shRNA沉默VEGF表达对肺癌细胞生长作用和机理的研究
摘要
背景与目的肿瘤的生长依赖于血管的新生,抑制肿瘤血管生成已经成为肿瘤治疗的一种策略。VEGF通过与血管内皮细胞膜的VEGF受体(VEGFR)结合,触发信号转导,转导级联反应,引起血管内皮细胞有丝分裂,刺激内皮细胞增殖和血管生成。VEGF/VEGFR通路可特异性促进内皮细胞增殖、参与血管形成、增加血管通透性及保护受损神经元等,不仅在胚胎发育、伤口愈合、炎症、肿瘤等多种生理及病理过程中发挥重要作用,而且参与缺血性脑血管病的损伤及抗损伤过程。VEGF具有增加微血管通透性、促进不同来源的内皮细胞分裂增殖和血管构建、促使内皮细胞迁移等多种作用。大多数肿瘤细胞高水平地表达VEGF,且其表达与肿瘤的恶性程度呈正相关,因此,VEGF及其受体成为目前抗肿瘤血管形成治疗最为成熟的靶分子。
RNA干扰(RNA interference,RNAi)是指由双链RNA(double stranded RNA,dsRNA)引发的转录后基因沉默机制,能够高效特异地沉默同源基因的表达,是一种转录后调控。RNAi技术不仅在功能基因组研究中发挥了重要的作用,而且是一种潜在的以特异性基因沉默为方法治疗疾病的手段。尤其在肿瘤的干预中更具有潜在意义。目前,通过RNAi技术干涉VEGF的表达能够抑制胰腺癌、喉鳞癌、大肠癌和肺癌等细胞株的增殖、生长、侵袭和迁移。
本课题,我们利用RNAi干涉技术,沉默肺癌细胞中VEGF的表达,从而达到干预肺癌生长增殖的目的,为肺癌的基因治疗提供新的理论依据。期望能够获得一个持续高效的抑制肺癌细胞中VEGF表达的RNAi片段,从体外、内观察其对肺癌细胞增殖和肺内癌肿生长的影响,并探讨其干预机制。
材料和方法通过筛选获得理想的能够沉默肺癌细胞中VEGF表达的shRNA片段,并将其插入到载体中,制备成pSilencer-VEGF-shRNA;体外利用该质粒对肺癌A549细胞系进行干预后,动态观察A549细胞中VEGF的表达、肺癌细胞的生长及内皮细胞小管结构的形成;用A549和pSilencer-VEGF-A549细胞分别接种裸鼠,观察荷瘤裸鼠体内肿瘤组织的生长及生存期。使用RT-PCR和免疫印迹的方法检测与内皮细胞相互作用后干涉VEGF的A549细胞内p53、Bcl-2和Bax转录和蛋白水平上的变化。
研究结果经RT-PCR和免疫印迹实验检测发现pSilencer-VEGF-2对肺癌细胞中VEGF的抑制率能够达到90%以上;在稳定转染的肺癌细胞株A549细胞中,2个月以后对VEGF的抑制率仍在80%以上;肺癌细胞生长干预实验结果显示,pSilencer-VEGF-2明显抑制了肺癌A549细胞系中VEGF的表达;虽然稳定转染pSilencer-VEGF-2的肺癌A549细胞株体外生长未发生明显变化,但是,它可以显著抑制内皮细胞的血管生成作用。体内荷瘤裸鼠实验结果显示:与接种对照组荷肺癌A549细胞株瘤的裸鼠相比,pSilencer-VEGF-2-A549荷瘤裸鼠体内肿瘤组织的生长显著减慢(P<0.05),生存期明显延长(P<0.05)。pSilencer-VEGF-2干预肺癌增殖生长的机制研究显示,稳定转染的人肺癌A549细胞株中P53,Bcl-2和Bax的表达无明显改变,也未见到明显的细胞凋亡现象。与对照组相比,在人HUVEC细胞系的作用下,稳定转染的人A549细胞中Bax表达无变化,但P53的表达明显增高,Bcl-2的表达下调,且有细胞凋亡现象。
结论我们筛选构建了理想的、能够有效抑制肺癌VEGF表达的shRNA质粒pSilencer-VEGF-2;该质粒在体外和体内均能够明显抑制肺癌的发展。其机制可能是通过shRNA沉默肺癌细胞VEGF的表达,抑制了血管内皮细胞的VEGF/VEGFR通路,从而引起肺癌细胞凋亡而实现的。
Bacground&Objective Angiogenesis is associated with several pathological disorders. Tumor growth and metastasis are dependent on angiogenesis. Components of vascular basement membrane are involved in regulating angiogenesis. The binding of VEGF to VEGFR expressing on the endothelial cells membrane, which triggered the signaling transduction, resulted in the mitosis and proliferation of endothelial cells, and promoted angiogenesis. The VEGF/VEGFR signaling pathway could enhance specially the penetration of blood vessel and protect the damaged neural cells, which plays an important role in the physiological and pathological process, including the embryonic development, inflammation and tumor. Previous studies showed that most solid tumor cloud secret the VEGF. What’s more, the VEGF expression was related with the malignant tumor development. Therefore, VEGF or VEGFR was the most potent target against tumor angiogenesis.
RNA interference (RNAi), which could silence the mRNA transcription specially, was a mechanism of post-transcription gene silence induced by the double stranded RNA. RNAi is not only a technology in study of genome function, but also an effective method to treat disease, especially, in gene therapy of the tumor. Studies demonstrated that the proliferation and invadation in many solid tumors could be inhibited with shRNA silencing the VEGF expression.In this study, to investigate the effects and mechanism of shRNA silencing VEGF gene expression in lung cancer.
Materials&Methods An effective and stable pSilencer-VEGF-shRNA plasmid to silence VEGF expression was screened and constructed successfully. A549 cells were transfected with pSilencer-VEGF-2 to observe its expression of VEGF, proliferation of the non small cell lung cancer and tubule formation of the endothelial cells in vitro. Finally the A549 cells and pSilencer-VEGF-A549 cells were injected subcutaneously into nude mice to observe the tumor growth and survival of the mice in vivo. Identify the changes of p53, Bcl-2 and Bax in VEGF interference A549 cells which interacted with HUVEC by RT-PCR and Western blot.
Results The results showed that the expression of VEGF in stable transfectant A549 cell line was obviously inhibited over 90% by pSilencer-VEGF-2. Two months later the inhibition rate was still above 80%. Although growth of the pSilencer-VEGF-A549 cells was hard inhibitedt, the angiogenesis of endothelial cells was decreased in vitro. Compared with the control, the tumor growth was significantly slow(P<0.05) and the survival was obviously enlonged in the mice injected pSilencer-VEGF-2-A549(P<0.05)in vivo.
