摘要
背景与目的:结肠癌是常见多发的恶性肿瘤之一,在全球男性、女性常见的恶性肿瘤中分别排第四位和第三位。在我国,结肠癌的发病率居全部恶性肿瘤的第四位。目前结肠癌的治疗以手术治疗为主,辅以化疗、放疗等。由于发病隐匿,大部分患者就诊时已属中晚期,失去手术机会,而化疗、放疗有严重的全身毒副作用,特异性差。因此,基因靶向治疗结肠癌是目前研究的热点。基因治疗的载体包括病毒载体和非病毒载体两类。病毒载体有免疫原性,可诱发机体产生免疫反应,在安全性方面存在潜在的危险。而非病毒载体无毒性,安全性高,故在研究中应用更多。磷酸钙纳米颗粒(CPNP)作为一种非病毒载体,具有转染效率高、对人体无毒副作用、使用安全性高等特点。自杀基因如CD/5-FC、HSV-TK/GCV系统对结肠癌有杀伤作用,且CD、TK两者联合之后的协同作用较之单独作用能更好地发挥对肿瘤的杀伤作用。肿瘤血管生成对肿瘤的生长、转移起到至关重要的作用,VEGF是促进肿瘤血管生成的重要因子,使用RNA干扰技术高度特异性地沉默VEGF表达,可有效抑制肿瘤血管生成,从而达到抑制肿瘤生长的目的。因此,本实验使用非病毒载体CPNP作为基因转染载体,应用CMV增强子、CEA启动子与融合自杀基因yCDglyTK整合,联合应用针对VEGF的RNA干扰技术,研究其对人结肠癌Lovo细胞的体外靶向性杀伤作用。
方法:将结肠癌Lovo细胞分五组,空白对照组(L1组),其它四组分别为用CPNP为载体瞬时转染pcDNA3.1(-) Null (L2组)、pcDNA3.1(-)CVyCDglyTK (L3组)、pGenesil-shVEGF(L4组)、pcDNA3.1(-) shVEGF/yCDglyTK (L5组)等,用RT-PCR、免疫荧光检测yCDglyTK、VEGF基因的表达,用MTT法和流式细胞仪检测5-FC对各组细胞的杀伤作用和凋亡效率。实验数据采用SPSS 13.0和Excel2003进行统计分析,P<0.05认为有统计学意义。
结果:1、yCDglyTK及VEGF基因表达:(1)L3组、L5组有yCDglyTK基因mRNA及蛋白表达的增强;(2)各组细胞均有VEGF基因mRNA及蛋白的表达,其中L4组、L5组的表达较其它三组减弱。以上说明CPNP能成功介导四种质粒转染Lovo细胞。
2、转染CPNP-DNA对各组Lovo细胞的增殖抑制作用:MTT结果显示L5组在5-FC的作用下生存率低于其他各组(P 3、转染CPNP-DNA对各组Lovo细胞的凋亡作用:流式细胞仪结果显示各组细胞凋亡率分别为:L1组1.92%,L2组5.57%,L3组43.8%,L4 20.2%,L5组67.8%,其中L5凋亡率最高,说明转染联合基因治疗体系pcDNA3.1(-)shVEGF/yCDglyTK对Lovo细胞的凋亡作用较单独转染自杀基因yCDglyTK或shVEGF强。
结论:1、磷酸钙纳米颗粒能成功介导pcDNA3.1(-)Null. pcDNA3.1(-)CVyCDglyTK、pGenesil-shVEGF、pcDNA3.1(-)shVEGF /yCDglyTK等四种质粒转染结肠癌Lovo细胞。
2、yCDglyTK基因能有效抑制Lovo细胞的增殖,VEGF-shRNA能有效抑制VEGF的表达。
3、联合基因治疗体系pcDNA3.1(-)shVEGF/yCDglyTK较单独的自杀基因治疗及RNA干扰更有效地杀伤结肠癌Lovo细胞。
Background and Aim:Colon carcinoma is one of most common malignancies, which is the fourth most common cancer in men and the third in women worldwide. In China, the incidence is in the fourth place of all malignancies. Nowadays the chief therapy of colon carcinoma is surgery, coupled with radiotherapy and chemotherapy. A majority of patients has been found in the advanced stage and loses the chance of surgery because the symptoms are occult. Furthermore, radiotherapy and chemotherapy have a severe toxic and side-effect and weak specificity. Consequently, the gene targeted therapy of colon carcinoma has become the focus of researches. The vectors of gene therapy consist of two kinds, viral vectors and non-viral vectors. Because of immunogenicity, viral vectors can induce immunological reaction of body and have potential risk of safety, while non-viral vectors are non-toxic and safe, which are applied more in researches. As a kind of non-viral vectors, the calcium phosphate nanoparticle (CPNP) has many nice characteristics like high transfection efficiency, nonpoisonous side effect to human body, and high safety and so forth. The suicide genes such as CD/5-FC and HSV-TK/GCV systems have a lethal effect on colon carcinoma cells. Furthermore, the synergistic action of the unity of CD and TK has a more powerful lethal effect on carcinoma cells than each of them. Tumor angiogenesis plays a vital role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is one of most important substances to