-65.
35. Tuschl T, et al. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 1999;13:3191-3197.
36. Tuschl T, et al. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 1999;13:3191-3197.
37. Dykxhoorn DM, Norina CD, sharp PA. Killing the messenger: short RNAs that silence gene expression. Nature Cell Biology 2003;4:457-466.
38. Chiu YL, Rana TM. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol Cell 2002;10:549-561.
39. Stein P, Svobada P, Anger M, et al. RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 2003;9:187-192.
40. Kapadia SB , Brideou2Andersen A , Chisari FV1 Interference of hepatitis C virus RNA replication by shore interfering RNA[J ]1 Proc Natl Acad Sci USA , 2003,100:20141.
41. 吴峰, 陈惠鹏. 通过 RNA 干涉沉默 SARS 病毒的研究前景生物技术通讯, 2003,3:252.
42. Susan L. Uprichard, Bryan Boyd, Alana Althage, and Francis V. Chisari , Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs ,MEDICAL SCIENCES, January 18, 2005 vol. 102 773-778
43. Cheng TL, Chang WW, Su IJ, Lai MD, Huang W, Lei HY, Chang WT, Therapeutic inhibition of hepatitiss B virus surfacee antigen expression by RNA interference, Biochem Biophys Res Commun. 2005 Oct 28;336(3): 820-30.
44. Morrissey DV, Lockridge JA, Shaw L, Blanchard K, Jensen K, Breen W,Hartsough K, Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.Nat Biotechnol. 2005 Aug;23(8):1002-7.
45. Soutschek, J. et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432,173-178.
46. Sorensen, D.R. et al. (2003) Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J. Mol. Biol. 327,761–766.
47. Chien, P.Y. et al. (2005) Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo. Cancer Gene Ther. 12, 321–328
48. Leng, Q. et al. (2005) Highly branched HK peptides are effective carriers of siRNA.J. Gene Med. 7, 977–986
49. Leng, Q. et al. (2005) Small interfering RNA targeting Raf-1 inhibits tumor
50. growth in vitro and in vivo. Cancer Gene Ther. 12, 682–690
51. Filleur, S. et al. (2003) SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res. 63, 3919–3922
52. Ge, Q. et al. (2004) Inhibition of influenza virus production in virus-infected
53. mice by RNA interference. Proc. Natl. Acad. Sci. U. S. A. 101, 8676–8681
54. Kariko, K. et al. (2004) Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3. J. Immunol. 172, 6545–6549.
55. Sledz, C.A. et al. (2003) Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839.
56. 56 Chi, J.T. et al. (2003) Genomewide view of gene silencing by small interfering RNAs. Proc. Natl. Acad. Sci. U. S. A. 100, 6343–6346.
57. Semizarov, D. et al. (2003) Specificity of short interfering RNA determinedthrough gene expression signatures. Proc. Natl. Acad. Sci. U. S. A. 100, 6347–6352.
58. Hornung, V. et al. (2005) Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270.
59. Judge, A.D. et al. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457–462
60. Liang weng, Dan liu, Yuanyuan Li, Shugui Cao, Yan Feng, An archaeal histone-like protein as an efficient DNA carrier in gene transfer. Biochimica et Biophysica Acta 1702(2004)209-216
61. Lewis, D.L. et al. (2002) Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat. Genet. 32,107–108.
62. McCaffrey, A.P. et al. (2002) RNA interference in adult mice. Nature 418, 38–39.
63. Song, E. et al. (2003) RNA interference targeting Fas protects mice from fulminant hepatitism. Nat. Med. 9,347-351.
64. Zender, L. et al. (2003) Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc. Natl. Acad. Sci. U. S. A. 100,7797-7802.
65. Layzer, J.M. et al. (2004) In vivo activity of nuclease-resistant siRNAs. RNA 10,766-771.
66. Giladi, H. et al. (2003) Small interfering RNA inhibits hepatitis B virus replication in mice. Mol. Ther. 8,769-776.
67. Sen, A. et al. (2003) Inhibition of hepatitis C virus protein expression by RNA interference. Virus Res. 96,27-35.
68. Lorenz, C. et al. (2004) Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells. Bioorg. Med. Chem. Lett. 14, 4975–4977.
69. Woodle, M.C. and Lu, P.Y. (2005). Nanoparticles deliver RNAi therapy. Nanotoday, 34–41.
70. Li, B.J. et al. (2005) Using siRNA in prophylactic and therapeutic regimens against SARS Coronavirus in Rhesus Macaque. Nat. Med. 11,944–951.
71. Morrissey, D. et al. (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23,1002–1007.
72. Lu, P.Y. et al. (2005) Modulation of angiogenesis with siRNA inhibitors for novel therapeutics. Trends Mol. Med. 11, 104–113.
73. osher JM, Labouesse M. RNA interference: genetic wand and genetic watchdog. Nature Cell Biology 2000;2:E31-36.