In the mechanism study, we found that the P53, Bcl-2 and Bax expression did not change in the stable transfected A549 cell lines. And we did not observe the apoptosis of stable transfected A549 cells. Compared with the control group, the expression of Bax was not changed and the Bcl-2 expression was decreased, however the P53 expression increased with apoptosis in the stable transfected A549 cell lines under the HUVEC.
Conclusion The shRNA vector pSilencer-VEGF which could silence the VEGF expression in lung cancer effectively was successfully constructed. Proliferation of the lung cancer cells and development of the lung cancer were inhibited with RNAi silencing VEGF expression both in vivo and vitro. The possible mechanism might be due to the block of VEGF/VEGFR resulting in apoptosis of the cancer cells.
RNA干扰(RNA interference,RNAi)是指由双链RNA(double stranded RNA,dsRNA)引发的转录后基因沉默机制,能够高效特异地沉默同源基因的表达,是一种转录后调控。RNAi技术不仅在功能基因组研究中发挥了重要的作用,而且是一种潜在的以特异性基因沉默为方法治疗疾病的手段。尤其在肿瘤的干预中更具有潜在意义。目前,通过RNAi技术干涉VEGF的表达能够抑制胰腺癌、喉鳞癌、大肠癌和肺癌等细胞株的增殖、生长、侵袭和迁移。
本课题,我们利用RNAi干涉技术,沉默肺癌细胞中VEGF的表达,从而达到干预肺癌生长增殖的目的,为肺癌的基因治疗提供新的理论依据。期望能够获得一个持续高效的抑制肺癌细胞中VEGF表达的RNAi片段,从体外、内观察其对肺癌细胞增殖和肺内癌肿生长的影响,并探讨其干预机制。
材料和方法通过筛选获得理想的能够沉默肺癌细胞中VEGF表达的shRNA片段,并将其插入到载体中,制备成pSilencer-VEGF-shRNA;体外利用该质粒对肺癌A549细胞系进行干预后,动态观察A549细胞中VEGF的表达、肺癌细胞的生长及内皮细胞小管结构的形成;用A549和pSilencer-VEGF-A549细胞分别接种裸鼠,观察荷瘤裸鼠体内肿瘤组织的生长及生存期。使用RT-PCR和免疫印迹的方法检测与内皮细胞相互作用后干涉VEGF的A549细胞内p53、Bcl-2和Bax转录和蛋白水平上的变化。
研究结果经RT-PCR和免疫印迹实验检测发现pSilencer-VEGF-2对肺癌细胞中VEGF的抑制率能够达到90%以上;在稳定转染的肺癌细胞株A549细胞中,2个月以后对VEGF的抑制率仍在80%以上;肺癌细胞生长干预实验结果显示,pSilencer-VEGF-2明显抑制了肺癌A549细胞系中VEGF的表达;虽然稳定转染pSilencer-VEGF-2的肺癌A549细胞株体外生长未发生明显变化,但是,它可以显著抑制内皮细胞的血管生成作用。体内荷瘤裸鼠实验结果显示:与接种对照组荷肺癌A549细胞株瘤的裸鼠相比,pSilencer-VEGF-2-A549荷瘤裸鼠体内肿瘤组织的生长显著减慢(P<0.05),生存期明显延长(P<0.05)。pSilencer-VEGF-2干预肺癌增殖生长的机制研究显示,稳定转染的人肺癌A549细胞株中P53,Bcl-2和Bax的表达无明显改变,也未见到明显的细胞凋亡现象。与对照组相比,在人HUVEC细胞系的作用下,稳定转染的人A549细胞中Bax表达无变化,但P53的表达明显增高,Bcl-2的表达下调,且有细胞凋亡现象。
结论我们筛选构建了理想的、能够有效抑制肺癌VEGF表达的shRNA质粒pSilencer-VEGF-2;该质粒在体外和体内均能够明显抑制肺癌的发展。其机制可能是通过shRNA沉默肺癌细胞VEGF的表达,抑制了血管内皮细胞的VEGF/VEGFR通路,从而引起肺癌细胞凋亡而实现的。
Bacground&Objective Angiogenesis is associated with several pathological disorders. Tumor growth and metastasis are dependent on angiogenesis. Components of vascular basement membrane are involved in regulating angiogenesis. The binding of VEGF to VEGFR expressing on the endothelial cells membrane, which triggered the signaling transduction, resulted in the mitosis and proliferation of endothelial cells, and promoted angiogenesis. The VEGF/VEGFR signaling pathway could enhance specially the penetration of blood vessel and protect the damaged neural cells, which plays an important role in the physiological and pathological process, including the embryonic development, inflammation and tumor. Previous studies showed that most solid tumor cloud secret the VEGF. What’s more, the VEGF expression was related with the malignant tumor development. Therefore, VEGF or VEGFR was the most potent target against tumor angiogenesis.
RNA interference (RNAi), which could silence the mRNA transcription specially, was a mechanism of post-transcription gene silence induced by the double stranded RNA. RNAi is not only a technology in study of genome function, but also an effective method to treat disease, especially, in gene therapy of the tumor. Studies demonstrated that the proliferation and invadation in many solid tumors could be inhibited with shRNA silencing the VEGF expression.In this study, to investigate the effects and mechanism of shRNA silencing VEGF gene expression in lung cancer.
Materials&Methods An effective and stable pSilencer-VEGF-shRNA plasmid to silence VEGF expression was screened and constructed successfully. A549 cells were transfected with pSilencer-VEGF-2 to observe its expression of VEGF, proliferation of the non small cell lung cancer and tubule formation of the endothelial cells in vitro. Finally the A549 cells and pSilencer-VEGF-A549 cells were injected subcutaneously into nude mice to observe the tumor growth and survival of the mice in vivo. Identify the changes of p53, Bcl-2 and Bax in VEGF interference A549 cells which interacted with HUVEC by RT-PCR and Western blot.
Results The results showed that the expression of VEGF in stable transfectant A549 cell line was obviously inhibited over 90% by pSilencer-VEGF-2. Two months later the inhibition rate was still above 80%. Although growth of the pSilencer-VEGF-A549 cells was hard inhibitedt, the angiogenesis of endothelial cells was decreased in vitro. Compared with the control, the tumor growth was significantly slow(P<0.05) and the survival was obviously enlonged in the mice injected pSilencer-VEGF-2-A549(P<0.05)in vivo.
In the mechanism study, we found that the P53, Bcl-2 and Bax expression did not change in the stable transfected A549 cell lines. And we did not observe the apoptosis of stable transfected A549 cells. Compared with the control group, the expression of Bax was not changed and the Bcl-2 expression was decreased, however the P53 expression increased with apoptosis in the stable transfected A549 cell lines under the HUVEC.
Conclusion The shRNA vector pSilencer-VEGF which could silence the VEGF expression in lung cancer effectively was successfully constructed. Proliferation of the lung cancer cells and development of the lung cancer were inhibited with RNAi silencing VEGF expression both in vivo and vitro. The possible mechanism might be due to the block of VEGF/VEGFR resulting in apoptosis of the cancer cells.