promote tumor angiogenesis. RNA interference can efficiently inhibit tumor angiogenesis by highly specific silence of VEGF gene expression, and eventually lead to the purpose of tumor growth inhibition. Hence, in this experiment, the non-virus vector CPNP is used as a gene transfection vector, and the integration of cytomegalovirus (CMV) enhancer, carcinoembryonic antigen (CEA) promoter and fusion suicide gene yCDglyTK combined with RNA interference of VEGF has a targeted lethal effect on human colon cancer cells Lovo in vitro.
Method:The Lovo cells were divided into five group, one of which was the blank controller (Group L1), and the rest were separately transiently transfected into pcDNA3.1 (-) Null (Group L2),pcDNA3.1 (-) CVyCDglyTK (Group L3),pGenesil-shVEGF (Group L4),pcDNA3.1(-)-shVEGF/yCDglyTK(Group L5) using CPNP as a vector. The expression of the yCDglyTK and VEGF gene was detected by RT-PCR and immunofluorescence. MTT assay and flow cytometry (FCM) were used to detect the cytotoxic effects and apoptosis rates of the yCDglyTK/ 5-FC system. SPSS 13.0 and Excel 2003 were used in data analysis. There was a statistical significance when P<0.05.
Results:1. The expression of yCDglyTK and VEGF genes:(1) The expression of yCDglyTK mRNA and protein was enhanced in Group L3 and L5; (2) All groups expressed VEGF mRNA and protein, but the expression was weakened in Group L4 and L5. It showed that CPNP can successfully induce the transfection of four plasmids into Lovo cells.
2. The anti-proliferative effect of transfection of CPNP-DNA in Lovo cells:MTT analysis showed that Group L5 had the lowest survival rate of all on the effect of 5-FC (P<0.05). It showed that transfection of the united gene therapic system pcDNA3.1(-)shVEGF/yCDglyTK had a more potential anti-proliferative effect on Lovo cells than yCDglyTK or shVEGF separately.
3. The apoptosis of transfection of CPNP-DNA in Lovo cells:The apoptosis rates of FCM were as follows:Group L1 1.92%, Group L2 5.57%, Group L3 43.8%, Group L4 20.2%, Group L5 67.8%. Group L5 had the highest apoptoticrate. It showed that the transfection of the united gene therapic system pcDNA3.1(-)shVEGF/yCDglyTK led to higher apoptosis in Lovo cells than yCDglyTK or shVEGF separately.
Conclusions:1.CPNP can successfully induce the transfection of pcDNA3.1(-)Null, pcDNA3.1(-)CVyCDglyTK, pGenesil-shVEGF and pcDNA3.1(-)shVEGF/yCDglyTK into colon cancer Lovo cells.
2. The yCDglyTK gene can effeciently inhibit the proliferation of Lovo cells, and VEGF-shRNA can efficiently inhibit the expression of VEGF.
3. The united gene therapic system of pcDNA3.1(-)shVEGF/yCD -glyTK is much more efficienct than suicide gene therapy or RNA interference separately in killing colon cancer Lovo cells.
引文
[1]Melissa M. Center, Ahmedin Jemal, Robert A. Smith, et al. Worldwide Variations in Colorectal Cancer. CA Cancer J Clin 2009;59;366-378.
[2]王强,郑海涛,丁德祥.结直肠癌的流行病学和筛查进展.中国现代医生,2008年6月第46卷第18期:103-104.
