【作者】 臧光祥；

【导师】 孙宏晨；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2009， 博士

【摘要】 涎腺腺样囊性癌是口腔颌面部较常见的恶性肿瘤,具有嗜神经和血管等特性,对放疗和化疗不敏感,传统治疗方法不能取得令人满意的治疗效果,随着分子生物学技术发展,转基因治疗涎腺腺样囊性癌成为新的策略,但转基因治疗肿瘤存在特异性差的问题。近年来研究发现hTERT在永生化细胞系和85%的人肿瘤组织中都具有较高的活性,而在大多数正常体细胞中则无活性,本课题组前期研究发现hTERT在涎腺腺样囊性癌阳性率100%,而在正常涎腺组织内阴性或弱阳性。肿瘤坏死因子(TNF)相关的凋亡诱导配体(TRAIL)是TNF家族成员,能引起多种肿瘤细胞凋亡,而对正常细胞影响较小,目前,TRAIL已被应用于多种肿瘤的转基因治疗研究。针对肿瘤转基因治疗特异性差的问题,根据hTERT和TRAIL均具有肿瘤特异性的特性,在国家自然基金面上项目和国家自然基金国际合作项目资金支持下,以同源重组法构建了AdCMV-TRAIL和AdTERT-TRAIL腺病毒载体,通过体外、体内转染涎腺腺样囊性癌细胞或正常细胞,探讨它们对涎腺腺样囊性癌的作用,结果显示通过双特异性AdTERT-TRAIL实现了TRAIL基因在SACC-83细胞中表达,进而对涎腺腺样囊性癌细胞具有肿瘤特异性杀伤作用:其对菏瘤裸鼠器官、肝脏功能和全血成分没有明显影响,这种特异性杀伤作用是由TRAIL基因激活了Caspase-3基因,从而启动了下游的凋亡途径。这就为AdTERT-TRAIL转基因治疗涎腺腺样囊性癌奠定了理论基础和实验技术基础。更多还原

【Abstract】 Adenoid cystic carcinoma is one of the most common malignant tumor in maxillofacial region,which has the ability to infiltrate nerve,vessel,fiber,and not sensitiveness to chemotherapy,radiotherapy. It is hot topic to study the development and treatment of adenoid cystic carcinoma.With the development of biomedicine,molecular biology and completion of human genome,gene therapy become a new strategy to treat adenoid cystic carcinoma,which is to transfer the external gene to cells,correct the heredity and get impairment of gene.After 20-year research and studying,there was great progression to treat the heredity disease,metabolism disease and malignant tumor using gene therapy,and some treatment were used in clinical case.Vector,promoter,target gene are the basic elements of gene therapy. The target expression of gene plays key role in gene therapy,which the target gene should be expressed in tumor cells,not common ones.Only in this way,the gene could become targeted expression.The common method of targeted gene expression is to control gene expression via tumor-specific promoters in specific tissues,cells or organs.Several promoters have been identified that are more active in particular tumor types than in the tissues from which they arise,and these promoters have been exploited to target gene expression in tumors,which include the AFP promoter,CEA promoter and Tyr enzyme promoter.But these promoters are limited to specific tumor histology and cannot be used universally in tumors of various origins and most of these promoters are much weaker than that commonly used viral promoters such as the CMV promoter.Consequently,their using is hampered by the problem of low expression.Recent years,telomerase was found to be responsible for cell-immortalization and tumor-genesis.It is a specialized DNA polymerase responsible for the replication of chromosomal ends,or telomeres.Telomerase is high active in immortalized cell lines and 85% human cancers,but is inactive in most somatic cells,therefore telomerase is to be very promising not only as a tumor-specific marker but also as a target for anticancer therapy.In summary,basing this characteristic of telomerase,if using the driver of telomerase to induce the target gene,the target gene should be express in tumor cells,not the somatic cells and targeted anticancer therapy will come true.The vector is another important part of gene therapy.With the development of gene therapy,adenovirus has become the most common one,which has several features:1.it is easy to prepare,because its structure and function are definitely understanding;2.it can infect both dividing cell and non-dividing cell;3.it can ingress different tissue;4.it can contain 10kb,or even 37 kb gene;5.high titer;6.the structure is stable and it is uneasy to insert the genome of host.So we use the adenovirus vector to transfer the target gene.For the purpose gene of gene therapy,at the beginning,researcher liked to select oncogene or anti-oncogene as target gene,using anti-sencetherapy or transfer anti-oncogene into tumor cell and induce apoptosis of tumor cell.Now apoptosis genes are selected as targeted gene and express in tumor to active the apoptosis cell signal to kill the tumor cells.Tumor necrosis factor-related apoptosis inducing ligand(TRAIL) is a new number of TNF family(named Apo-2L),and many researchers found the TRAIL gene could induce apoptosis of many types of tumor cells,while almost no effect of normal cells.According to overview above,we used two types of adenovirus vector,one of which contain CMV driver and TRAIL target gene in adenovirus vector and another one contain hTERT driver and TRAIL target gene,according to the feature of hTERT activity and TRAIL gene. In vitro,after transferred the AdCMV-TRAIL and AdTERT-TRAIL into adenoid cystic carcinoma cells,the proliferation and apoptosis of cells were detected by histological,molecular,MTT,FCM techniques in adenoid cystic carcinoma cells and/or HEL.In vivo,after AdTERT-TRAIL was injected into tumors of adenoid cystic carcinoma in nude mice,the tumor volumes,the survive time of nude mouse,the morphology of organs and elements of blood in nude mouse were detected,in order to illuminate the function and safety of AdTERT-TRAIL.Result showing:1.AdCMV-TRAIL could inhibit the proliferation and promote apoptosis of SACC-83 cells and ACC-2 cells of adenoid cystic carcinoma.There was no difference of proliferation and apoptosis between two cell lines;2.AdTERT-TRAIL could inhibit proliferation and enhance apoptosis of SACC-83,while not obviously effect in somatic cells of HEL;3.AdTERT-TRAIL inhibit the tumor growth of adenoid cystic carcinoma and prolong the survive time of nude mice,and there were not obviously change of morphology of organs and elements of blood in nude mice;4.The mechanism of AdTERT-TRAIL to inhibit proliferation and promote apoptosis of adenoid cystic carcinoma cells is because the transfer of TRAIL activated Caspase-3 gene and then induced a series of apoptosis of cell signal.The innovation of this study was that dual-targeted AdTERT-TRAIL has targeted-effect to the cells of adenoid cystic carcinoma, AdTERT-TRAIL is not harmfulness to the organs,the function of liver and the elements blood system of nude mice,and AdTERT-TRAIL induces apoptosis signals by actived Caspase-3 gene after transferred into adenoid cystic carcinoma.Our research achievements provided a foundation for us to investigate the tumor-specific transgene expression ability of hTERT promoter in salivary gland tumors,as well as theory basis and practice experience for us to conduct gene therapy clinically with high efficiency,targeting effect and no trauma,which will develop a new way to treat salivary gland tumors.更多还原