摘要
鼻咽癌(Nasopharyngeal carcinoma，NPC)是我国最为常见的头颈部恶性肿瘤，我国是世界上鼻咽癌的高发区，而国内又以广东、广西、湖南、福建和江西南方5省发病率最高。鼻咽癌的原发部位深而隐蔽，不易观察，与鼻腔、鼻窦及颅内毗邻，出现的早期症状各异，转移率高，易早期转移，常常容易误诊或漏诊，延误治疗。鼻咽癌的发病人群以30～50岁的青壮年多见，一旦发病，可对社会、经济和家庭造成较大影响。因此，鼻咽癌早期诊断及治疗的相关研究一直是我国耳鼻咽喉科研工作的重点。
目前鼻咽癌的病因尚未完全明确，普遍认为其发病与EB病毒的感染、环境及遗传因素有关。鼻咽癌的治疗主要是以放射治疗为主，对中晚期鼻咽癌患者、鼻咽癌放疗后复发患者以及放疗效果欠佳的患者，可采取化学药物治疗，但仅属辅助性治疗或姑息性治疗。因受鼻咽腔部位隐蔽、腔道狭小、四周结构复杂、鼻咽癌低分化癌多见、易早期转移、难以完整切除等特点的限制，鼻咽癌的手术治疗仅仅在某些特殊情况下作为辅助方法采用。为了提高鼻咽癌的治疗效果，特别是为了解决对放疗不敏感、放疗后病灶残留或放疗后复发患者的治疗难题，国内外研究者近年来相继开展了各种生物治疗研究，包括主动及被动免疫治疗、基因治疗、抗肿瘤血管生成治疗、免疫调节剂和生物反应调节剂治疗等，取得了一定的成果，但仍还不能达到临床应用的水平，我们仍需要不断努力，寻找更有效的治疗手段。目前，依据肿瘤本身生物学特点及发生机制来制定基因治疗方案已成为肿瘤治疗的热点，尤其是RNA干扰技术在哺乳动物体内的应用，为包括鼻咽癌在内的肿瘤的基因治疗开辟了一个新的领域。
端粒的长短与细胞寿命及增殖关系密切，端粒酶可以阻止端粒随细胞分裂而逐渐缩短，从而稳定染色体末端。端粒酶的激活可使细胞获得永生，研究表明，端粒酶活性与恶性肿瘤关系密切。绝大部分恶性肿瘤细胞包括鼻咽癌细胞在内均可检测到端粒酶的活性表达，而在正常组织和良性肿瘤端粒酶活性绝大部分为阴性，hTERT是端粒酶的活性亚单位，因此，hTERT基因是一个很好的鼻咽癌基因治疗的靶基因。
血管内皮因子VEGF是唯一能特异作用于内皮细胞的有丝分裂原，与鼻咽癌局部复发、淋巴结转移、远处转移等有密切的联系。绝大多数恶性肿瘤中均呈现VEGF及其受体的高表达。研究表明，VEGF的高表达与肿瘤患者生存期及转移有关，即VEGF高表达者易转移，生存期短。鼻咽癌患者中VEGF阳性高的病人，区域淋巴结转移机会也高。目前利用VEGF基因开展抗肿瘤血管新生治疗越来越受到研究者的重视，恶性肿瘤中VEGF及其受体的协同表达，旁／自分泌作用机制的确立也为肿瘤的基因治疗提供了新的靶点。抑制VEGF及其受体，阻断VEGF与其受体相互作用，可以达到阻止肿瘤血管新生，抑制肿瘤生长转移的目的，目前，鼻咽癌相关研究中VEGF与鼻咽癌的关系以及VEGF在鼻咽癌治疗中的应用日益受到关注。
RNA干扰是近年来发现的研究生物体基因表达、调控与功能的新技术，它利用了由小干扰RNA引起的生物细胞内同源基因的特异性沉默现象，其本质是siRNA与对应的mRNA特异结合、降解，从而阻止mRNA的翻译。它普遍存在于各种生物，是一种集经济、快捷、高效、特异性强等特点于一体的抑制基因表达的技术手段，可广泛用于基因功能测定及基因治疗等方面。
本研究的目的是利用RNA干扰技术抑制hTERT基因和VEGF基因的表达，开展对鼻咽癌的基因治疗研究。整个研究内容分三部分进行，第一部分主要是siRNA的体外合成及鼻咽癌细胞内RNA干扰效应的观察，目的是确定针对hTERT基因和VEGF基因进行RNA干扰的有效作用位点，观察设计的sihTERT和siVEGF在鼻咽癌中的RNA干扰效应，为后续的质粒构建和体内实验奠定基础。通过利用在线网站的生物软件设计多个针对hTERT基因和VEGF基因的RNA干扰片段，利用体外转录法快速合成相应的siRNA，然后利用脂质体瞬时转染到鼻咽癌CNE-2细胞中，通过荧光显微镜观察、半定量RT-PCR、Western Blot、流式细胞学检查及TRAP-银染法观察RNA干扰效应，确定RNA干扰最佳作用位点。结果确定了hTERT基因和VEGF基因各2个有效作用位点，观察到对hTERT基因和VEGF基因的有效RNA干扰可以将对应基因的mRNA水平分别下调89％～92％、80％～95％，同时使其表达的蛋白水平下降。对hTERT基因的有效干扰还可以使肿瘤细胞增殖受到抑制，使其细胞周期受阻于G_0～G_1期，诱导肿瘤细胞凋亡。本研究的第二部分主要是针对第一部分确定的hTERT基因和VEGF基因RNA干扰的有效作用位点，设计并构建能在肿瘤细胞内表达siRNA的质粒载体。方法是利用pGenesil-1 Vector载体，分别构建针对hTERT基因的RNA干扰质粒pGenezil-hTERT、针对VEGF基因的pGenesil-VEGF质粒、同时针对上述两种基因的pGenesil-hTERT-VEGF质粒，然后将构建的质粒转染CNE-2细胞，通过RT-PCR、Western Blot、MTT法来观察RNA干扰效应，结果成功构建了pGenesil-hTERT、pGenesil-VEGF及pGenesil-hTERT-VEGF质粒，观察到上述质粒在CNE-2细胞可以表达siRNA，发挥RNA干扰效应。本研究第三部分主要是进行RNA干扰的裸鼠体内实验，为今后的临床实验奠定基础。方法是将裸鼠接种CNE-2细胞建立人鼻咽癌裸鼠移植瘤模型，然后利用pGenesil-hTERT、pGenesil-VEGF及pGenesil-hTERT-VEGF质粒分别进行siRNA治疗鼻咽癌的基因治疗，通过显微镜下形态学观察、测量肿瘤大小绘制肿瘤生长曲线、TUNEL法计算肿瘤细胞凋亡指数以及免疫组化等方法，观察RNA干扰体内实验的效果，结果发现3种质粒都能有效地抑制裸鼠移植瘤的生长，促进细胞的凋亡，而pGenesil-hTERT-VEGF质粒的RNAi效应要强于其他2个质粒载体。