[3]Blagbrough IS, Zara C. Animal models for target diseases in gene therapy—using DNA and siRNA delivery strategies. Pharm Res.2009 Jan; 26(1):1-18.
[4]Liu L, Wang S, Shan B, et al. Advances in viral-vector systemic cytokine gene therapy against cancer. Vaccine.2010 Apr 4.
[5]Collins SA, Guinn BA, Harrison PT, et al. Viral vectors in cancer immunotherapy:which vector for which strategy? Curr Gene Ther.2008 Apr; 8 (2):66-78.
[6]Nayak S, Herzog RW. Progress and prospects:immune responses to viral vectors. Gene Ther.2010 Mar;17(3):295-304.
[7]Andrieu-Soler C, Bejjani RA, de Bizemont T, et al. Ocular gene therapy: a review of nonviral strategies. Mol Vis.2006 Oct 30;12:1334-47.
[8]Karmali PP, Chaudhuri A. Cationic liposomes as non-viral carriers of gene medicines:resolved issues, open questions, and future promises. Med Res Rev.2007 Sep;27(5):696-722.
[9]Li SD, Huang L. Gene therapy progress and prospects:non-viral gene therapy by systemic delivery. Gene Ther.2006 Sep;13(18):1313-9.
[10]Kodama K, Katayama Y, Shoji Y, et al. The features and shortcomings for gene delivery of current non-viral carriers. Curr Med Chem. 2006:13(18):2155-61.
[11]Xu G, Zhang N. Nanoparticles for gene delivery:a brief patent review. Recent Pat Drug Deliv Formul.2009;3(2):125-36.
[12]Stephen L. Hart. Multifunctional nanocomplexes for gene transfer and gene therapy. Cell Biol Toxicol.2010 Feb;26(1):69-81.
[13]Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev. 2009 Feb;109(2):259-302.
[14]Liu T, Tang A, Zhang G, et al. Calcium phosphate nanoparticles as a novel nonviral vector for efficient transfection of DNA in cancer gene therapy. Cancer Biother Radiopharm.2005 Apr;20(2):141-9.
[15]Khurana B, Goyal AK, Budhiraja A, et al. siRNA delivery using nanocarriers-an efficient tool for gene silencing. Curr Gene Ther. 2010 Apr;10(2):139-55.
[16]Agrawal N, Dasaradhi PV, Mohmmed A, et al. RNA interference:biology, mechanism, and applications. Microbiol Mol Biol Rev.2003 Dec; 67(4):657-85.
[17]程杉,熊英,贺俊崎.RNA干扰及其在肿瘤研究中的应用.首都医科大学学报,2008年2月第29卷第1期:87-95.
[18]He S, Zhang D, Cheng F, et al. Applications of RNA interference in cancer therapeutics as a powerful tool for suppressing gene expression. Mol Biol Rep.2009 Nov;36 (8):2153-63.
[19]Rassouli FB, Matin MM. Gene silencing in human embryonic stem cells by RNA interference. Biochem Biophys Res Commun.2009 Dec 25;390(4):1106-10.
[20]Shinkaruk S, Bayle M, Lain G, et al. Vascular endothelial cell growth factor (VEGF), an emerging target for cancer chemotherapy. Curr Med Chem Anticancer Agents.2003 Mar;3(2):95-117.
[21]徐伟,刘政,季国忠.血管内皮生长因子在消化系肿瘤中的研究进展.医学研究生学报,2009年10月第22卷第10期:1084-1087.
[22]Moreira IS, Fernandes PA, Ramos MJ. Vascular endothelial growth factor (VEGF) inhibition—a critical review. Anticancer Agents Med Chem.2007 Mar;7(2):223-45.
[23]Ellis LM, Hicklin DJ. VEGF-targeted therapy:mechanisms of anti-tumour activity. Nat Rev Cancer.2008 Aug;8(8):579-91.
[24]Hsu JY, Wakelee HA, Monoclonal antibodies targeting vascular endothelial growth factor:current status and future challenges in cancer therapy. BioDrugs.2009;23(5):289-304.
[25]Tie-Jun Li, Jian-Ning Song, Kai Kang, et al. RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells. World J Gastroenterol 2007 October 28; 13(40): 5312-5316.
[26]张树春,白松,邵佳发,等.大肠癌基因治疗的研究进展.河南外科学杂志,2009年11月第15卷第6期:59-61.
