摘要
研究背景
原发性肝癌是我国常见的恶性肿瘤，死亡率很高，占恶性肿瘤死亡的第二位，在部分农村中占第一位，而且呈上升趋势。手术切除仍是治疗肝癌的首选方法，但由于切除率低，术后复发率高，所以其总体疗效不太理想，而放疗和化疗常难彻底消灭肿瘤细胞，又易损伤正常组织，因此目前迫切需要研究和发展新的治疗方法。近年来生物治疗已成为肿瘤治疗的第四种模式。肝癌的生物治疗策略主要包括抑制细胞增殖和促进细胞凋亡两个方面，其中反义技术因其靶向性强、易操作、可控性等优势，在肝癌的生物治疗中具有重要意义。目前肝癌反义基因治疗主要是采用反义核苷酸技术。由于肝细胞癌的发生具有多因素、多阶段、多基因、多突变的特点，所以选择反义治疗的靶基因至关重要。Altieri等于1997年首先克隆出Survivin基因，其编码的蛋白是目前发现的作用最强的凋亡抑制因子，被称为生存素或生存蛋白。目前发现其生物学效应为：①抑制细胞凋亡，②调节细胞增殖，③诱导血管生成。在人的生长发育中，Survivin在胚胎组织中表达，而在分化成熟的组织中表达丧失。Survivin广泛表达于人类多种肿瘤组织，在100多种肿瘤特异性表达基因中，其表达的特异性列前4位。由于Survivin在正常成熟组织中几乎不表达，而特异性的在肿瘤组织表达，因而被认为是“肿瘤的理想反义靶向治疗标志”，具有良好的靶向性、特异性和安全性。
目前针对Survivin与原发性肝癌的发生及生物学行为关系的研究为数不多，尤其Survivin和Caspase-9的关系及其在肝癌细胞凋亡和新生血管中作用的研究未见报道，以Survivin作为反义靶基因行肝癌反义治疗的研究也很少见。本实验在证实“Survivin在肝癌中稳定高效表达可抑制细胞凋亡和诱导肝癌新生血管生成，并可作为肝癌独立的不良预后因子”的前提下，以Survivin为靶基因，针对Survivin mRNA翻译起始密码子区设计Survivin反义寡核苷酸片断，转染SMMC-7721细胞，研究其对肝癌细胞增殖和凋亡的影响，为肝癌综合评估和生物治疗开辟一条新的途径。
研究目的
1、研究Survivin和Caspase-9在原发性肝癌中的表达及其与细胞凋亡和新生血管的关系，探讨Survivin和Caspase-9在肝癌发生及生物学行为中的作用，为肝癌综合评估提供一个新的指标。
2、以Survivin为靶基因，设计合成针对Survivin的反义寡核苷酸片断，在阳离子脂质体介导下转染SMMC-7721细胞，研究其对肝癌细胞增殖和凋亡的影响，为肝癌生物治疗提供一条新的途径。
主要材料和方法
一、主要材料
1、研究对象
（1）组织标本 42例原发性HCC均为吉林大学第一医院普外科1998-2002年手术切除的标本，其中男33例，女9例，≤60岁16例，>60岁26例。按照Edmondson分级标准，Ⅰ级8例，Ⅱ级7例，Ⅲ级17例，Ⅳ级10。肿瘤直径≤5cm16例，>5cm26例。有肝内外转移倾向或转移者（肝门淋巴结转移、门静脉癌栓形成、远处转移、肿瘤数目>2个、癌周有卫星结节）19例。42例癌旁组织均为相应原发性肝癌病人的距癌肿病灶≤2cm的组织，
其中肝硬化组织28例，肝炎组织10例，4例为正常肝组织。12例正常肝组织均为吉林大学基础医学院尸检标本。以上所有标本均经10%中性甲醛溶液固定，4μm石蜡连续切片后贴在涂有切片粘合剂的载玻片上备染。
（2）细胞株 人肝癌细胞株SMMC7721购置中科院上海细胞研究所。
2、主要器材 切片机、孵育箱、超净工作台、紫外光光度仪、PCR扩增仪、流式细胞仪、酶标仪、透射电子显微镜等。
3、主要试剂 兔抗人Survivin多克隆抗体、鼠抗人Caspase-9单克隆抗体、鼠抗人CD34mAb试剂盒、TUNEL检测试剂盒、S-P免疫组化试剂盒、阳离子脂质体、RPMI1640培养液、噻唑蓝、RT-PCR 相关试剂盒等。
二、主要方法
（一）免疫组织化学染色和细胞凋亡的检测
S-P法检测Survivin和Caspase-9表达和微血管密度。肝癌细胞浆或细胞核中出现棕黄色颗粒者为免疫组化染色阳性，参照Caide等的计数方法定量Survivin和Caspase-9表达的阳性程度；参照Weidner等报道的方法判定微血管密，统计学处理MVD采用X±S表示；原位末端标记法检测细胞凋亡。
（二）反义核苷酸的设计合成
应用RNA structure3.5对Survivin的二级结构进行模拟，针对Survivin mRNA翻译起始密码子区设计Survivin反义寡核苷酸（ASODN），其序列为：5/-GGCAACGTCGGGGCACCCAT-3/。正义寡核苷酸（SODN）序列：5/-GCCATCGTAGGGGATCGCTT-3/。两条核苷酸序列各有5个硫代硷基修饰，由上海生工公司合成，-20℃保存备用。
（三）实验分组和细胞转染
实验分3组：空白对照组(control)：以无血清的RPMI1640培养液代替转染复合物作为空白对照组。正义对照组（SODN）：SODN转染作为正义对照。实验组（ASODN）：根据ASODN浓度分为100、200、400、800nmol/L转染组。SMMC-7721细胞用含10%小牛血清的RPMI1640培养液在37℃、5%CO2培养箱中培养，在阳离子脂质体介导下以100、200、400、800nmol/L ASODN、SODN分别转染肝癌细胞，每组设4个复孔，转染24、48、72h后收集各组细
胞用于后续实验。
（四）反义核苷酸转?