本研究最后得到以下结论：(1)体外转录合成的siRNA可在鼻咽癌中发挥特异性的RNA干扰效应；(2)hTERT基因的位点的335～357、2324～2346，VEGF基因的1130～1152、1504～1526为RNA干扰的有效作用位点；(3)针对hTERT基因的有效RNA干扰可导致鼻咽癌肿瘤细胞生长缓慢，细胞增殖受抑，hTERT基因的mRNA水平下调，蛋白表达下降，端粒酶活性降低，细胞周期在G_0～G_1期受阻，并不同程度诱导细胞凋亡；(4)针对VEGF基因的有效RNA干扰可使VEGF基因mRNA水平下降，蛋白表达下调，但不抑制肿瘤细胞增殖，也不诱导肿瘤细胞凋亡；(5)利用pGenesil-1Vector载体成功构建了针对hTERT基因和VEGF基因的质粒载体pGenesil-hTERT、pGenesil-VEGF和pGenesil-hTERT-VEGF，均能在细胞中长期表达siRNA，起到RNA干扰作用；(6)成功建立了人鼻咽癌裸鼠移植瘤模型；(7)pGenesil-hTERT、pGenesil-VEGF和pGenesil-hTERT-VEGF的裸鼠体内实验表明这3种质粒载体均能有效地抑制鼻咽癌肿瘤的生长，促进肿瘤细胞的凋亡，达到治疗鼻咽癌的目的；(8)首次联合干扰端粒酶逆转录酶基因hTERT和血管内皮生长因子基因VEGF成功地进行了鼻咽癌的基因治疗，证明联合干扰hTERT基因和VEGF基因的RNA干扰效果强于对单个基因的干扰效应，证实了联合抑制多个基因治疗肿瘤的可行性和有效性，为其它肿瘤的基因治疗探索了一条新的途径，这也是本研究最大的创新点所在。
Nasopharyngeal carcinoma (NPC) is the most common carcinoma of the head-neck in our country. The incidence of NPC in China is the highest of the world. The incidence of NPC in Guangdong, Guangxi, Hunan, Fujian and Jiangxi province is higher than the other provinces' in China. The place NPC arises from is covert and it is difficult to be found. Those places are often neighbor to nose, nasal sinuses and basilaris cranii. The early signs of NPC are multiple. NPC often have early metastases. NPC is easy to be misdiagnosed and missed diagnosed. The maximum age incidence is in the forth decade and the fifth decade. NPC strikes the patients and their family greatly. The researches of the early diagnosis and therapy of NPC are the emphases of the otolaryngologists' researches in our country.
The etiology of NPC is not clear up to now. Doctors think it is related with the infection of EB virus, environment pollutions and genetic factors. The major therapy of NPC is radiotherapy. The chemotherapy only use in the patients who isn't sensitive with radiotherapy or the patients in recurrence as the adjunctive therapy, and it also used in the patients of later period as the alleviative treatment. The NPC patients rarely have the chance to be operated, because, the place of NPC arises from is difficulty to reach and NPC's early metastasis. Operation can only be used in some special conditions as a adjunctive therapy. Scientists made many attempts to get better therapeutic effects to the NPC patients especially the patients who are