[27]Luo Q, Lu YZ, Liu GY, et al. Anti-tumor and bystander effects of cationic liposome-mediated CD/5-FC suicide gene system combined with interferon-gamma in vivo. Nan Fang Yi Ke Da Xue Xue Bao.2008 Aug;28 (9):1621-5.
[28]Armen Azatian,Hong Yu, Wande Dai, et al. Effectiveness of HSV-tk suicide gene therapy driven by the Grp78 stress-inducible promoter in esophagogastric junction and gastric adenocarcinomas. J Gastrointest Surg.2009 Jun;13(6):1044-51.
[29]Xia K, Liang D, Tang A, et al. A novel fusion suicide gene yeast CDglyTK plays a role in radio-gene therapy of nasopharyngeal carcinoma. Cancer Gene Ther.2004 Dec;11(12):790-6.
[30]Liu T, Zhang G, Chen YH, et al. Tissue specific expression of suicide genes delivered by nanoparticles inhibits gastric carcinoma growth. Cancer Biol Ther.2006 Dec;5(12):1683-90.
[31]Goldstein MJ, Mitchell EP. Carcinoembryonic antigen in the staging and follow-up of patients with colorectal cancer. Cancer Invest. 2005:23(4):338-51.
[32]Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LM02-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science.2003 Oct 17; 302(5644):415-9.
[33]Li SD, Huang LJ. Non-viral is superior to viral gene delivery. Control Release.2007 Nov 20; 123(3):181-3.
[34]Edelstein ML, Abedi MR, Wixon J, et al. Gene therapy clinical trials worldwide 1989-2004-an overview. J Gene Med.2004 Jun; 6(6):597-602.
[35]Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2007--an update. J Gene Med.2007 Oct; 9(10):833-42.
[36]Maitra A. Calcium phosphate nanoparticles:second-generation nonviral vectors in gene therapy. Expert Rev Mol Diagn.2005 Nov; 5(6):893-905.
[37]Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003; 3:401-410.
[38]Carmeliet P. VEGF as a key mediator of angiogenesis in cancer. Oncology.2005; 69 Suppl 3:4-10.
[39]Roskoski RJr. Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol.2007 Jun; 62 (3):179-213.
[40]黎明,彭勃.血管内皮生长因子与结直肠癌淋巴结转移的研究进展.中国老年学杂志,2009年11月第29卷页码2990-2992.
[41]Willett CG, Boucher Y, di Tomaso E, et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004; 10:145-147.
[42]Tokatlian T, Segura T.siRNA applications in nanomedicine. Wiley Interdiscip. Rev Nanomed Nanobiotechnol.2010 May;2(3):305-15.
[43]Bertrand J-R, Pottier M, Vekris A, et al. Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem Biophys Res Commun 2002,296:1000-1004.
[44]靳西凤,冉志华.RNA干扰与结肠癌.世界华人消化杂志,2006年7月18日,14(20):2003-2008.
[45]Takahashi Y, Nishikawa M, Takakura Y. Nonviral vector-mediated RNA interference:its gene silencing characteristics and important factors to achieve RNAi-based gene therapy. Adv Drug Deliv Rev.2009 Jul 25;61(9):760-6.
[46]Takahashi Y, Yamaoka K, Nishikawa M, et al. Quantitative and temporal analysis of gene silencing in tumor cells induced by small interfering RNA or short hairpin RNA expressed from plasmid vectors. J Pharm Sci. 2009 Jan;98(1):74-80.
[47]Niculescu-Duvaz I, Springer CJ. Introduction to the background, principles, and state of the art in suicide gene therapy. Mol Biotechnol.2005 May;30(1):71-88.
[48]Kievit E, Nyati MK, Ng E, et al. Yeast cytosine deaminase improves radiosensitization and bystander effect by 5-fluorocytosine of human colorectal cancer xenografts. Cancer Res.2000 Dec 1; 60 (23):6649-55.
[49]Portsmouth D, Hlavaty J, Renner M. Suicide genes for cancer therapy. Mol Aspects Med.2007 Feb;28(1):4-41.
[50]徐文贵,鲍润贤.肿瘤自杀基因靶向治疗的研究进展.中国肿瘤临床,2009 年第36卷第13期:777-780.
[51]屈二军,张现青,陈兰英.自杀基因治疗在消化道肿瘤中的应用.癌症进展杂志2008年11月第6卷第6期:591-594.