引文
1. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003,9(6):669-676
2. Folkman J. Tumor angiogenesis: Therapeutic implication. N Engl J Med. 1971,285:1182- 1186
3. Fidler IJ, Ellis LM. The implications of angiogenesis for the biology and therapy of cancer metastasis. Cell. 1994,79(2):185-188
4. Folkman J. The vascula rization of tumors. Sci Am. l976,234(5): 58-64, 70-73
5. Baillie CT, Winslet MC, Bradley NJ. Tumor Vasculatur-apotential therapeutic target. Br J Cancer. 1995,72(2):257-267
6. 26.Weidner N. Tumour vascularity and proliferation: clear evidence of a close relationship. J Pathol. 1999,189(3):297-299
7. van der Schaft DW, Seftor RE, Seftor EA, Hess AR, Gruman LM, Kirschmann DA, Yokoyama Y, Griffioen AW, Hendrix MJ. Effects of angiogenesis inhibitors on vascular network formation by human endothelial and melanoma cells. J Natl Cancer Inst. 2004,96(19):1473-1477
8. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis; an imbalance of positive and negative regulation. Cell. 1991,64(2):327-336
9. Yoshiji H, Kuriyama S, Hicklin DJ, Huber J, Yoshii J, Miyamoto Y, Kawata M, Ikenaka Y, Nakatani T, Tsujinoue H, Fukui H. KDR/Flk-1 is a major regulator of vascular endothelial growth factor-induced tumor development and angiogenesis in murine hepatocellular carcinoma cells. Hepatology.1999,30(5):1179-1186
10. Folkman J, Beckner K. Angiogenesis imaging. Acad Radiol. 2000,7(10):783-785
11. Barbareschi M, Gasparini G, Morelli L, Forti S, Dalla Palma P. Novel methods for the determination of the angiogenic activity of human tumors. Breast Cancer Res Treat. 1995,36(2):181-192
12. Folberg R, Hendrix MJ, Maniotis AJ. Vasculogenic mimicry and tumor angiogenesis. Am J Pathol. 2000,156(2):361-381
13. Chang YS, di Tomaso E, McDonald DM, Jones R, Jain RK, Munn LL. Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. Proc Natl Acad Sci USA. 2000,97(26):14608-14613
14. Lee TY, Purdie TG, Stewart E. CT imaging of angiogenesis. Q J Nucl Med. 2003,47(3):171-187
15. Abeloff MD, Armitage JO, Lichter AS, Niederhuber JE. Clinical oncology, 2nd, Beijing Science Press. 2000,29-53
16. Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res. 1990,50(19):6130-6138
17. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev. 1992,13(1):18-32
18. Kieran MW, Billett A: Antiangiogenesis therapy, current and future agents. Hematol Oncol Clin North Am. 2001. 15(5): 835
19. Gospodarowicz D, Abraham JA, Schilling J. Isolation andcharacterization of a vascular endothelial growth factor cellmitogen produced by pituitary 2 derived folliculo stellate cells. Proc Natl Acad Sci USA, 1989, 86:7311- 7315.
20. Houck KA, Leung DW, Rowland AM, et al. Dual regulationof vascularendothelial growth factor bioavailability by goneticand proteolytic mechanisms. Biol Chem , 1992, 267:26031-26037
21. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and apotential target for diagnosis and therapy. Clin Oncol, 2002, 20:4368-4380
22. Vander Zee R , Murohara T, Luo Z, et al. Vascular endothelial growth factor/ vascular permeability factor augments nitric oxide recease from quiescent rabbit and human vascular endothelium. Circulation, 1997,95 (4):1030-1037
23. West A F, O′Donnell M, Charlton R G, et al. Correlation of vascular endothelial growth factor expression with fibroblast growth factor-8 exp- ression and clinico-pathologic parame2ters in human prostate cancer. Br J Cancer, 2001, 85(4): 576-583
24. Eisma RJ, Spiro JD, Krentzer DL. Vascular endothelialgrowth factor expression in head and neck squamous cellcarcinoma. Am J Surg, 1997, 174: 513-517
25.胡映波,习小庆. VEG在前列腺癌和良性前列腺增生组织中的表达.江西医学院学报, 2006, 46 ( 6): 12-16
26.李杨.血管内皮生长因子作为结直肠癌新生血管标志的临床价值.中国医药生物技术, 2006, 1 (1): 42-45
27. Stacker SA, Caesar C, Baldwin ME, et al. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med, 2001, 7(2): 186-191
28. Shariat SF, Anwuri VA, Lamb DJ , et al. Association of preoperative plasma levels of vascular endothelial growth factor and soluble vascular cell adhesion molecule-1 with lymph node status and biochemical progression after radi2cal prostatectomy. J Clin Oncol, 2004, 22(9): 1655-1663
29. Juttner S, Wissmann C, Jons T, et al. Vascular endothelial growth factor-D and its receptor VEGFR-3: two novel independent prognostic markers in gastric adenocarcinoma. J Clin Oncol, 2006, 24 (2): 228-240
30.肖继平,张莉,齐云,等. uPA及VEGF在乳腺癌中的表达及意义.中国普通外科杂志, 2007, 16 (1): 93-96
31. Cianchi F, Cortesini C, Bechi P, et al. Up-regulation of cyclooxygenase- gene expression correlate with tumor angiogenesis in human colorectal cancer. Gast roenterology, 2001, 121 (6): 1339
32. Hasegawa Y, Takanashi S, Okudera K, Kumagai M, Hayashi A, Morimoto T, Okumura K. Vascular endothelial growth factor level as a prognostic determinant of small cell lung cancer in Japanese patients. Intern Med. 2005,44(1):26-34
33. Shaheen RM, Davis DW, Liu W, Zebrowski BK, Wilson MR, Bucana CD, McConkey DJ, McMahon G, Ellis LM. Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis. Cancer Res. 1999,59(21):5412-5416
34. Riedel F, G?tte K, Goessler U, Sadick H, H?rmann K. Targeting chemotherapy-induced VEGF up-regulation by VEGF antisense oligonucleotides in HNSCC cell lines. Anticancer Res. 2004,24(4):2179-2183
35. Kamiyama M, Ichikawa Y, Ishikawa T, Chishima T, Hasegawa S, Hamaguchi Y, Nagashima Y, Miyagi Y, Mitsuhashi M, Hyndman D, Hoffman RM, Ohki S, Shimada H. VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice. Cancer Gene Ther. 2002,9(2):197-201
36. Hoekman K. SU6668, a multitargeted angiogenesis inhibitor. Cancer J. 2001,7 Suppl 3:S134-138
37. Ran S, Huang X, Downes A, et al. Evaluation of novel antimouse VEGFR2 antibodies as potential antiangiogenic or vascular targeting agents for tumor therapy. Neoplasia. 2003, 5(4):297-307