insensitive to the radioactive ray, who are in recurrence and who have residual tumor in recent years. They paid more attention to the biotherapy of NPC such as immunotherapy, gene therapy, anti-vascular therapy, immunomodulator therapy and the therapy of biological response modifier. Although there are many achievements in these areas, it is still difficulty to use in clinic medicine and we still need to work hard to get more efficient therapeutic methods. Now the gene therapies based on the pathogenesis and the biology characters of carcinomas are popular. The application of RNAi in mammal provides a new way of gene therapy of carcinomas.
The length of telomere is related to the proliferation and life-span of cells. Telomerase can prevent telomeres shortening with cell division and stabilize the terminal of chromosome. Cells can be immortalized by the activation of telomerase. The study of the relations between telomerase and the carcinomas is helpful to our research. Most of the carcinomas including NPC can be detected with the active of telomerase. And the activity of telomerase in normal tissue and benign tumors is negative. As the activity subunit of telomerase, hTERT gene can be a good target of NPC's gene therapy.
VEGF is the only mitogen which can act especially on the endothelial cells. VEGF is related with the recurrence of NPC. The lymph nodes' metastases and distant metastates are also related with it. VEGF and VEGF receptor express highly in most carcinomas. Scientists find there are some relations between the expression of VEGF and the patients' life-span and distant metastases. The carcinoma patients with higher expression of VEGF have the lower life-span and more chances of distant metastases. The NPC patients with higher VEGF expression have more chances of lymph nodes metastases. Now more and more attention is paid to the tumor's anti-bloodvessel newborn therapy. The co-express of VEGF and VEGF receptor and the establishment of paracrine/autocrine mechanism provide new target of the gene therapy of carcinoma. The inhibitation of the interaction of VEGF and VEGF receptor can prevent the newborn of carcinoma bloodvessel, the proliferation and the metastases of carcinomas. Now, more and more researchers of otolaryngology study the relations between VEGF and NPC and they wish it can help to the treatment of NPC.