[52]Barton KN, Paielli D, Zhang Y, et al. Second-generation replication-competent oncolytic adenovirus armed with improved suicide genes and ADP gene demonstrates greater efficacy without increased toxicity. Mol Ther.2006 Feb;13(2):347-56.
[1]Zhang, SH; Cui, Y; Weng, ZJ, et al. Changes on the disease pattern of primary colorectal cancers in Southern China:a retrospective study of 20 years. INTERNATIONAL JOURNAL OF COLORECTAL DISEASE,2009,24(8): 943-949.
[2]Peng JJ, Cai SJ, Lu HF, et al. Predicting prognosis of rectal cancer patients with total mesorectal excision using molecular markers[J]. World J Gastroenterol,2007,13(21):3009-3015.
[3]Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst.2001 Jul 18;93(14):1062-74.
[4]Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer,2003 Jan;3(1):11-22.
[5]Friday, B.B. et al. K-ras as a target for cancer therapy. Biochim. Biophys. Acta,2005 Nov 25;1756(2):127-44.
[6]Tsang WP, Kwok TT. The miR-18a* microRNA functions as a potential tumor suppressor by targeting on K-Ras. Carcinogenesis.2009 Jun;30(6): 953-9.
[7]Pelengaris S, Khan M, Evan G. c-Myc:More than just a matter of life and death. Nat Rev Cancer 2002;2:764.
[8]Doisneau-Sixou SF, Sergio CM, et al. Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells. Endocr Relat Cancer 2003;10:179.
[9]Hongxing Z, Nancai Y, Wen S, et al. Depletion of c-Myc inhibits human colon cancer colo 320 cells'growth. Cancer Biother Radiopharm.2008 Apr; 23 (2):229-37.
[10]Palacios G, Crawford HC, Vaseva A, et al. Mitochondrially targeted wild-type p53 induces apoptosis in a solid human tumor xenograft model. Cell Cycle.2008 Aug 15;7(16):2584-90.
[11]谭进富,赵振献,黄文生,等.Fas基因对裸鼠腹腔移植结肠癌的抑制作用.现代医药卫生,2008年24卷第20期:3011-3013.
[12]Kievit E, Bershad E, Ng E, et al. Superiority of yeast over bacterial cytosine deaminase for enzyme/prodrug gene therapy in coloncancer xenografts[J]. Cancer Res,1999,59(5):1417-1421.
[13]Kievit E, Nyati MK, Ng E, et al. Yeast cytosine deaminase improves radiosensitization and bystander effect by 5-fluorocytosine of human colorectal cancer xenografts[J]. Cancer Res,2000,60(23):6649-6655.
[14]Shen LZ, Wu WX, Xu DH, et al. Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene.World J Gastroenterol.2002 Apr;8(2):270-5.
[15]Wolfgang Walther, Ulrike Stein, 1Iduna Fichtner, et al. Nonviral Jet-Injection Gene Transfer for Efficient in Vivo Cytosine Deaminase Suicide Gene Therapy of Colon Carcinoma. Mol Ther.2005 Dec; 12 (6):1176-84.
[16]徐文贵.肿瘤自杀基因靶向治疗的研究进展.中国肿瘤临床,2009年第36卷第13期:777-780.
[17]王东平.腺病毒介导自杀基因对消化系统肿瘤的杀伤作用.中华胃肠外科杂志,2002年6月第5卷第2期:137-139.
[18]Xia K, Liang D, Tang A, et al. A novel fusion suicide gene yeast CDglyTK plays a role in radio-gene therapy of nasopharyngeal carcinoma. Cancer Gene Ther.2004 Dec;11(12):790-6.
[19]Liu T, Zhang G, Chen YH, et al. Tissue specific expression of suicide genes delivered by nanoparticles inhibits gastric carcinoma growth. Cancer Biol Ther.2006 Dec;5(12):1683-90.
[20]李尊岭.WTP53联合双自杀基因CD和TK抑制结肠癌细胞SW480生长.基础医学与临床,2006年12月第26卷第12期:1340-1344.
[21]吴斌文,李友佳,张凯军.FCU1重组腺病毒载体构建及对结肠癌细胞的杀伤作用.世界华人消化杂志,2008年8月18日;16(23):2599-2603.