74. Pasquinelli AE. MicroRNAs: deviants no longer. Trends Genet 2002;18:171-173.
75. Chiu YL, Rana TM. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol Cell 2002;10:549-561.
76. Stein P, Svobada P, Anger M, et al. RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 2003;9:187-192.
77. Svoboda P, stoin P, Schultz RM. RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA. Biochem Biophys Res Commun 2001;287:1099-1104.
78. Tavernarakis N, Wang SL, Dorovkov M, et al. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genet 2000;24:180-183.
79. Brummelkamp TR, Bernard R, Agami R, A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-553.
80. McManus MT, Petersen CP, Haines BB, et al. Gene silencing using micro-RNA designed hairpins. RNA 2002;8:842-850.
81. Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expressionby RNAi in mammalian cells. PNAS USA 2002;99:1443-1448.
82. Paddison PJ, Caudy AA, Bernstein E, etal. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 2002;16:948-958.
83. Paul CP, Good PD, Winer I, et al. Effective expression of small interfering RNA in human cells. Nature Biotechnol 2002;20(5):505-508.
84. Sui G.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. PNAs. USA 2002;99(8):5515-5526.
85. Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnol 2002;20(5):500-505.
86. Yokota T, et al. Inhibition of intracellular hepatitis V virus replication by synthetic and vector-derived small interfering RNAs. EMBO J 2003;4(6):1-7
87. Hutvagner G, Zamore PD, A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002;297:2056-2060.
88. Kavasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNAVa promoter significantly induce RNAi mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 2003;31:700-707.
89. Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics? Cancer Cell 2002;2:17-23.
90. ammalian cells. PNAs. USA 2002;99(8):5515-5526.
91. 马鑫, 鲁晓春, 彭建强, 等. AAV 载体质粒介导的荧光素酶 shRNA 抑制其在哺乳动物细胞中的表达. 中华实验和临床病毒学杂志 2002;16: 253-255.
92. Hijikata M, Kato N, Ootsuyama Y, et al. Gene mapping of the putative structural region of the hepatitis C genome by in vitro processing analysis. Proc Natl Acad Sci USA. 1991;88:5547-51.
93. Sharookh B. Kapadia, Amy Brideau-Andersen, and Francis V. Chisari* Interference of hepatitis C virus RNA replication by short interfering RNAs Proc Natl Acad Sci U S A. 2003 February 18; 100 (4): 2014–2018.
94. Joyce A. Wilson,* Sumedha Jayasena, RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells Proc Natl Acad Sci U S A. 2003 March 4; 100 (5): 2783–2788
95. Joyce A. Wilson1 and Christopher D. Richardson1,2* Hepatitis C Virus Replicons Escape RNA Interference Induced by a Short Interfering RNA Directed against the NS5b Coding Region, J Virol. 2005 June; 79(11): 7050–7058.
96. Das, A. T., T. R. Brummelkamp, E. M. Westerhout, M. Vink, M. Madiredjo, R. Bernards, and B. Berkhout. 2004. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J. Virol. 78:2601-2605.
97. Bertolini L., S. Lacovacci., A. Ponzetto. et al. (1993): The human bone-marrow-derived B-cell line CE. susceptible to hepatitis C virus infection. Res. Virol., 144: 281-285.
98. Carloni G., S. Lacovacci, M. Sargiacomo, et al. (1993): Susceptibility of human liver cell cultures to hepatitis C virus infection. Arch. Virol. Suppl., 8: 31-39.
99. Lanford R.E., C. Sureau, J.R. Jacob, et al. (1994): Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand- specific RT/PCR. Virology, 202: 606-614.
100. V. Lohmann, F. K?rner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager , Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line science.vol285. 110-113.
101. Thomas Pietschmann,Volker Lohmann, Gabriel Rutter, KatharinaKurpanek,and Ralf Bartenschlager Characterization of Cell Lines Carrying Self-Replicating Hepatitis C Virus RNAs Journal of Virology, February 2001, Vol75, 1252-1264.
102. JL. Workman, and RE Kingston. Alteration of Nucleosome Structure as a Mechanism of Transcriptional Regulation. Ann Rev Biochem. 1998;67: 545-579.
103. AP Wolffe, and JJ Hayes. Chromation Disruption and Modification. Nucl Acids Res. 1999;27:711-720.
104. G Orphanides, and D Reinberg. RNA polymerase II elongation through chromation. Nature. 2000; 407:471-475.
105. Aiuti A, Slavin S, et al. Science. 2002; 296: 2410-2413.
106. Audouy S, Molema G, deleij L, Hoekstra D. Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability. Journal of Gene Med. 2002;2:465-476.
107. Jan Kr?nke,1 Ralf Kittler,2 Frank Buchholz,2 Marc P. Windisch,1 Thomas Pietschmann,1 Ralf Bartenschlager,1* and Michael Frese, Alternative Approaches for Efficient Inhibition of Hepatitis C Virus RNA Replication by Small Interfering RNAs. J Virol. 2004 April; 78(7): 3436–3446.