RNAi is a new technique discovered in recent years and it is useful in the researches of gene express, gene control and gene function. It works by the short interference RNAs' specificity silence of the homology genes. The siRNAs combinate with corresponding mRNA and made it degradation, then the translation of mRNA is terminated. RNAi present in every biology. It is economic and works quickly. At the same time, RNAi is of high efficiency and works in high specificity. RNAi can be used to study the function of genes, and it also can be used in carcinomas' gene therapy.
Our research will study the gene therapy of NPC by the application of RNA interference by inhibiting the expression of hTERT gene and VEGF gene. We divided our research in three parts. The first, we will observe the RNA interference in NPC. Our objects are to confirm the efficient positions of RNAi of hTERT gene and VEGF gene and observe the result after RNAi. We design the siRNA by software of online web, synthesize the siRNA by transcription in vitro, and instant transfect to CNE-2 cells by liposome.Then we observe the results by semiquantitative RT-PCR, Western Blot, FCM and TRAP to find the efficient RNAi positions of those two genes. We find two efficient RNAi positions of hTERT gene and two of VEGF gene. The levels of mRNA of those two genes were down-regulated by 89%～92% and 80%～95% separately. And the expression of protein of hTERT gene and VEGF gene were down-regulated in different degree too. The efficient RNAi of hTERT gene resulted the cells' detention at G_0～G_1 stage, and different degrees of cell apoptosis were induced by different siRNAs. As the second part of our researches, we constructed three plasmids of siRNA which can work stablely in carcinoma cells with the information from part one. The plasmids were all constructed on the base of pGenesil-1 Vector. pGenesil-hTERT was constructed to silence hTERT gene. pGenesil-VEGF was constructed to silence VEGF gene. pGenesil-hTERT-VEGF was constructed to silence both hTERT gene and VEGF gene. We transfected this plasmids into CNE-2 cells and observe the RNA interference effect of them. These three plasmids were constructed successfully and they can synthesize siRNAs in CNE-2 cells stably. The plasmids we constructed can silence corresponding gene in CNE-2 cells effectively. The last part of our research is the in vivo RNAi test of nude mice. We expected the test in mice can help the application in human body in future. We transplanted CNE-2 cells to nude mice and established the animal model of human NPC. We transfected plasmid pGenesil-hTERT, pGenesil-VEGF and pGenesil-hTERT-VEGF to nude mice respectively. The tumor volume and tumor morphology were observed. The Apoptosis Index was calculated. And we observed the protein express by immunohistochemisty. We found these three plasmids all can restrain the proliferation of the transplanted tumor in nude mice and induced CNE-2 cell's apoptosis. pGenesil-hTERT-VEGF is the most effective plasmid in this three plasmids.
At last, we come to some conclusions: (1) The siRNA synthesized in vitro can silence corresponding gene in NPC cells and their are RNAi in NPC. (2) The siRNA of hTERT gene from position 335 to 357 and from position 2324 to 2346 are effective to silence hTERT gene. The siRNA of VEGF gene from position 1130 to 1152 and from position 1504 to 1526 are effective to silence VEGF gene. (3) The effective siRNAs of hTERT gene can slow down the cell growth and restrain the proliferation. It also can down regulate the mRNA level of hTERT gene and depress the express of hTERT protein. It can result the cells detented at G_0～G_1 stage, and induce cell apoptosis. (4) The effective siRNAs of VEGF can down regulate the mRNA level of VEGF gene and depress the express of VEGF protein. But it will not restrain the proliferation of carcimoma cells and will not induce cell apoptosis. (5) We constructed three plasmids by pGenesil-1 vector and they all can silence corresponding genes stably in NPC cells. (6) We established animal model of human nasopharyngeal carcinoma in nude mouse. (7) The in vivo test proved that pGenesil-hTERT, pGenesil-VEGF and pGenesil-hTERT-VEGF all can inhibite the growth of the transplanted tumor in nude mice and induce the apoptosis of cells of NPC. They are all can be used to the gene therapy of nasopharyngeal carcinoma. (8) It is the first time to combinate hTERT gene and VEGF gene to one plasmid--pGenesil-hTERT-VEGF and the plasmid silenced these two genes successfully in the gene therapy of NPC. We had proved the silence effect of pGenesil-hTERT-VEGF is strong than pGenesil-hTERT and pGenesil-VEGF. Our results certified the feasibility and availability of multiple genes combinated therapy. It will provide a new approach to gene therapy of other genes, and it also the biggest innovation of our researches.