Background
Primary liver cancer is a common malignant tumor in China. It is the second cause which leads to death, especially in countryside. At present its prevalence has an increase tendency. Now the surgical operation is still the first choice, however, the effect isn’t very satisfying because the rate of postoperation relapse is very high. On the other hand, radiotherapy and chemotherapy can’t kill all tumor cells, and they are easy to impair the normal tissues. So we need to seek new treatments. Recently biological therapy has become the fouth model in treating tumors which mainly includes inhibiting cell proliferation and promoting cell apoptosis. Anyway, anti-sense gene therapy plays an important role because it is easy to operate and control. At present it means anti-sense oligonucleotides technology. Because the pathogenesis of hepatocellular cancer is characterized as multi-factor, multi-stage, multi-gene and multi-mutation.The choice of exact targeting gene is very crucial. Altieri et al first clones the gene of Survivin in 1997. The protein is the strongest apoptosis inhibitor at present called survivin protein. Its biological effects includ: ①inhibit cell apopt, ②regulate cell proliferation, ③induce angiogenesis. Survivin can express in embryonic tissues, but undetectable in differentiated adult tissues It can be detected in many human tumors. And it is the fouth of over 100 tumor-specific genes. Survivin can’t express in normal tissues, so it is regarded as
the ideal anti-sense therapeutic target. At present the research on the relationship of Survivin and the pathogenesis of liver cancer is few. Especially there is no report about the relationgship about Survivin, Caspase-9 and its effect in inhibiting liver cell apoptosis and angiogenesis. Anti-sense Survivin gene therapy in the field of liver cancer is very limited. This study proves that Survivin can stably express in liver cancer to inhibit cell apoptosis, and induce angiogenesis. It can function as a kind of poor prognosis index. We design anti-sense oligonucleotides sequences targeting Survivin mRNA translation initiation coden which can transfect SMMC-7721 cells to study the proliferation and apoptosis of liver cancer. This can open a new way for the comprehensive evaluation and biological therapy.
Objective
1. We study the expression of Survivin and Caspase-9 in primary live cancer and their effect on cell apoptosis and angiogenesis, and discuss their effect on the pathogenesis of liver caner and biological action. Finally, we offer a new index for liver cancer comprehensive evaluation. 2.Anti-sense oligonucleotides sequences targeting Survivin can transfect SMMC-7721 with cation liposome to examine its effect on liver cancer cell proliferation and apoptosis, this offer a new way for biological therapy of liver cancer finally.
Materials and Methods
Materials
1.Target
(1) tissues specimen: 42 primary liver cancer tissues were obtained by the surgical resection from the department of general surgery, the first hospital of Jilin University from1998 to 2000: 33 male and 9 female, 16 (<60yr) and 26 (>0yr). According to Edmondson Grade Criteria, Grade I is 8 cases, Grade II is 10, Grade III is 16, Grade IV is 8. The diameter of tumor (<5cm) is 16 cases, >cm is
26.Metastasis tendency (lymph node metastasis, portal vein cancer thrombosis, distant metastasis, the tumor amounts >2, satellite node around cancer) is 19 cases. 42 nontumor tissues were those which were 2cm away from the lesion, including 28 cirrotic tissues, 10 hepatitis tissues and 4 normal liver tissues. 12 normal liver tissues were obtained by autopsy from Faculty of medicine of Jilin University. All tissues were fixed in 10% formalin for slices staining.
(2) Cell lines: SMMC-7721 is purchased from Shanghai cell research institute of Chinese Academy of science.
2.Equipment: Microtome, Incubator, Hyperclean Working platform, Ultraviolet Spectrophotometer, PCR Flycyte Meter, Enzyme Labelling Meter, Transmission Electron Mic
引文
Kerr JF, Wyllie AH, Currie AR. Apoptois: a basic biological phenomenon with Wide-ranging implications in tissue kinetics. Br J Cancer, 1972, 26(4):239-257.
Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol, 1999, 17:2941-2953.
Dveraux QL, Reed JC. IAP family Proteins: suppressors of apoptosis. Gene Dev, 1999, 13:239-252.
T. Yamamoto, N. Tanidawa. The role of survivin as a new target of diagnosis and treatment in human cancer. Med Electron Microsc, 2001, 34:207-212.
Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med, 1997, 3(8): 917-921.
Ambrosini G, Adida C, Altieri DC. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting.J Biol Chem, 1998, 273(18):11177-11182.
Zwicker J, Lucibello FC, Wolfraim La, et al. Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes based on a common mechanism of transcriptional repression. EMBO J, 1995, 14:4514-4522.
Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by surviving. Nature, 1998, 369:580-584.
Lacasse EC, Baird S, Korneluk, RG, et al. The inhibitors of apoptosis and their emerging role in cancer. Onco-gene, 1998, 17(25):3247-3259.
Duckett CS, Nava VE, Gedrich RW, et al. A conserved family of cellular genes related to the baculovirus iap gene and encoding
apoptosis inhibitors. EMBO J, 1996, 14:2685-2694.
Clem RJ, Miller LK. Control of programmed cell death by the baculovirus genes p35 and iap. Mol Biol Cell, 1994, 14:5212-5222.
Hay BA, Wssarman DA, Rubin GM. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell, 1995, 83:1253-1262.
Takahashi R, Deveraux QL, Tamm I, et al. A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem, 1998, 273:7787-7790.
Reed JC. The survivin saga goes in vivo. J Clin Invest, 2001, 108(7):965-969.
Mahotka C, Wenzel M, Springre E, et al. Survivin-δEx3 and Survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptosis properties. Cancer Res, 1999, 59:6097-6102.
Jiang X, Wilford C, Duensing R, et al. Participation of survivin in mitotic and apoptosis activities of normal and tumor-derived cells. J Cell Biochem, 2001, 83(2):342-354.
Zhao J, Tenev T, Martins LM, et al. The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner. J Cell Sci, 2000, 113(23):4363-4371.
Rohayem J, Diestelkoetter P, Weigle B, et al. Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients. Cancer Res, 2000, 60(7):1815-1817.
Hirohashi Y, Torigoe T, Maeda A, et al. An HLA-A24-restricted cytotoxic T lymphocyte epitope of a tumor-associated protein, survivin. Clin Cancer Res, 2002, 8(6):1731-1739.
Sarela AI, Macadam RC, Farmery SM, et al. Expression of the
antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma. Gut, 2000, 46(5):645-650.
Lehner R, Enomoto T, McGregor JA, et al. Correlation of survivin mRNA detection with histologic diagnosis in normal endometrium and endometrial carcinoma.Acta Obstet Gynecol Scand, 2002, 81(2):162-167.
Koch CA, Vortmeyer AO, diallo R, et al. Survivin: a novel neuroendocrine marker for pheochromocytoma. Eur J Endocrinol, 2002, 146(3):381-388.
Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res, 1998, 58(23):5315-5320.
Velculescu VE, Madden SL, Zhang l, et al. Analysis of human transcriptomes. Nat Genet, 1999, 23(4):387-388.
Ikeguchi M, Ueda T, Sakatani T, Hirooka Y, Kaibara N. Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma. Diagn Mol Pathol, 2002, 11(1):33-40.
Aida C, Berrebi D, Peuchmaur M, et al. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma.Lancet, 1998, 351(9106):882-883.
Lu CD, Altieri N, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res, 1998, 58(9):1808-1812.
Kawasaki H, Altieri DC, Lu CD, et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res, 1998, 58(22):5071-5074.
Swana HS, Grossman D, Anthony JN, et al. Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer. N
Engl J Med, 1999, 341(6):452-453.
Shin S, Sung BJ, Cho YS, et al. An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and –7. Biochemistry. 2001, 40(4):1117-1123.
Chakravarti A, Noll E, Black PM, et al. Quantitatively determined survivin expression levels are of prognostic value in human gliomas. J Clin Oncol. 2002, 20(4):1063-1068.
Borriello A, Roberto R, Della Ragione F, et al. Proliferate and survive: cell division cycle and apoptosis in human neuroblastoma. Haematologica. 2002, 87(2):196-214.
Mesri M, Wall NR, Li J, et al. Cancer gene therapy using a survivin mutant adenovirus. J Clin Invest, 2001, 108(7):981-990.
O'Connor DS, Grossman D, Plescia J, et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of surviving. Proc Natl Acad Sci U S A, 2000, 97(24):13103-13107.
Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell, 2000, 102(1):43-53.
Reed JC. The surviving saga goes in vivo. J Clin Invest, 2001, 108(7):965-969.
Suzuki A, Ito T, Kawano H, et al. Survivin initiates cell cycle entry by the competitive interaction with Cdk4/p16 (INK4a) and Cdk2/cyclin E complex activation [J]. Oncogene. 2000, 19(29):3225-3234.
Shankar SL, Mani S, O'Guin KN, et al. Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways. J Neurochem, 2001, 79(2):426-436.
Uren AG, Rourke K, Aravid LA, et al. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of
which plays a key role in MALT lymphoma. Mol Cell, 2000, 6(4):961-967.
Chen J, Wu W, Tahir SK, et al. Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-independent growth. Neoplasia, 2000, 2(3):235-241.
Li F, Ackermann EJ, Bennett CF, et al. Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat Cell Biol, 1999, 1(8):461-466.
Giodini A, Kallio MJ, Wall NR, et al. Regulation of microtubule stability and mitotic progression by surviving. Cancer Res, 2002, 62(9):2462-2467.
O'Connor DS, Schechner JS, Adida C, et al. Control of apoptosis during angiogenesis by survivin expression in endothelial cells. Am J Pathol, 2000, 156(2):393-398.