38. Ciardiello F, Caputo R, Damiano V, Caputo R, Troiani T, Vitagliano D, Carlomagno F, Veneziani BM, Fontanini G, Bianco AR, Tortora G. Antitumoreffects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase. Clin Cancer Res. 2003,9(4):1546-1556
39. Hess-Stumpp H, Haberey M, Thierauch KH. PTK 787/ZK 222584, a tyrosine kinase inhibitor of all known VEGF receptors, represses tumor growth with high efficacy. Chembiochem. 2005,6(3):550-557
40. Sengupta S, Sellers LA, Matheson HB, Fan TP. Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms. Br J Pharmacol. 2003,139(2):219-231
41. Huang DS, Shen KZ, Wei JF, Liang TB, Zheng SS, Xie HY. Specific COX-2 inhibitor NS398 induces apoptosis in human liver cancer cell line HepG2 through BCL-2. World J Gastroenterol. 2005,11(2):204-207
42. Fire A,Xu SQ,Montgomery M, et a1.Potentand specif genetic interference by double.stranded RNA in Camorhabditis elegans.Nature.1998.39l:806.1l
43. Jorgenson R.Altered gone expression in plants due to trans -interactions between homologous genes[J].Trends Biotechnol,1990,8(12):340-4
44. Guo S,Kemphues K J.Par-1,a gone required for establishing polarity in C.elegans embryos encodes a putative Ser/Thr kinase that is asymmetrically distributed[J].Cell,1995,81(4):611-20
45. Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR:siRNA primers conve~ mRNA into dsRNAs that are degraded to generatenew siRNAs[J].Cell,2001,107(3):297-307
46.林少微,王雪华,郑高哲,陈淑贞RNAi的研究进展[J]. 2007,4(29)
47. Petr Svoboda.Of targeting and other non.specific effects of RNAi experimeuts in nlanlnlalian cells[J] . Current Opinion in Molecular Therapeutics,2007,9(3):248
48. Fire A,Xu S,Montgonmry MK.et al Potent and specific genetic interfereneeby double stranded RNA in Caenorhabditis elegans[J].Nature.1998.391:806
49. Hannon G J.RNA interference[J].Nature,2002,418(6894):244
50. Trevor Stokes.RNAi-Based gene expression nmnipulation[J].Genetic Engineering News 2007,27(2):28
51. Mark SD,Edward EW.RNA interference:a practical approach[J].J Surg Res,2004,t17(2) 339
52.闵敏,高国兰. RNA干扰及其在肿瘤治疗中的研究进展[J].实用癌症杂志. 2008,23(5):537-39
53.史毅,金由辛. RNA干扰与siRNA(小干扰RNA)研究进展[J].生命科学, 2008, 20(2):196-201
54.秦玉新,蒙凌华,丁健. RNA干扰技术的研究进展[J]. Chinese Pharmacological Bulletin 2007 Apr;23(4):421-4
55. Overhof M , Alken M ,Far RK,et al. Local RNA target structure influences siRNA eficacy:a systematic global analysis.J Mol Biol,2005,348:871-881
56. Ui-Tei K,Nalto Y,Takahashi F,et a1.Guidelines for tbe selection of highly efective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res,2004,32:936-948
57. Ian RG,Stephen‘PF,Andrew JH,et a1.The design and exogenous delivery of siRNA for post-transcriptional gene silencing. J Drug Target,2004,12:315-340
58. Akhtar S,Hughes MD,Khan A,et a1.Th e delivery of antisense therapeutics.Adv Drug Deliv Rev,2000,44:3-21
59. Smith L,Andersen KB,Hovgaard L,et a1.Rational selection of antisenseoligonucleotide sequences.Eur J Pharm Sci,2000,11 l9l-l98
60. Aronin N.Target selectivity in mRNA silencing. Gene Ther,2006,13:509-516
61. Kim DH, Behlke MA, Rose SD, et a1. Synthetic dsRNA Dicer substrates enhance RNAi potency an d efficacy. Nat Biotechnol,2005,23:222-226
62. Pham JW ,Pellino儿,Lee YS,et a1. A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell,2004,117:83-94
63. Amarzguioui M, Rossi J, Kim D. Approaches for chemicalsynthesized siRNA and vector-mediated RNAi.FEBS Lett,2005,579:5794-5981
64. Reynolds A,Leake D,Boese Q,et a1.Rational siRNA design for A interference.Nat Biotechnol,2004,22:326-330
65. Th azha PP, Charies RA , Prasad D, et a1. Positional efect of chemical modification on short Interference RNA activity in mammalian cells.J Med Chem,2005,48:4247-4253
66. Harborth J, Elbashir SM, Vandenburgh K, et al. Sequence,chemical,and structural variation of small interfering RNAs an short hairpin RNAs and the efect on mam malian gene silencing.Antisense Nucleic Acid Drug Dev,2003,13:83-105
67. Muratovska A,Eccles MR.Conjugate for eficient delivery of shortinterfering RNA (siRNA)into mam malian cells.FEBS Lett,2004 ,558:63-68
68.胡颖,叶枫,谢幸. RNA干扰技术中siRNA设计原则的研究进展[J]. Int J Genet Dec 15,2007,Vol 30,No.6:419-23
69. Caplen N J.RNAi as a genetherapy approach[J].Expert Opin biol Ther,2003,3(4):575-86
70. Miyagishi M ,Taira K.Development and application of siRNA expression vector[J].Nucl Acids Res,2002,2(supp1):113-4
71. Audouy S A,Leij L F,Hoekstra D,Molema G.In vivo characteristics of cationic liposomes as delivery vectors for gene therapy[J].Pharm Res,2002,19(11):1599-605
72. Tousignant J D,Gates A L,Ingram L A,et a1.Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice[J].Hum C.ene Ther,2000,11(18):2493-513
73. MeCafrey A P,Meuse L,Pham T T,et a1.RNA interference in adult mice[J].Nature,2002 418(6893):38-9
74. Fu A L , Wang Y X , Sun M J . Naked DNA prevents soman intoxi-eatlon[J].Biochem Biophy Res Commun,2005,328(4):901-5
75. SongE , Lee S K , Wang J , et a1 . RNA interference targeting Fasprotectsmicefromfulminant hepatitis[J].Nat Med,2003,9(3):347-51
76. MeCafrey A P,Nakai H,Pandey K,et a1.Inhibition of hepatitisB virus in mice by RNA interference[J].Nat Biotechnol,2003,21(6):639-44
77. Filleur S,Courtin A,Ait.Si A1i S,et a1.SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor re-sistance to an tian giogenic thrombospondin-1 an d slows tumor vas-cularlzation and growth[J].Cancer Res,2003,63(14):3919-22
78. Judge A D,Bola G,Lee A C,et a1.Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo[J].MolTher,2006,13(3):494-505
79. Sorensen D R,Leirdal M,Sioud M.Gene silencing by systemic delivery of synthetic siRNAs in aduh mice[J].J Mol Biol,2003,327(4):761-6
80. Yin C,Xi L,Wang X,et a1.Silencing heat shock factor 1 by small interfering RNA abrogates heat shoc k-·induced cardioprotection againstischemia-reperfusion injury in mice[J].J Mol Cell Cardiol,2005,39(4):681-9