引文
[1] Thorley-Lawson DA, Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas. N Engl J Med, 2004, 350(13): 1328～1337
[2] Castro CY, Ostrowski ML, Barrios R, et al. Relationship between Epstein-Barr virus and lymphoepithelioma-like carcinoma of the lung: a clinicopathologic study of 6 cases and review of the literature. Hum Pathol, 2001, 32(8): 863～872
[3] Chang MS, Lee HS, Kim HS, et al. Epstein-Barr virus and microsatellite instability in gastric carcinogenesis. J Pathol, 2003, 199(4): 447～452
[4] Xue SA, Lampert IA, Haldane JS, et al. Epstein-Barr virus gene expression in human breast cancer: protagonist or passenger? Br J Cancer, 2003, 89(1): 113～119
[5] Rowe M, Young LS, Crocker J, et al. Epstein-Barr virus (EBV)-associated lymphoproliferative disease in the SCID mouse model: implications for the pathogenesis of EBV-positive lymphomas in man. J Exp Med, 1991, 173(1): 147～158
[6] Wilson JB, et al: Transgenic model of EBV associated neoplasia. 7th International EBV Symposium, 1996, E2. 3
[7] Raab-Traub N, et al: EBV LMP1: Strain variation, molecular, and biologic properties. 7th International EBV Symposium, 1996, HE4. 2
[8] 赵素萍，王承龙．血管内皮生长因子在鼻咽癌中的表达及其临床意义．湖南医科大学学报，2003，28(2)：114～116
[9] 王行炜，肖健云．端粒酶组分亚单位研究进展．中国耳鼻咽喉颅底外科杂志，2000，6(1)：60-63
[10] 张俊毅，赵素萍，肖健云，等．yCDgly TK融合自杀基因前药系统对鼻咽癌CNE-2细胞株放射增敏作用的研究．中国耳鼻咽喉颅底外科杂志，2005，11(2)：72～77
[11] 王承龙，赵素萍，肖健云，等．紫杉醇对鼻咽癌CNE-1细胞株作用的研究．中国现代医学杂志，2005，15(2)：221～223
[12] 黄东海，田勇泉，肖健云，等．组织微阵列研究eIF4E在鼻咽癌中的表达及其临床意义．中国耳鼻咽喉颅底外科杂志，2005，11(1)：1～4
[13] 唐瑶云，邓雪娥，肖健云，等．自杀基因质粒表达载体pcDNA3.1(-)CMV．TK在鼻咽癌细胞中的构建及转染研究．中国耳鼻咽喉颅底外科杂志，2004，10(6)：339～342
[14] 蒋卫红，肖健云，赵素萍，等．EB病毒基因BARF1在鼻咽癌中的表达及其意义。中国耳鼻咽喉颅底外科杂志，2004，10(6)：333～335
[15] 黄东海，范松青，田勇泉，等．鼻咽癌组织中survivin、bcl-2和p53蛋白的表达及其相关性．实用癌症杂志，2004，19(6)：585～587
[16] 王承龙，邓雪娥，肖健云，等．紫杉醇对鼻咽癌上皮细胞株HNE-1生长抑制和凋亡的影响．中国耳鼻咽喉颅底外科杂志，2004，10(5)：263～266
[17] 张帅，肖健云，田勇泉，等．凋亡抑制基因Survivin与鼻咽癌预后关系的研究．中国耳鼻咽喉颅底外科杂志，2004，10(4)：196～200
[18] 张帅，肖健云，田勇泉，等．凋亡抑制基因Survivin mRNA在鼻咽癌中的表达及其临床意义．中国耳鼻咽喉颅底外科杂志，2004，10(1)：1～4
[19] 唐瑶云，肖健云，赵素萍，等．自杀基因质粒表达载体pcDNA3.1(-)CMV．CD的构建及进行喉癌Hep-2细胞株转染研究．临床耳鼻咽喉科杂志，2005，19(21)：988～991
[20] 邱元正，肖健云，田勇泉，等．维甲酸诱导后CK13基因在鼻咽癌中的表达研究．中国耳鼻咽喉颅底外科杂志，2003，9(5)：290～293
[21] 王承龙，赖金平，王行炜，等．光动力作用诱导人鼻咽癌细胞凋亡超微结构形态改变．中华理疗杂志，2001，24(5)：270～273
[22] 邱元正，文忠，林功标，等．CK13基因与EB病毒DNA在鼻咽癌中的表达及临床意义．中国耳鼻咽喉颅底外科杂志，2001，7(1)：18～20
[23] 王行炜，肖健云，赵素萍，等．鼻咽癌端粒酶各组分基因表达与端粒酶活性的关系．中华医学杂志，2001，81(9)：553～556
[24] 王行炜，肖健云，赵素萍，等．鼻咽癌组织端粒酶各组分基因表达的研究．癌症，2001，20(3)：245～248
[25] 王行炜，肖健云，赵素萍，等．鼻咽癌细胞株γ射线照射后端粒酶活性及各组分表达的研究．中国耳鼻咽喉颅底外科杂志，2000，6(4)：221～225
[26] 文忠，肖健云，唐发清，等．鼻咽癌HNE1细胞株端粒酶的表达．中国耳鼻咽喉颅底外科杂志，2000，6(2)：76～79
[27] 王承龙，赖金平，唐发清，等．HNE1细胞株光动力学治疗前后形态学的动态变化．中国耳鼻咽喉颅底外科杂志，2000，6(1)：32～36
[28] 邱元正，田勇泉，肖健云，等．细胞角蛋白基因13在鼻咽癌中的表达研究．中华肿瘤杂志，2000，22(2)：129-132
[29] 罗均利，肖健云，田勇泉，等．MDM2基因在鼻咽癌中的表达及其与p53蛋白表达、EB病毒潜伏感染的关系．临床耳鼻咽喉科杂志，2000，14(11)期507-509
[30] 王行炜，赵素萍，肖健云，等．Bcl-2、Bax蛋白产物在鼻咽癌中的表达、分布及意义．中国耳鼻咽喉颅底外科杂志，1999，5(4)：213～216
[31] 田勇泉，文忠，肖健云，等．hTR反义寡核苷酸诱导鼻咽癌细胞凋亡的研究．中国耳鼻咽喉颅底外科杂志，1999，5(4)：193～197