Mesri M, Morales-Ruiz M, Ackermann EJ, et al. Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting. Am J Pathol, 2001, 158(5):1757-1765.
Papapetropoulos A, Fulton D, Mahboubi K, et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem, 2000, 275(13):9102-9105.
Altieri DC. Survivin in apoptosis control and cell cycle regulation in cancer. Prog Cell Cycle Res, 2003, 5:447-452.
Altieri DC. Survivin and apoptosis control. Adv Cancer Res, 2003, 88:31-52.
LaCasse EC, Baird S, Korneluk RG, et al. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene, 1998, 17(25):3247-3259.
Deveraux QL, Reed JC. IAP family proteins--suppressors of apoptosis.
Genes Dev, 1999, 13(3):239-252.
Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med, 1997, 3(8):917-21.
Kato J, Kuwabara Y, Mitani M, et al. Expression of survivin in esophageal cancer: correlation with the prognosis and response to chemotherapy. Int J Cancer, 2001, 95(2):92-95.
Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res, 1998, 58(9): 1808-1812.
Sarela AI, Macadam RC, Farmery SM, et al. Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma. Gut, 2000, 46(5):645-650.
Ito T, Shiraki K, Sugimoto K. Survivin promotes cell proliferation in human hepatocellular carcinoma. Hepatology, 2000, 31(5):1080-1085.
Ikeguchi M, Ueda T, Sakatani T, et al. Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma. Diagn Mol Pathol, 2002, 11(1):33-40.
Satoh K, Kaneko K, Hirota M, et al. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer, 2001, 92(2):271-278.
Kennedy SM, O'Driscoll L, Purcell R, et al. Prognostic importance of survivin in breast cancer. Br J Cancer, 2003, 88(7):1077-1083.
Nasu S, Yagihashi A, Izawa, ET AL. Survivin mRNA expression in patients with breast cancer. Anticancer Res, 2002, 22(3):1839-1843.
Monzo M, Rosell R, Felip E, et al. A novel anti-apoptosis gene:
Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol, 1999, 17(7):2100-2104.
Falleni M, Pellegrini C, Marchetti A, et al. Survivin gene expression in early-stage non-small cell lung cancer. J Pathol, 2003, 200(5):620-626.
Swana HS, Grossman D, Anthony JN, Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer. N Engl J Med, 1999, 341(6):452-453.
Schultz IJ, Kiemeney LA, Witjes JA, et al. Survivin mRNA expression is elevated in malignant urothelial cell carcinomas and predicts time to recurrence. Anticancer Res, 2003, 23(4):3327-3331.
Saitoh Y, Yaginuma Y, Ishikawa M. Analysis of Bcl-2, Bax and Survivin genes in uterine cancer. Int J Oncol, 1999, 15(1):137-141.
Takai N, Miyazaki T, Nishida M, et al. Expression of survivin is associated with malignant potential in epithelial ovarian carcinoma. Int J Mol Med, 2002, 10(2):211-216.
Trieb K, Lehner R, Stulnig T, et al. Survivin expression in human osteosarcoma is a marker for survival. Eur J Surg Oncol, 2003, 29(4):379-382.
Adida C, Berrebi D, Peuchmaur M, et al. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet, 1998, 351(9106):882-883.
Kajiwara Y, Yamasaki F, Hama S, et al. Expression of survivin in astrocytic tumors: correlation with malignant grade and prognosis. Cancer, 2003, 97(4):1077-1083.
Dong Y, Sui L, Watanabe Y, Survivin expression in laryngeal squamous cell carcinomas and its prognostic implications. Anticancer Res, 2002, 22(4):2377-83.
Mori A, Wada H, Nishimura Y, et al. Expression of the antiapoptosis gene survivin in human leukemia. Int J Hematol, 2002, 75(2):161-165.
Grossman D, McNiff JM, Li F, Expression of the apoptosis inhibitor, survivin, in nonmelanoma skin cancer and gene targeting in a keratinocyte cell line. Lab Invest, 1999, 79(9):1121-1126.
Grossman D, McNiff JM, Li F, et al. Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma. J Invest Dermatol, 1999, 113(6):1076-1081.
Kawasaki H, Toyoda M, Shinohara H, et al. Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis. Cancer, 2001, 91(11):2026-32.
Lin LJ, Zheng CQ, Jin Y, et al. Expression of survivin protein in human colorectal carcinogenesis. World J Gastroenterol, 2003, 9(5):974-977.
Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res, 1998, 58(23):5315-5320.
Kawasaki H, Altieri DC, Lu CD, et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res, 1998, 58(22):5071-5074.
Chen J, Wu W, Tahir SK, et al. Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-independent growth. Neoplasia, 2000, 2(3):235-241.
Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J Cancer, 2002, 86(6):886-892.
Ikehara M, Oshita F, Kameda Y, et al. Expression of survivin correlated with vessel invasion is a marker of poor prognosis in small adenocarcinoma of the lung. Oncol Rep, 2002, 9(4):835-838.
Takai N, Miyazaki T, Nishida M, et al. Survivin expression correlates with clinical stage, histological grade, invasive behavior and survival
rate in endometrial carcinoma. Cancer Lett. 2002, 184(1):105-116.
Ikeguchi M, Yamaguchi K, Kaibara N. Survivin gene expression positively correlates with proliferative activity of cancer cells in esophageal cancer. Tumour Biol, 2003, 24(1):40-45.