81. Dorn G,Patel S,Wotherspoon G,et a1. siRNA relieves chronic neuropathic pain[J].Nucleic Acids Res,2004,32(5):223-8
82. Thakker D R,Natt F,Husken D,et a1. Neurochemical and behavioral consequences of widespread gene knoc kdown in the adult mouse brain by using nonviral RNA interference[J].Proc Natl Acad Sci U ,20o4,101(49):17270-5
83. Thakker D R ,Natt F,Husken D ,et a1.siRNA.mediated knock down of the serotonin transporter in the adult mouse brain[J].Mol Psychiatry,2005,10(8):782-9,714
84. LuoM C,ZhangDQ,Ma SW,et a1.An eficientintrathecal de1ivery of small interfering RNA to the spinal cord an d peripheral neurons[J].Mol ,2005,1:29
85. Maeda Y,Fukushima K,Nishizaki K。et a1.In vitro and n vivo suppression of GJB2 expression by RNA interference[J].Hum Mol C.enet,2o05,14(12):1641-50
86. Schiffelers R M,Xu J,Storm G,et a1.Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis[J].Arthritis Rheum,2005,52(4):1314-8
87. Bitko V,Musiyenko A,Shulyayeva O ,et a1.Inhibition of respiratory viruses by nasally administered siRNA[J].Nat Med,2005,11(1):50-5
88. Brummelkamp TR,Niiman SM,Dirac AM,et a1.Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB[J].Nature,2003,424:797
89. Sumimoto H,Yamagata S,Shimizu A,et a1.Gene therapy for humansmall-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference[J].Gene Ther,2005,12(1):95
90. Sumimoto H,Hirata K,Yamaqata S,et a1.Effective inhibition of cell growth and invasion of melanoma by cornbined suppression of BRAF(V599E)and Skp2 with 1enti viral RNAi[J].Int J Cancer,2006,118(2):472
91. Zeng P,Wagoner HA,Pescovitz OH,et a1.RNA interference(RNAi)for extracellular signal-regulated kinase 1 (ERK1)alone is sufficient to suppress cell viability in ovarian cancer cellsEJ].Cancer Biol Ther,2005,4(9):961
92. Faltus T,Yuan J,Zimmer B,et a1.Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells [J].Neoplasia,2004,6(6):786
93. Ren W ,Duan Y,Ji Y,et a1.Down-regulation of Stat3 induces apoptosis of human glioma cell:a potential method to treat brain cancer[J].Neurol Res,2007,22
94. Li T,Chang CY,Jin DY,et a1.Identification of the gene for vitamin K epoxide reductase[J].Nature,2004,427(6974):541
95. Berns K,Hijmans EM,Mullenders J,et a1.A large-scale RNAi screen in human cells identifies new components of the p53 pathway[J].Nature,2004,428:431
96. Brummelkamp TR , Bernards R , Agami R . Stable suppression of tumorigenicity by virus ediated RNA interference[J].Cancer Cell,2002,2:243
97. Koldehoff M,Steckel NK,Beelen DW,et a1.Therapeutic application of small interfering RNA directed against bcr-ab1 transcripts to a patient with imatinib-resistant chronic myeloid leukaemia[J].Clin Exp Med,2007,7(2):47
98. Lima RT,Martins LM,Guimaraes JE,et a1.Specific downregulation of bcl一2 and xlAP by RNAi enhances the effects of chemotherapeutic agents in M CF-7 human breast cancer cells[J].Cancer Gene Ther,2004,11(5):309
99. van de Wetering M,Oving I,Muncan V,et a1.Specific inhibition of gene expression using a stably integrated , iducible small interfering-RNA vector[J].EMBO Rep,2003,4:609
100. Sciamanna I,Landriscina M,Pittoggi C,et a1.Inhibition of endogenous reverse transcriptase antagonizes human tumor growth[J].Oncogene,2005,24(24):3923
101. Wang R,Wang X,Gao P,et a1.Suppression of bcl—xL expression by a novel tumor-specific RNA interference systern inhibits proliferation and enhances radiosensitivity inprostatic carcinoma cells[J].Cancer Chemother Pharmacol,2007。26[Epub ahead of print]
102. Schulze-Berqkamen H,Fleischer B,Schuchmann M,et a1.Suppression of M cl—-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction[J].BMC Cancer,2006,6:232
103. Simpson KJ,Dugan AS,Mercurio AM.Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma[J].Cancer Res,2004,64(23):8694
104. Pille JY,Denoyelle C,Varet J,et a1.Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDAMB-231 breast cancer cells in vitro and in vivo[J].M o1 Ther,2005,11(2):267
105. Gondi CS,Lakka SS,Dinh DH,et a1.RNAi—mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion,angiogenesis and tumor growth in gliomas[J].Oncogene,2004,23(52):8486
106. Gondi CS,Lakka SS,Dinh DH,et a1.Intraperitoneal injection of a hairpin RNA-expressing plasmid targeting urokinase~type plasminogenactivator(uPA ) receptor and uPA retards angiogenesis and inhibits intracranial tumor growth in nude mice[J].Clin Cancer Res,2007,13(14):4051
107. Wang T,Tamae D.The role of peroxiredoxin 1I in radiation resistant MCF-7 breast cancer cells[J].Cancer Res,2005,65(22):10338
108. Pichler A,Zelcer N,Prior JL,et a1.In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein[J].Clin Cancer Res,200,511(12):4487
109. DiTullio RA,Mochan TA,Venere M,et a1.53BPI functions in an ATM-depo ndent checkpoint pathway that is constitutively vated in human cancer.Nat Cel Biol,2002,4(12):998
110. Giilin L'Karelsky S,An dino R.Short interfering RNA corLfers intracelular antiviral immunity in human cells.Nature,2002,418(6896):430
111. Coburn GA,Cullen BR.Potent an d specific inhibition of human unodeflcieney virus type l replication by RNA interference.J Viml,2002,76(18):9225
112. Ge Q,MeManus biT,Nguyen T'et a1.RNA interference of influenza virus production by directly targeting mRNA for gvariation and indirectly inhibiting all viral RNA transcription.Proe Natl Acad Sci U S A,2003,100(5):2718
113. Shan J, Brooks C, Kon N, et al. Dissecting roles of ubiquitination in the p53 pathway. Ernst Schering Found Symp Proc. 2008, (1):127-36
114. Saetta AA, Korkolopoulou P, Karlou M, et al. TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 alterations are not associated with microsatellite instability or p53 mutations in invasive urothelial carcinoma of the urinary bladder. Pathology. 2007, 39(4):425-32
115. Kroemer G, Petit P, Zamzami N, et al. The biochemistry of programmed cell death.FASEB J, 1995, 9:1277-1287