[32] 邱元正，肖健云，田勇泉，等．细胞连接蛋白基因在鼻咽癌中的表达．中国耳鼻咽喉颅底外科杂志，1999，5(1)：1～5
[33] 邱元正，田勇泉，肖健云，等．细胞角蛋白基因13在鼻咽癌中的作用研究．中华肿 瘤杂志，1999，21(6)：444～446
[34] 罗非君，胡智，曾亮，等．EB病毒编码的潜伏膜蛋白1参与鼻咽癌恶性演进的转录调控实验研究．生物化学与生物物理进展，2002，29(4)：567～571
[35] 胡智，曾亮，陶永光，等．EB病毒编码的潜伏膜蛋白1通过TRAF／TRADD激活JNK信号途径．生物化学与生物物理进展，2002，29(4)：562～565
[36] Cheng ST, Huang DP, Hui AB, et al. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer, 1999, 83: 121～126
[37] 宋立兵，汪慧民，曾木圣，等．鼻咽癌细胞株SUNE-1异质性研究．癌症，1998，17(5)：324～327
[38] 蓝轲，姚开泰．EB病毒BNLF-1基因研究的新进展(综述)．生命科学，2001，17(1)：18～21
[39] Hu LF, Chen F, Zheng X, et al. Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1. Oncogene, 1993, 8: 1575～1583
[40] Li SN, Chang YS, Liu ST, et al. Effect of a 10-amino acid deletion on the oncogenic activity of latent membrane protein 1 of Epstein-Barr virus. Oncogene, 1996, 12: 2129～2135
[41] Miller WE, Cheshire JL, Raab-Traub N, et al. The NPCderived C 15 lmp1 protein confers enhanced activation of NF-kappa B and induction of the EGFR in epithelial cells. Oncogene, 1998, 16: 1869～1877
[42] Nicolson NL, Yalpaz M, Nicolson GL. Chromatin nucleoprotein complexes containing tightly bound c-abl, p53 and bcl-2 gene sequences: correlation with progression of chronic myelogenous leukemia. Gene, 1996, 169(2): 173～178
[43] Murono S, Yoshizaki T, Park CS, et al. Association of Epstein-Barr virus infection with p53 protein accumulation but not bcl-2 protein in nasopharyngeal carcinoma. Histopathology, 1999, 34(5): 432～438
[44] Arac S, Akyol MU, Kanbur B, et al. Bcl-2 and LMP1 expression in nasopharyngeal carcinomas. Am J Otolaryngol, 2001, 22(6): 377～382
[45] Khabir A, Ghorbel A, Daoud J, et al. Similar BCL-X but different BCL-2 levels in the two age groups of north African nasopharyngeal carcinomas. Cancer Detect Prev, 2003, 27(4): 250～255
[46] Dawson CW, Eliopoulos AG, Dawson J, et al. BHRF1, a viral homologue of the Bcl-2 oncogene, disturbs epithelial cell differentiation. Oncogene, 1995, 10(1): 69～77
[47] McCarthy NJ, Hazlewood SA, Huen DS, et al. The Epstein-Barr virus gene BHRF1, a homologue of the cellular oncogene Bcl-2, inhibits apoptosis induced by gamma radiation and chemotherapeutic drugs. Adv Exp Med Biol, 1996, 406: 83～97
[48] Nicholls J, Kremmer E, Meseda CA, et al. Comparative analysis of the expression of the Epstein-Barr virus (EBV) anti-apoptotic gene BHRF1 in nasopharyngeal carcinoma and EBV-related lymphoid diseases. J Med Virol, 2001, 65(1):105～113
[49] Bernstein E, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001, 409(6818):363～366
[50] Sijen T, Simmer F, Fire A.et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell. 2001 ;107(4):465-476.
[51] Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 2001, 107(3):297～307
[52] Billy E, Zhang HD, Filipowicz W. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA, 2001, 98 (25): 14428-14433