Sela B. [Survivin: anti-apoptosis protein and a prognostic marker for tumor progression and recurrence]. Harefuah, 2002, 141(1):103-107, 123.
Smith SD, Wheeler MA, Plescia J, et al. Urine detection of survivin and diagnosis of bladder cancer. JAMA, 2001, 285(3):324-328.
Sharp JD, Hausladen DA, Maher MG. Bladder cancer detection with urinary survivin, an inhibitor of apoptosis. Front Biosci, 2002, 7:e36-41.
Rohayem J, Diestelkoetter P, Weigle B, et al. Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients. Cancer Res, 2000, 60(7):1815-1817.
Yagihashi A, Asanuma K, Nakamura M, et al. Detection of anti-survivin antibody in gastrointestinal cancer patients. Clin Chem, 2001, 47(9):1729-1731.
Grabowski P, Kuhnel T, Muhr-Wilkenshoff F Prognostic value of nuclear survivin expression in oesophageal squamous cell carcinoma. Br J Cancer, 2003, 88(1):115-119.
Gradilone A, Gazzaniga P, Ribuffo D, et al. Survivin, bcl-2, bax, and bcl-X gene expression in sentinel lymph nodes from melanoma patients. J Clin Oncol, 2003, 21(2):306-312.
Rodel C, Haas J, Groth A, et al. Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: survivin as a radioresistance factor. Int J Radiat Oncol Biol Phys, 2003, 55(5):1341-1347.
Ikeguchi M, Liu J, Kaibara N. Expression of survivin mRNA and protein in gastric cancer cell line (MKN-45) during cisplatin treatment. Apoptosis, 2002, 7(1):23-29.
Ikeguchi M, Kaibara N. Changes in survivin messenger RNA level during cisplatin treatment in gastric cancer. Int J Mol Med, 2001，8(6):661-666.
Asanuma K, Moriai R, Yajima T, et al. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res, 2000, 91(11):1204-1209.
O'Connor DS, Grossman D, Plescia J. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of surviving. Proc Natl Acad Sci U S A, 2000, 97(24):13103-13107.
Grossman D, Kim PJ, Schechner JS, et al. Inhibition of melanoma tumor growth in vivo by survivin targeting. Proc Natl Acad Sci U S A, 2001, 98(2):635-640.
Mesri M, Wall NR, Li J,et al. Cancer gene therapy using a survivin mutant adenovirus. J Clin Invest, 2001, 108(7):981-990.
Wall NR, O'Connor DS, Plescia J, et al. Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis. Cancer Res, 2003, 63(1):230-235.
Yamamoto T, Manome Y, Nakamura M, et al. Downregulation of survivin expression by induction of the effector cell protease receptor-1 reduces tumor growth potential and results in an increased sensitivity to anticancer agents in human colon cancer. Eur J Cancer, 2002, 38(17):2316-2324.
Olie RA, Simoes-Wust AP, Baumann B, et al. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res, 2000, 60(11):2805-2809.
Xu Z, Yuan X, Uematsu K, et al. Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides. Mol Cancer Ther, 2002, 1(9): 687-694.
O'Connor DS, Schechner JS, Adida C, et al. Control of apoptosis during angiogenesis by survivin expression in endothelial cells. Am J Pathol, 2000, 156(2): 393-398.
Mesri M, Morales-Ruiz M, Ackermann EJ, et al. Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting. Am J Pathol, 2001, 158(5): 1757-1765.
Petropoulos A, Fulton D, Mahboubi K, et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem, 2000, 275(13): 9102-9105.
Schmitz M, Diestelkoetter P, Weigle B, et al. Generation of survivin-specific CD8+ T effector cells by dendritic cells pulsed with protein or selected peptides. Cancer Res, 2000, 60(17): 4845-4849.
Andersen MH, Pedersen LO, Becker JC. Identification of a cytotoxic T lymphocyte response to the apoptosis inhibitor protein survivin in cancer patients. Cancer Res, 2001, 61(3): 869-872.
Andersen MH, Pedersen LO, Capeller B, et al. mSpontaneous cytotoxic T-cell responses against survivin-derived MHC class I-restricted T-cell epitopes in situ as well as ex vivo in cancer patients. Cancer Res, 2001, 61(16):5964-5968.
Zamecnik PD, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A, 1978, 75(1): 280-284.
Zamecnik PD, Stephenson ML. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A, 1978, 75(1): 285-288.
Izant JG, Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell, 1984, 36(4):1007-1015.
Uhlmann E. Peptide nucleic acids (PNA) and PNA-DNA chimeras: from high binding affinity towards biological function. Biol Chem, 1998, 379(8-9):1045-1052.
Sommer W, Heilig M. Antisense oligonucleotides are clinically tested. They inhibit the expression of disease-related genes. Lakartidningen, 1999, 96(4):348-354.
Agrawal S, Kandimalla ER. Antisense therapeutics: is it as simple as complementary base recognition? Mol Med Today, 2000, 6(2):72-81.
Cook PD. Medicinal chemistry of antisense oligonucleotides-future opportunities. Anticancer Drug Des, 1991, 6(6):585-607.
Zhao Q, Matson S, Herrera CJ. Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides. Antisense Res Dev, 1993, 3(1):53-66.