[22]Moreira IS, Fernandes PA, Ramos MJ. Vascular endothelial growth factor (VEGF) inhibition—a critical review. Anticancer Agents Med Chem.2007 Mar; 7 (2):223-45.
[23]Ho QT, Kuo CJ. Vascular endothelial growth factor:biology and therapeutic applications. Int J Biochem Cell Biol.2007;39(7-8): 1349-57.
[24]Yin Y, Cao LY, Wu WQ, et al. Blocking effects of siRNA on VEGF expression in human colorectal cancer cells. World J Gastroenterol. 2010 Mar 7;16(9):1086-92.
[25]李铁军,王茜,宋建宁,等.RNA干扰抑制血管内皮生长因子介导结肠癌细胞凋亡.生物技术通报2007年第6期:125-128.
[26]Gray MJ, Van Buren G, Dallas NA, et al. Therapeutic targeting of neuropilin-2 on colorectal carcinoma cells implanted in the murine liver. J Natl Cancer Inst.2008 Jan 16;100(2):109-20.
[27]Abdollahi A, Hahnfeldt P, Maercker C, et al. Endostatin's antiangiogenic signaling network. Mol Cell.2004 Mar 12; 13(5):649-63.
[28]Folkman J. Antiangiogenesis in cancer therapy--endostatin and its mechanisms of action. Exp Cell Res.2006 Mar 10;312(5):594-607.
[29]Lan KL,Ou-Yang F, Yen SH, et al. Cationic liposome coupled endostatin gene for treatment of peritoneal colon cancer. Clin Exp Metastasis. 2010 Apr 7.
[30]Matsumoto K, Nakamura T. NK4 (HGF-antagonist/angiogenesis inhibitor) in cancer biology and therapeutics. Cancer Sci 2003; 94:321-327.
[31]Maehara N, Matsumoto K, Kuba K, et al. NK4, a four-kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells. Br J Cancer 2001; 84:864-873.
[32]Matsumoto K, Nakamura T. Mechanisms and significance of bifunctional NK4 in cancer treatment. Biochem Biophys Res Commun 2005; 333:316-327.
[33]Kuba K, Matsumoto K, Date K, et al. HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice. Cancer Res 2000; 60:6737-6743.
[34]Jian-Zheng Jie, Jian-Wei Wang, Jian-Guo Qu, et al. Suppression of human colon tumor growth by adenoviral vector-mediated NK4 expression in an athymic mouse model. World J Gastroenterol 2007 April 7; 13(13): 1938-1946.
[35]Peng SP, Fang WY, Jiang RC, et al. Prokaryotic expression of vascular basement membrane-derived multifunctional peptide and anti-tumor activity assay. Chinese Pharmacological Bulletin 2003,19:678-672.
[36]Cao JG, Peng SP, Sun L, et al. Vascular basement membrane-derived multifunctional peptide, a novel inhibitor of angiogenesis and tumor growth. Acta Biochim Biophys Sin (Shanghai).2006 Jul;38(7):514-21.
[37]Pamey IF, Chang LJ. Cancer immunogene thempy:a review[J]. J Biomed Sci,2003,10(1):37-43.
[38]Parney IF, Hao C, Petruk KC. Glioma immunologay and immunotheyapy [J]. Neurosurgery,2000,46(3):778-792.
[39]Zhao L, Gu J, Dong A, et al. Potent antitumor activity of oncolytic adenovirus expressing mda-7/IL-24 for colorectal cancer. Hum Gene Ther. 2005 Jul;16(7):845-58.
[40]Kanagawa N, Niwa M, Hatanaka Y, et al. CC-chemokine ligand 17 gene therapy induces tumor regression through augmentation of tumor-infiltrating immune cells in a murine model of preexisting CT26 colon carcinoma. Int J Cancer.2007 Nov 1;121 (9):2013-22.
[41]Iida T, Shiba H, Misawa T, et al. Immunogene therapy against colon cancer metastasis using an adenovirus vector expressing CD40 ligand. Surgery.2010 Apr 6.
[42]Oba S, Wang Y, Song J, et al. Genomic structure and loss of heterozygosity of EphB2 in colorectal cancer. Cancer Lett.2001; 164:97-104.
[43]Dodelet V, Pasquale E. Eph receptors and Ephrin ligands: embryogenesis to tumorgenesis. Oncogene.2000;19:5614-5619.