[53] Elbashir SM, Lendeckel W, Tuschl T, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411(6836):428～429
[54] Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 2002,16(8): 948～958
[55] Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33(3):401-406
[56] Barton GM, Medzhitov R. Retro viral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A, 2002, 99(23): 14943-14945
[57] Lee NS, Bauer G, Rossi J. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol, 2002, 20(5):500-505
[58] Capodici J, Kariko K, Weissman D. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol, 2002,169(9):5196-5201
[59] Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci U S A, 2003,100(4):2014～2018
[60] Wilson JA, Jayasena S, Khvorova A, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci U S A, 2003,100(5):2783～2788
[61] Shlomai A, Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology, 2003, 37(4):764～770
[62] McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003, 21(6):639～644
[63] Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene, 2002, 21(37):5716～5724
[64] Wohlbold L, Van Der Kuip H, Miething C, et al. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571). Blood,2003, 102(6):2236～223
[65] Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene, 2002, 21(39):6041～6048
[66] Hall AH, Alexander KA. RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells. J Virol, 2003, 77(10):6066～6069
[67] Kamath RS, Fraser AG, Dong Y, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 2003, 421(6920):231-237
[68] Kamath RS, Ahringer J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods, 2003, 30(4):313-321
[69] Lee SS, Lee RY, Fraser AG, et al. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet, 2003, 33(1):40～48
[70] Wolkow CA, Munoz MJ, Riddle DL, et al. Insulin receptor substrate and p55 orthologous adaptor proteins function in the Caenorhabditis elegans daf-2/insulin-like signaling pathway. J Biol Chem, 2002, 277(51):49591～49597
[71] Li K, Lin SY, Brunicardi FC, et al. Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma. Cancer Res, 2003, 63(13):3593～3597
[72] Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature, 2002,418(6896):435～438
[73] Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA, 2002, 9(9): 6047 -6052
[74] Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res, 2002, 30(10):e46
[75] Yang D, Buchholz F, Huang Z, et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci U S A, 2002, 99(15):9942～9947
[76] Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol, 2002, 20(5):497～500
[77] Paul CP, Good PD, Winer I, et al. Effective expression of small interfering RNA in human cells. Nat Biotechnol, 2002, 20(5):505～508
[78] Sui G, Soohoo C, Affar EB, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc NatI Acad Sci U S A, 2002, 99(8): 5515～5520
[79] McCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice. Nature, 2002, 418: 38～39