Eckstein F. Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev, 2000, 10(2):117-121.
Agrawal S, Jiang Z, Zhao Q, et al. Mixed-backbone oligonucleotides as second generation antisense oligonucleotides: in vitro and in vivo studies. Proc Natl Acad Sci U S A, 1997, 94(6):2620-2625.
Lambert G, Fattal E, Couvreur P. Nanoparticulate systems for the delivery of antisense oligonucleotides. Adv Drug Deliv Rev, 2001, 47(1):99-112.
Braasch DA, Corey DR. Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry, 2002,
41(14):4503-4510.
Krieg AM, Tonkinson J, Matson S, et al. Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy. Proc Natl Acad Sci U S A, 1993, 90(3):1048-1052.
张礼和. 以核酸为作用靶的药物研究. 北京大学报(医学版)，2002, 34(5): 418-426.
Weiss B, Davidkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci, 1999, 55(3):334-358.
Yoshikawa M, Oka T. Specific regulation of gene expression in brain by antisense oligodeoxynucleotides. Nippon Yakurigaku Zasshi, 1997, 109(4):187-191.
Oberbauer R. Not nonsense but antisense-applications of antisense oligonucleotides in different fields of medicine. Wien Klin Wochenschr, 1997, 109(2):40-46.
Faria M, Spiller DG, Dubertret C, et al. Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo. Nat Biotechnol, 2001, 19(1):40-44.
Baker BF, Monia BP. Novel mechanisms for antisense-mediated regulation of gene expression. Biochim Biophys Acta, 1999, 1489(1):3-18.
Godard G, Boutorine AS, Saison-Behmoaras E, et al. Antisense effects of cholesterol-oligodeoxynucleotide conjugates associated with poly (alkylcyanoacrylate) nanoparticles. Eur J Biochem, 1995, 232(2): 404-410.
Schwab G, Chavany C, Duroux I，et al. Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit
mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice. Proc Natl Acad Sci U S A, 1994, 91(22):10460-10464.
Lambert G, Fattal E, Pinto-Alphandary H, et al. Polyisobutyl- cyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides. Int J Pharm, 2001, 214(1-2):13-16.
Varga LV, Toth S, Novak I, et al. Antisense strategies: functions and applications in immunology. Immunol Lett, 1999, 69(2):217-224.
Garcia-Chaumont C, Seksek O, Grzybowska J, et al. Delivery systems for antisense oligonucleotides. Pharmacol Ther, 2000, 87(2-3):255-277.
Buck AC, Shen C, et al. Schirrmeister HLiposomal delivery of antisense oligonucleotides for efficient downregulation of Bcl-2 and induction of apoptosis. Cancer Biother Radiopharm, 2002, 17(3):281-289.
Sugano M, Makino N. Changes in plasma lipoprotein cholesterol levels by antisense oligodeoxynucleotides against cholesteryl ester transfer protein in cholesterol-fed rabbits. J Biol Chem, 1996, 271(32): 19080-19083.
Sugano M, Makino N, Sawada S, et al. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits. J Biol Chem, 1998, 273(9):5033-5036.
Citro G, Perrotti D, Cucco C, et al. Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides. Proc Natl Acad Sci USA. 1992, 89(15): 7031-7035.
Citro G, Ginobbi P, Candiloro A, et al. Chemical modification of ligands for cell receptors to introduce foreign compounds into the cells.
Dis Colon Rectum, 1994, 37(2 Suppl):S127-132.
Sarti P, Ginobbi P, D'Agostino I, et al. Liposomal targeting of leukaemia HL60 cells induced by transferrin-receptor endocytosis. Biotechnol Appl Biochem, 1996, 24 (Pt 3):269-276.
Reddy JA, Low PS. Folate-mediated targeting of therapeutic and imaging agents to cancers. Crit Rev Ther Drug Carrier Syst, 1998, 15(6):587-627.
Hofland HE, Masson C, Iginla S, et al. Folate-targeted gene transfer in vivo. Mol Ther, 2002, 5(6):739-744.
Ginobbi P, Geiser TA, Ombres D, et al. Folic acid-polylysine carrier improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (M14) cells. Anticancer Res, 1997, 17(1A):29-35.
Galderisi U, Cascino A, Giordano A. Antisense oligonucleotides as therapeutic agents. J Cell Physiol, 1999, 181(2):251-257.
Tamm I, Dorken B, Hartmann G. Antisense therapy in oncology: new hope for an old idea? Lancet, 2001, 358(9280):489-497.
Cho-Chung YS. Antisense DNAs as targeted therapeutics for cancer: no longer a dream. Curr Opin Investig Drugs, 2002, 3(6):934-939.
Westermann P, Gross B, Hoinkis G. Inhibition of expression of SV40 virus large T-antigen by antisense oligodeoxyribonucleotides. Biomed Biochim Acta, 1989, 48(1):85-93.
Player MR, Barnard DL, Torrence PF. Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA. Proc Natl Acad Sci U S A, 1998, 95(15):8874-8879.
Offensperger WB, Offensperger S, Blum HE. Antisense therapy of hepatitis B virus infection. Mol Biotechnol, 1998, 9(2):161-170.
Liu S, Sun W, Cao Y. Study on anti-HBV effects by antisense
oligodeoxynucleotides in vitro. Zhonghua Yu Fang Yi Xue Za Zhi, 2001, 35(5):338-340.