2. Folkman J. Tumor angiogenesis: Therapeutic implication. N Engl J Med. 1971,285:1182- 1186
3. Fidler IJ, Ellis LM. The implications of angiogenesis for the biology and therapy of cancer metastasis. Cell. 1994,79(2):185-188
4. Folkman J. The vascula rization of tumors. Sci Am. l976,234(5): 58-64, 70-73
5. Baillie CT, Winslet MC, Bradley NJ. Tumor Vasculatur-apotential therapeutic target. Br J Cancer. 1995,72(2):257-267
6. 26.Weidner N. Tumour vascularity and proliferation: clear evidence of a close relationship. J Pathol. 1999,189(3):297-299
7. van der Schaft DW, Seftor RE, Seftor EA, Hess AR, Gruman LM, Kirschmann DA, Yokoyama Y, Griffioen AW, Hendrix MJ. Effects of angiogenesis inhibitors on vascular network formation by human endothelial and melanoma cells. J Natl Cancer Inst. 2004,96(19):1473-1477
8. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis; an imbalance of positive and negative regulation. Cell. 1991,64(2):327-336
9. Yoshiji H, Kuriyama S, Hicklin DJ, Huber J, Yoshii J, Miyamoto Y, Kawata M, Ikenaka Y, Nakatani T, Tsujinoue H, Fukui H. KDR/Flk-1 is a major regulator of vascular endothelial growth factor-induced tumor development and angiogenesis in murine hepatocellular carcinoma cells. Hepatology.1999,30(5):1179-1186
10. Folkman J, Beckner K. Angiogenesis imaging. Acad Radiol. 2000,7(10):783-785
11. Barbareschi M, Gasparini G, Morelli L, Forti S, Dalla Palma P. Novel methods for the determination of the angiogenic activity of human tumors. Breast Cancer Res Treat. 1995,36(2):181-192
12. Folberg R, Hendrix MJ, Maniotis AJ. Vasculogenic mimicry and tumor angiogenesis. Am J Pathol. 2000,156(2):361-381
13. Chang YS, di Tomaso E, McDonald DM, Jones R, Jain RK, Munn LL. Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. Proc Natl Acad Sci USA. 2000,97(26):14608-14613
14. Lee TY, Purdie TG, Stewart E. CT imaging of angiogenesis. Q J Nucl Med. 2003,47(3):171-187
15. Abeloff MD, Armitage JO, Lichter AS, Niederhuber JE. Clinical oncology, 2nd, Beijing Science Press. 2000,29-53
16. Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res. 1990,50(19):6130-6138
17. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev. 1992,13(1):18-32
18. Kieran MW, Billett A: Antiangiogenesis therapy, current and future agents. Hematol Oncol Clin North Am. 2001. 15(5): 835
19. Gospodarowicz D, Abraham JA, Schilling J. Isolation andcharacterization of a vascular endothelial growth factor cellmitogen produced by pituitary 2 derived folliculo stellate cells. Proc Natl Acad Sci USA, 1989, 86:7311- 7315.
20. Houck KA, Leung DW, Rowland AM, et al. Dual regulationof vascularendothelial growth factor bioavailability by goneticand proteolytic mechanisms. Biol Chem , 1992, 267:26031-26037
21. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and apotential target for diagnosis and therapy. Clin Oncol, 2002, 20:4368-4380
22. Vander Zee R , Murohara T, Luo Z, et al. Vascular endothelial growth factor/ vascular permeability factor augments nitric oxide recease from quiescent rabbit and human vascular endothelium. Circulation, 1997,95 (4):1030-1037
23. West A F, O′Donnell M, Charlton R G, et al. Correlation of vascular endothelial growth factor expression with fibroblast growth factor-8 exp- ression and clinico-pathologic parame2ters in human prostate cancer. Br J Cancer, 2001, 85(4): 576-583
24. Eisma RJ, Spiro JD, Krentzer DL. Vascular endothelialgrowth factor expression in head and neck squamous cellcarcinoma. Am J Surg, 1997, 174: 513-517
25.胡映波,习小庆. VEG在前列腺癌和良性前列腺增生组织中的表达.江西医学院学报, 2006, 46 ( 6): 12-16
26.李杨.血管内皮生长因子作为结直肠癌新生血管标志的临床价值.中国医药生物技术, 2006, 1 (1): 42-45
27. Stacker SA, Caesar C, Baldwin ME, et al. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med, 2001, 7(2): 186-191
28. Shariat SF, Anwuri VA, Lamb DJ , et al. Association of preoperative plasma levels of vascular endothelial growth factor and soluble vascular cell adhesion molecule-1 with lymph node status and biochemical progression after radi2cal prostatectomy. J Clin Oncol, 2004, 22(9): 1655-1663
29. Juttner S, Wissmann C, Jons T, et al. Vascular endothelial growth factor-D and its receptor VEGFR-3: two novel independent prognostic markers in gastric adenocarcinoma. J Clin Oncol, 2006, 24 (2): 228-240
30.肖继平,张莉,齐云,等. uPA及VEGF在乳腺癌中的表达及意义.中国普通外科杂志, 2007, 16 (1): 93-96
31. Cianchi F, Cortesini C, Bechi P, et al. Up-regulation of cyclooxygenase- gene expression correlate with tumor angiogenesis in human colorectal cancer. Gast roenterology, 2001, 121 (6): 1339
32. Hasegawa Y, Takanashi S, Okudera K, Kumagai M, Hayashi A, Morimoto T, Okumura K. Vascular endothelial growth factor level as a prognostic determinant of small cell lung cancer in Japanese patients. Intern Med. 2005,44(1):26-34
33. Shaheen RM, Davis DW, Liu W, Zebrowski BK, Wilson MR, Bucana CD, McConkey DJ, McMahon G, Ellis LM. Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis. Cancer Res. 1999,59(21):5412-5416
34. Riedel F, G?tte K, Goessler U, Sadick H, H?rmann K. Targeting chemotherapy-induced VEGF up-regulation by VEGF antisense oligonucleotides in HNSCC cell lines. Anticancer Res. 2004,24(4):2179-2183
35. Kamiyama M, Ichikawa Y, Ishikawa T, Chishima T, Hasegawa S, Hamaguchi Y, Nagashima Y, Miyagi Y, Mitsuhashi M, Hyndman D, Hoffman RM, Ohki S, Shimada H. VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice. Cancer Gene Ther. 2002,9(2):197-201
36. Hoekman K. SU6668, a multitargeted angiogenesis inhibitor. Cancer J. 2001,7 Suppl 3:S134-138
37. Ran S, Huang X, Downes A, et al. Evaluation of novel antimouse VEGFR2 antibodies as potential antiangiogenic or vascular targeting agents for tumor therapy. Neoplasia. 2003, 5(4):297-307