[44]Bittner M,-Meltzer P, Chen Y, et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature. 2000:406:536-540.
[45]Pawel D, Kathryn H, Susan J,-et al. Antiangiogenic and antitumor efficacy of EphA2 receptor antagonist. Cancer Res.2004;64:910-919.
[46]Cheng N, Brantley DM, Chen J. The ephrins and Eph receptors in angiogenesis. Cytokine Growth Factor Rev.2002;13:75-85.
[47]Saito T, Masuda N, Miyazaki T, et al. Expression of EphA2 and E-cadherin in colorectal cancer with cancer metastasis. Oncol Rep. 2004; 11:605-611.
[48]Yamaguchi S, Tatsumi T, Takehara T, et al. Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines. Cancer.2007 Oct 1; 110 (7):1469-77.
[49]付前锋,刘连新.肿瘤的耐药基因和耐药基因治疗研究发展.中华实验外 科杂志,2007年12月第24卷第12期:1613-1618.
[50]Perez-Tomas R. Multidrug resistance:retrospect and prospects in anti-cancer drug treatment. Curr Med Chem.2006;13(16):1859-76.
[51]Ramachandran C, Wellham LL. Effect of MDR1 phosphorothioate antisense oligodeoxynucleotides in multidrug-resistant human tumor cell lines and xenografts. Anticancer Res.2003 May-Jun 23 (3B):2681-90.
[52]Lee KH, Piao H, Son BR, et al. Herpes simplex virus thymidine kinase and granulocyte macrophage colony-stimulating factor combination gene therapy in a murine CT26 cell colon cancer model. Cancer Gene Ther. 2004 Aug;11(8):570-6.
[53]Yao B, He QM, Tian L, et al. Enhanced antitumor effect of the combination of tumstatin gene therapy and gemcitabine in murine models. Hum Gene Ther.2005 Sep; 16(9):.1075-86.
[54]Yu-Kyoung Oh, Tae Gwan Park. siRNA delivery systems for cancer treatment. Advanced Drug Delivery Reviews,2009 Aug 10;61 (10):850-62.
[55]N. Doi, S. Zenno, R. Ueda, et al. Short-interfering RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors. Curr. Biol.2003 Jan 8;13(1):41-6.
[56]S. M. Hammond, E. Bernstein, D. Beach, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature,2000 Mar 16;404(6775):293-6.
[57]A. de Fougerolles, H. P. Vornlocher, J. Maraganore, et al. interfering with disease:a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov.2007 Jun;6(6):443-53.
[58]D. Bumcrot, M. Manoharan, V. Koteliansky, et al. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol.2006 Dec; 2 (12):711-9.
[59]N. Manjunath, Haoquan Wu, Sandesh Subramanya, et al. Lentiviral delivery of short hairpin RNAs. Advanced Drug Delivery Reviews 2009 Jul 25;61(9):732-45. [60] Liu Q, Muruve DA. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther 2003 Jun; 10 (11):935-40.
[61]Marta Lo'pez-Fraga, Tamara Martinez, Ana Jime'nez. RNA Interference Technologies and Therapeutics:From Basic Research to Products. Biodrugs 2009; 23 (5):305-332.
[62]Xu G, Zhang N. Nanoparticles for gene delivery:a brief patent review. Recent Pat Drug Deliv Formul.2009;3(2):125-36.
[63]Stephen L. Hart. Multifunctional nanocomplexes for gene transfer and gene therapy. Cell Biol Toxicol.2010 Feb;26(1):69-81.
[64]Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev. 2009 Feb;109(2):259-302.
[65]Khurana B, Goyal AK, Budhiraja A, et al. siRNA delivery using nanocarriers-an efficient tool for gene silencing. Curr Gene Ther. 2010 Apr; 10(2):139-55.
[66]Liu T, Tang A, Zhang G, et al. Calcium phosphate nanoparticles as a novel nonviral vector for efficient transfection of DNA in cancer gene therapy. Cancer Biother Radiopharm.2005 Apr;20(2):141-9.
[67]Zhang G, Liu T, Chen YH, et al. Tissue specific cytotoxicity of colon cancer cells mediated by nanoparticle-delivered suicide gene in vitro and in vivo. Clin Cancer Res.2009 Jan 1;15(1):201-7.
[68]陶铭.基因治疗及其研究[J].生物学通报,2008,43(4):16-18.