Lund OS, Hansen JE. Inhibition of HIV-1 replication by chimeric phosphorothioate oligodeoxynucleotides applied in free solution. Intervirology, 1998, 41(2-3):63-68.
Field AK. Oligonucleotides as inhibitors of human immunodeficiency virus. Curr Opin Mol Ther, 1999, 1(3):323-331.
Galderisi U, Cascino A, Giordano A. Antisense oligonucleotides as therapeutic agents. J Cell Physiol, 1999, 181(2):251-257.
Iwase R, Yamayoshi A, Nishida J, et al. Photodynamic antisense regulation using psoralen-conjugated oligo (nucleoside phosphorothioate) s (I). Growth regulation of cervical carcinoma cells. Nucleic Acids Symp Ser, 1999, (42):223-224.
Venturini F, Braspenning J, Homann M, et al. Kinetic selection of HPV 16 E6/E7-directed antisense nucleic acids: anti-proliferative effects on HPV 16-transformed cells. Nucleic Acids Res. 1999, 27(7):1585-1592.
Epper C, Hooper K, Thomas A, Hoy T, Bentley P. Bcl-2 antisense oligonucleotides enhance the cytotoxicity of chlorambucil in B-cell chronic lymphocytic leukaemia cells. Leuk Lymphoma. 2001, 42(3): 491-498.
Zhang Y, Lei XY. Effect of bcl-2 antisense oligodeoxynucleotides on drug sensitivity of leukemic cells. Hematol J, 2003, 4(3):187-197.
Lei XY, Zhang H, He DM. Bcl-2 antisense oligodeoxynucleotide enhances araninosyl cytosine-induced apoptosis of primary leukemia cells. Ai Zheng, 2002, 21(12):1301-1304.
Brignole C, Pagnan G, Marimpietri D. Targeted delivery system for antisense oligonucleotides: a novel experimental strategy for neuroblastoma treatment. Cancer Lett, 2003, 197(1-2):231-235.
Sun HZ, Wu SF, Tu ZH. Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity. Cell Res, 2001, 11(2):107-115.
Iyer R, Ding L, Batchu RB, et al. Antisense p53 transduction leads to overexpression of bcl-2 and dexamethasone resistance in multiple myeloma. Leuk Res, 2003, 27(1):73-78.
Liu Q, Gazitt Y. Potentiation of dexamethasone-, paclitaxel-, and Ad-p53-induced apoptosis by Bcl-2 antisense oligodeoxynucleotides in drug-resistant multiple myeloma cells. Blood, 2003, 101(10):4105-4114.
Roychowdhury D, Lahn M. Antisense therapy directed to protein kinase C-alpha (Affinitak, LY900003/ISIS 3521): potential role in breast cancer. Semin Oncol, 2003, 30(2 Suppl 3):30-33.
Yang DC, Jiang X, Elliott RL, et al. Antisense ferritin oligonucleotides inhibit growth and induce apoptosis in human breast carcinoma cells. Anticancer Res, 2002, 22(3):1513-1524.
Yonemura Y, Endo Y, Fujita H, et al. Inhibition of peritoneal dissemination in human gastric cancer by MMP-7-specific antisense oligonucleotide. Inhibition of peritoneal dissemination in human gastric cancer by MMP-7-specific antisense oligonucleotide. J Exp Clin Cancer Res, 2001, 20(2):205-212.
Jiang YA, Luo HS, Zhang YY, et al. Telomerase activity and cell apoptosis in colon cancer cell by human telomerase reverse transcriptase gene antisense oligodeoxynucleotide. World J Gastroenterol, 2003, 9(9):1981-1984.
Xia C, Xu Z, Yuan X, et al. Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides. Mol Cancer Ther, 2002, 1(9):687-694.
Pujol JL, Jacot W. Antisense oligonucleotides in lung cancer. Suppl
Tumori, 2002, 1(4):S50-51.
Hu Y, Cherton-Horvat G, Dragowska V, et al. Antisense oligonucleotides targeting XIAP induce apoptosis and enhance chemotherapeutic activity against human lung cancer cells in vitro and in vivo. Clin Cancer Res, 2003, 9(7):2826-2836.
Gutierrez AA, Lemoine NR, Sikora K. Gene therapy for cancer. Lancet. 1992, 339(8795):715-721.
Chana JS, Grover R, Tulley P, ET AL. The c-myc oncogene: use of a biological prognostic marker as a potential target for gene therapy in melanoma. Br J Plast Surg, 2002, 55(8):623-627.
Stahel RA, Zangemeister-Wittke U. Antisense oligonucleotides for cancer therapy-an overview. Lung Cancer, 2003, 41 Suppl 1:S81-88.
Koty PP, Lei W, Levitt ML. Induction of programmed cell death with an antisense Bcl-2 oligonucleotide. Methods Mol Med, 2003; 75:671-684.
Hopkins-Donaldson S, Cathomas R, et al. Induction of apoptosis and chemosensitization of mesothelioma cells by Bcl-2 and Bcl-xL antisense treatment. Int J Cancer, 2003, 106(2):160-166.