38. Ciardiello F, Caputo R, Damiano V, Caputo R, Troiani T, Vitagliano D, Carlomagno F, Veneziani BM, Fontanini G, Bianco AR, Tortora G. Antitumoreffects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase. Clin Cancer Res. 2003,9(4):1546-1556
39. Hess-Stumpp H, Haberey M, Thierauch KH. PTK 787/ZK 222584, a tyrosine kinase inhibitor of all known VEGF receptors, represses tumor growth with high efficacy. Chembiochem. 2005,6(3):550-557
40. Sengupta S, Sellers LA, Matheson HB, Fan TP. Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms. Br J Pharmacol. 2003,139(2):219-231
41. Huang DS, Shen KZ, Wei JF, Liang TB, Zheng SS, Xie HY. Specific COX-2 inhibitor NS398 induces apoptosis in human liver cancer cell line HepG2 through BCL-2. World J Gastroenterol. 2005,11(2):204-207
42. Fire A,Xu SQ,Montgomery M, et a1.Potentand specif genetic interference by double.stranded RNA in Camorhabditis elegans.Nature.1998.39l:806.1l
43. Jorgenson R.Altered gone expression in plants due to trans -interactions between homologous genes[J].Trends Biotechnol,1990,8(12):340-4
44. Guo S,Kemphues K J.Par-1,a gone required for establishing polarity in C.elegans embryos encodes a putative Ser/Thr kinase that is asymmetrically distributed[J].Cell,1995,81(4):611-20
45. Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR:siRNA primers conve~ mRNA into dsRNAs that are degraded to generatenew siRNAs[J].Cell,2001,107(3):297-307
46.林少微,王雪华,郑高哲,陈淑贞RNAi的研究进展[J]. 2007,4(29)
47. Petr Svoboda.Of targeting and other non.specific effects of RNAi experimeuts in nlanlnlalian cells[J] . Current Opinion in Molecular Therapeutics,2007,9(3):248
48. Fire A,Xu S,Montgonmry MK.et al Potent and specific genetic interfereneeby double stranded RNA in Caenorhabditis elegans[J].Nature.1998.391:806
49. Hannon G J.RNA interference[J].Nature,2002,418(6894):244
50. Trevor Stokes.RNAi-Based gene expression nmnipulation[J].Genetic Engineering News 2007,27(2):28
51. Mark SD,Edward EW.RNA interference:a practical approach[J].J Surg Res,2004,t17(2) 339
52.闵敏,高国兰. RNA干扰及其在肿瘤治疗中的研究进展[J].实用癌症杂志. 2008,23(5):537-39
53.史毅,金由辛. RNA干扰与siRNA(小干扰RNA)研究进展[J].生命科学, 2008, 20(2):196-201
54.秦玉新,蒙凌华,丁健. RNA干扰技术的研究进展[J]. Chinese Pharmacological Bulletin 2007 Apr;23(4):421-4
55. Overhof M , Alken M ,Far RK,et al. Local RNA target structure influences siRNA eficacy:a systematic global analysis.J Mol Biol,2005,348:871-881
56. Ui-Tei K,Nalto Y,Takahashi F,et a1.Guidelines for tbe selection of highly efective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res,2004,32:936-948
57. Ian RG,Stephen‘PF,Andrew JH,et a1.The design and exogenous delivery of siRNA for post-transcriptional gene silencing. J Drug Target,2004,12:315-340
58. Akhtar S,Hughes MD,Khan A,et a1.Th e delivery of antisense therapeutics.Adv Drug Deliv Rev,2000,44:3-21
59. Smith L,Andersen KB,Hovgaard L,et a1.Rational selection of antisenseoligonucleotide sequences.Eur J Pharm Sci,2000,11 l9l-l98
60. Aronin N.Target selectivity in mRNA silencing. Gene Ther,2006,13:509-516
61. Kim DH, Behlke MA, Rose SD, et a1. Synthetic dsRNA Dicer substrates enhance RNAi potency an d efficacy. Nat Biotechnol,2005,23:222-226
62. Pham JW ,Pellino儿,Lee YS,et a1. A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell,2004,117:83-94
63. Amarzguioui M, Rossi J, Kim D. Approaches for chemicalsynthesized siRNA and vector-mediated RNAi.FEBS Lett,2005,579:5794-5981
64. Reynolds A,Leake D,Boese Q,et a1.Rational siRNA design for A interference.Nat Biotechnol,2004,22:326-330
65. Th azha PP, Charies RA , Prasad D, et a1. Positional efect of chemical modification on short Interference RNA activity in mammalian cells.J Med Chem,2005,48:4247-4253
66. Harborth J, Elbashir SM, Vandenburgh K, et al. Sequence,chemical,and structural variation of small interfering RNAs an short hairpin RNAs and the efect on mam malian gene silencing.Antisense Nucleic Acid Drug Dev,2003,13:83-105
67. Muratovska A,Eccles MR.Conjugate for eficient delivery of shortinterfering RNA (siRNA)into mam malian cells.FEBS Lett,2004 ,558:63-68
68.胡颖,叶枫,谢幸. RNA干扰技术中siRNA设计原则的研究进展[J]. Int J Genet Dec 15,2007,Vol 30,No.6:419-23
69. Caplen N J.RNAi as a genetherapy approach[J].Expert Opin biol Ther,2003,3(4):575-86
70. Miyagishi M ,Taira K.Development and application of siRNA expression vector[J].Nucl Acids Res,2002,2(supp1):113-4
71. Audouy S A,Leij L F,Hoekstra D,Molema G.In vivo characteristics of cationic liposomes as delivery vectors for gene therapy[J].Pharm Res,2002,19(11):1599-605
72. Tousignant J D,Gates A L,Ingram L A,et a1.Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice[J].Hum C.ene Ther,2000,11(18):2493-513
73. MeCafrey A P,Meuse L,Pham T T,et a1.RNA interference in adult mice[J].Nature,2002 418(6893):38-9
74. Fu A L , Wang Y X , Sun M J . Naked DNA prevents soman intoxi-eatlon[J].Biochem Biophy Res Commun,2005,328(4):901-5
75. SongE , Lee S K , Wang J , et a1 . RNA interference targeting Fasprotectsmicefromfulminant hepatitis[J].Nat Med,2003,9(3):347-51
76. MeCafrey A P,Nakai H,Pandey K,et a1.Inhibition of hepatitisB virus in mice by RNA interference[J].Nat Biotechnol,2003,21(6):639-44
77. Filleur S,Courtin A,Ait.Si A1i S,et a1.SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor re-sistance to an tian giogenic thrombospondin-1 an d slows tumor vas-cularlzation and growth[J].Cancer Res,2003,63(14):3919-22
78. Judge A D,Bola G,Lee A C,et a1.Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo[J].MolTher,2006,13(3):494-505
79. Sorensen D R,Leirdal M,Sioud M.Gene silencing by systemic delivery of synthetic siRNAs in aduh mice[J].J Mol Biol,2003,327(4):761-6
80. Yin C,Xi L,Wang X,et a1.Silencing heat shock factor 1 by small interfering RNA abrogates heat shoc k-·induced cardioprotection againstischemia-reperfusion injury in mice[J].J Mol Cell Cardiol,2005,39(4):681-9