[80] Tiscomia G, Singer O, Ikawa M, et al. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci U S A, 2003, 100(4): 1844～1848
[81] Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products. RNA, 2002, 8(11): 1454～1460
[82] 张钦明，孙宁，陈小毅，等．EBV-LMP1对人高分化鼻咽癌细胞株(CNE1)体外增殖能力的影响．广东医学院学报，1999，16(1-2)：19～22
[83] 张钦明，孙宁，陈小毅，等．EB病毒潜伏膜蛋白对人高分化鼻咽癌细胞系CNE1生长分化的影响．中华病理学杂志，1999，28(4)：285～289
[84] Li G, Li XP, Peng Y, et al. Effect of inhibition of EBV-encoded latent membrane protein-1 by small interfering RNA on EBV-positive nasopharyngeal carcinoma cell growth. Di Yi Jun Yi Da Xue Xue Bao, 2004, 24(3): 241～246
[85] Rosse T, Olivier R, Monney L, et al. Bcl-2 prolongs cell survival after Bax-induced release of cytochromec. Nature, 1998, 391: 496～499
[86] Voehringer DW, McCondey DJ, McDonneil TJ, et al. Bcl-2 expression causes redistribution of glutathiome to the nucleus. Proc Natl Acad Sci USA, 1998, 95(6): 2956～2960
[87] Sheu LF, Chen A, Merg CL. Analysis ofbcl-2 expression in normal, inflamed, dysplaslic nasopharygeak epithelia and nasopharyngeal carcinoma association with p53 expression. Hum Pathol, 1997, 28(5): 556～562
[88] Sheu LF, Chen A, Hsu HY, et al. Cooperative effects of p53, bcl-2, and Epstein-Barr virus latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma. Cancer Detection and Prevention, 2000, 24(Supplement 1): 101～104
[89] Zhang Y, Lei XY. Effect of bcl-2 antisense oligodeoxynucleotides on drug sensitivity of leukemic cells. Hematol J, 2003, 4(3): 187～97
[90] Lopes de Menezes DE, Hu Y, Mayer LD. Combined treatment of Bcl-2 antisense oligodeoxynucleotides (G3139), p-glycoprotein inhibitor (PSC833), and sterically stabilized liposomal doxorubicin suppresses growth of drug-resistant growth of drug-resistant breast cancer in severely combined immunodeficient mice. J Exp Ther Oncol, 2003, 3(2): 72～82
[91] Rudin CM, Kozloff M, Hoffman, et al. Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell lung cancer.J Clin Oncol, 2004, 22(6): 1110-1117
[92] Dr. Jill Lacy, Yale University. Bcl-2 Antisense Oligonucleotide (G3139,Genasense) Chemosensitizes Human Nasopharyngeal Carcinoma to Cisplatin In Vivo in a SCID Xenotransplanatation Model. The 95th Annual Meeting the American Association of Clinical Research (AACR)
[93] Yin JQ, Gao J, Shao R, et al. siRNA agents inhibit oncogene expression and attenuate human tumor cell growth. J Exp Ther Oncol, 2003, 3(4): 194-204
[94] Jiang M, Milner J. Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells. Genes Dev, 2003, 17(7):832～837
[95] Futami T, Miyagishi M, Seki M, et al. Induction of apoptosis in HeLa cells with siRNA expression vector targeted against bcl-2. Nucleic Acids Res Suppl,2002, (2):251-252
[96] Cioca DP, Aoki Y, Kiyosawa K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther,2003, 10(2): 125-133
[97] Lima RT, Martins LM, Guimaraes JE, et al. Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells. Cancer Gene Ther, 2004, 11(5):309～316