Lambert G, Bertrand JR, Fattal E, et al. EWS fli-1 antisense nanocapsules inhibits ewing sarcoma-related tumor in mice. Biochem Biophys Res Commun, 2000, 279(2):40140-40146.
Wang J, Liu X, Jiang W. Co-transfection of MRP and bcl-2 antisense S-oligodeoxynucleotides reduces drug resistance in cisplatin-resistant lung cancer cells. Chin Med J (Engl). 2000, 113(10):957-960.
Brigui I, Djavanbakht-Samani T, et al. Minimally modified phosphodiester antisense oligodeoxyribonucleotide directed against the multidrug resistance gene mdr1. Biochem Pharmacol, 2003, 65(5):747-754.
Tortora G, Ciardiello F. Antisense strategies targeting protein kinase C: preclinical and clinical development. Semin Oncol, 2003, 30 (4 Suppl 10):26-31.
Fu J, Zheng J, Fang W, Wu B. Effect of interleukin-6 on the growth of human lung cancer cell line. Chin Med J (Engl). 1998, 111(3):265-268.
Nita ME, Ono-Nita SK, Tsuno N, et al. Bcl-X (L) antisense sensitizes human colon cancer cell line to 5-fluorouracil. Jpn J Cancer Res, 2000, 91(8):825-32.
Stephan D, Nabel EG. Gene and other biological therapies for vascular diseases. Fundam Clin Pharmacol, 1997, 11(2):97-110.
Weiss B, Davidkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci, 1999, 55(3):334-358.
Makino N, Sugano M, Ohtsuka S, et al. Intravenous injection with antisense oligodeoxynucleotides against angiotensinogen decreases blood pressure in spontaneously hypertensive rats. Hypertension, 1998, 31(5):1166-1170.
Makino N, Sugano M, Ohtsuka S, et al. Chronic antisense therapy for angiotensinogen on cardiac hypertrophy in spontaneously hypertensive rats. Cardiovasc Res, 1999, 44(3):543-548.
Edmondson AH, Steiner PE. Primary carcinoma of liver. A study of 100 cases among 48900 necropsies. Cancer, 1954, 7:462.
Caide Lu, Dario CA, Nobuhiko T. Expression of a novel antiapoptosis gene, survivin, correlated with turmor cell apoptosis and p53 accumulation in gastric carcinoma. Cancer Res, 1998, 58(9): 1808-1812.
Weidner N, Folkman J, Pozza F, et al. Tumor angiogenesis: a new signiticant and independent prognostic indicator in early-stage breast
carcinoma. J Natl Cancer Metastasis Rev, 1993, 12(3):303-324.
Caide Lu, Nobuhiko T.Spontaneous apoptosis is inversely related to intratumoral microvessel density in gastric carcinoma. Cancer Res, 1997, 57(2): 221-224.
Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin , a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J Cancer 2002, 86:886-892.
Mari A, Wada H, Nishimura Y, et al. Expression of antiapoptosis gene survivin in human leukemia. Int J Hematol 2002, 75:161-165.
Das A, Tan WL, Teo J, Smith DR. Expression of survivin in primary glioblastomas. J Cancer Res Clin Oncol .2002, 128:302-306.
Rus C, Gores GJ. Apoptosisi and liver disease. Am J Med, 2000, 108(3): 567-574.
Hannun YA. Apoptosis and the dilemma of cancer therapy. Blood, 1997, 89(6): 1846-1853.
Rus C, Gores GJ. Apoptosisi and liver disease. Am J Med, 2000, 108(3): 567-574.
Okada E, Murai Y, Matsui K, et al. Survivin expression in tumor cell nuclei is predictive of a favorable prognosis in gastric cancer patients. Cancer Lett, 2001, 163(9): 109-116.
Frtugno P, Wall NR, Giodini A, et al. Survivin exits in imunochemically distinct subcellular pools and is involved in spindle microtubule function. J Cell Sci, 2002, 115(3): 575-585.
Graslk B, Ruttkay NB, Mullauer L, et al. Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression. Hepa-tology, 1997, 25: 906-912.
Terada T, Nakanuma Y. Expression of apoptosis, proliferating cell nuclear antigen, and apoptosis-related antigen (bcl-2, c-myc, Fas,
lewisy, and p53) in human cholangiocarcinomas and hepatocellular carcinomas. Pathol Int, 1996, 46: 764-770.
Hino N, Higashi T, Nouso K, et al. Apoptosis and proliferation of human HCC. Liver, 1996, 16(2): 123-127.
Folkman J. Tumor angiogenesis: therapeutic implication. N Engl J Med, 1971, 285(21):1182-1186.
李春海, 李克勤. 肿瘤微血管生成的机制与肿瘤浸润和转移. 中华肿瘤学杂, 2000, 22:181-183。
金冬雁主译. 精编分子生物学实验指南. 第一版.北京: 科学出版社, 1998, 284.
Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000, 21(3):485-495.
Hikita H, Vaughan J, Pitot HC. The effect of two periods of short-term fasting during the promotion stage of hepatocarcinogenesis in rat: the role of apoptosis and proliferation. Carcino-genesis, 1997, 18(1): 159-166.
Wong YF, Chung TK, Cheung TH, et al. Effects of human papillomavirus type-specific antisense oligonucleotides on cervical cancer cells containing papillomavirus type 16. Med Oncol, 1994, 11(3-4):149-151.