81. Dorn G,Patel S,Wotherspoon G,et a1. siRNA relieves chronic neuropathic pain[J].Nucleic Acids Res,2004,32(5):223-8
82. Thakker D R,Natt F,Husken D,et a1. Neurochemical and behavioral consequences of widespread gene knoc kdown in the adult mouse brain by using nonviral RNA interference[J].Proc Natl Acad Sci U ,20o4,101(49):17270-5
83. Thakker D R ,Natt F,Husken D ,et a1.siRNA.mediated knock down of the serotonin transporter in the adult mouse brain[J].Mol Psychiatry,2005,10(8):782-9,714
84. LuoM C,ZhangDQ,Ma SW,et a1.An eficientintrathecal de1ivery of small interfering RNA to the spinal cord an d peripheral neurons[J].Mol ,2005,1:29
85. Maeda Y,Fukushima K,Nishizaki K。et a1.In vitro and n vivo suppression of GJB2 expression by RNA interference[J].Hum Mol C.enet,2o05,14(12):1641-50
86. Schiffelers R M,Xu J,Storm G,et a1.Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis[J].Arthritis Rheum,2005,52(4):1314-8
87. Bitko V,Musiyenko A,Shulyayeva O ,et a1.Inhibition of respiratory viruses by nasally administered siRNA[J].Nat Med,2005,11(1):50-5
88. Brummelkamp TR,Niiman SM,Dirac AM,et a1.Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB[J].Nature,2003,424:797
89. Sumimoto H,Yamagata S,Shimizu A,et a1.Gene therapy for humansmall-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference[J].Gene Ther,2005,12(1):95
90. Sumimoto H,Hirata K,Yamaqata S,et a1.Effective inhibition of cell growth and invasion of melanoma by cornbined suppression of BRAF(V599E)and Skp2 with 1enti viral RNAi[J].Int J Cancer,2006,118(2):472
91. Zeng P,Wagoner HA,Pescovitz OH,et a1.RNA interference(RNAi)for extracellular signal-regulated kinase 1 (ERK1)alone is sufficient to suppress cell viability in ovarian cancer cellsEJ].Cancer Biol Ther,2005,4(9):961
92. Faltus T,Yuan J,Zimmer B,et a1.Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells [J].Neoplasia,2004,6(6):786
93. Ren W ,Duan Y,Ji Y,et a1.Down-regulation of Stat3 induces apoptosis of human glioma cell:a potential method to treat brain cancer[J].Neurol Res,2007,22
94. Li T,Chang CY,Jin DY,et a1.Identification of the gene for vitamin K epoxide reductase[J].Nature,2004,427(6974):541
95. Berns K,Hijmans EM,Mullenders J,et a1.A large-scale RNAi screen in human cells identifies new components of the p53 pathway[J].Nature,2004,428:431
96. Brummelkamp TR , Bernards R , Agami R . Stable suppression of tumorigenicity by virus ediated RNA interference[J].Cancer Cell,2002,2:243
97. Koldehoff M,Steckel NK,Beelen DW,et a1.Therapeutic application of small interfering RNA directed against bcr-ab1 transcripts to a patient with imatinib-resistant chronic myeloid leukaemia[J].Clin Exp Med,2007,7(2):47
98. Lima RT,Martins LM,Guimaraes JE,et a1.Specific downregulation of bcl一2 and xlAP by RNAi enhances the effects of chemotherapeutic agents in M CF-7 human breast cancer cells[J].Cancer Gene Ther,2004,11(5):309
99. van de Wetering M,Oving I,Muncan V,et a1.Specific inhibition of gene expression using a stably integrated , iducible small interfering-RNA vector[J].EMBO Rep,2003,4:609
100. Sciamanna I,Landriscina M,Pittoggi C,et a1.Inhibition of endogenous reverse transcriptase antagonizes human tumor growth[J].Oncogene,2005,24(24):3923
101. Wang R,Wang X,Gao P,et a1.Suppression of bcl—xL expression by a novel tumor-specific RNA interference systern inhibits proliferation and enhances radiosensitivity inprostatic carcinoma cells[J].Cancer Chemother Pharmacol,2007。26[Epub ahead of print]
102. Schulze-Berqkamen H,Fleischer B,Schuchmann M,et a1.Suppression of M cl—-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction[J].BMC Cancer,2006,6:232
103. Simpson KJ,Dugan AS,Mercurio AM.Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma[J].Cancer Res,2004,64(23):8694
104. Pille JY,Denoyelle C,Varet J,et a1.Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDAMB-231 breast cancer cells in vitro and in vivo[J].M o1 Ther,2005,11(2):267
105. Gondi CS,Lakka SS,Dinh DH,et a1.RNAi—mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion,angiogenesis and tumor growth in gliomas[J].Oncogene,2004,23(52):8486
106. Gondi CS,Lakka SS,Dinh DH,et a1.Intraperitoneal injection of a hairpin RNA-expressing plasmid targeting urokinase~type plasminogenactivator(uPA ) receptor and uPA retards angiogenesis and inhibits intracranial tumor growth in nude mice[J].Clin Cancer Res,2007,13(14):4051
107. Wang T,Tamae D.The role of peroxiredoxin 1I in radiation resistant MCF-7 breast cancer cells[J].Cancer Res,2005,65(22):10338
108. Pichler A,Zelcer N,Prior JL,et a1.In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein[J].Clin Cancer Res,200,511(12):4487
109. DiTullio RA,Mochan TA,Venere M,et a1.53BPI functions in an ATM-depo ndent checkpoint pathway that is constitutively vated in human cancer.Nat Cel Biol,2002,4(12):998
110. Giilin L'Karelsky S,An dino R.Short interfering RNA corLfers intracelular antiviral immunity in human cells.Nature,2002,418(6896):430
111. Coburn GA,Cullen BR.Potent an d specific inhibition of human unodeflcieney virus type l replication by RNA interference.J Viml,2002,76(18):9225
112. Ge Q,MeManus biT,Nguyen T'et a1.RNA interference of influenza virus production by directly targeting mRNA for gvariation and indirectly inhibiting all viral RNA transcription.Proe Natl Acad Sci U S A,2003,100(5):2718
113. Shan J, Brooks C, Kon N, et al. Dissecting roles of ubiquitination in the p53 pathway. Ernst Schering Found Symp Proc. 2008, (1):127-36
114. Saetta AA, Korkolopoulou P, Karlou M, et al. TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 alterations are not associated with microsatellite instability or p53 mutations in invasive urothelial carcinoma of the urinary bladder. Pathology. 2007, 39(4):425-32
115. Kroemer G, Petit P, Zamzami N, et al. The biochemistry of programmed cell death.FASEB J, 1995, 9:1277-1287