【作者】 潘建青；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 背景和目标：鼻咽癌（nasopharyngeal carcinoma，NPC）是我国南方的常见肿瘤。EB病毒（Epstein Barr virus，EBV）是鼻咽癌最重要的病因。鼻咽癌的组织中有EB病毒潜伏期膜蛋白1、2不同程度的表达。研究发现，很少的EB病毒携带者能产生LMP1特异性细胞毒性T淋巴细胞（cytotoxic T lymphocyte，CTL），这提示我们建立一个针对LMP1和LMP2的特异性细胞毒性T淋巴细胞反应，对鼻咽癌患者应该有长期的治疗效果。腺相关病毒（adeno-associated virus，AAV）是目前最常用的基因治疗载体，它拥有一些独特的优点：可以携带基因转染分裂期和非分裂期细胞、无致病性、能驱动基因在体内长期、稳定的表达。在本研究中，我们利用腺相关病毒载体，以EB病毒潜伏期膜蛋白1、2的基因作为表位，并融合了热休克蛋白作为佐剂，构建了一种鼻咽癌疫苗。方法：1．我们首先克隆了EB病毒LMP1、LMP2基因的编码序列，并构建pAAV-LMP2／1-hsp重组质粒，然后与重组腺相关病毒包装质粒pXX₂，辅助质粒phelper，通过磷酸钙沉淀法共转染293细胞，包装制备rAAV-LMP2／1-hsp病毒，同时包装制备对照病毒rAAV-GFP，病毒经纯化后以斑点杂交方法检测其病毒滴度。2．SP2／0细胞转染了pCDNA3．1-LMP2质粒，然后通过G418（400ug／ml）筛选，得到稳定表达LMP2的阳性细胞株SP 2／0-LMP2。3．LMP2_228-242肽注射于新西兰白兔的皮下，6周后，收集血清、纯化后得到兔抗LMP2多克隆抗体。4．为确认rAAV转染细胞后嵌合基因是否有表达，我们用1×10⁹的病毒颗粒感染293细胞，2天后提取蛋白，通过免疫印迹法检测嵌合基因的表达。5．我们采用了两个不同的免疫策略。第一种方法：首先肌肉注射rAAV-LMP2／1-hsp或rAAV GFP免疫BALB／c小鼠。3周后，动物皮下注射SP2／0-LMP2或SP2／0细胞成瘤，观察30天，定期测量肿瘤直径。第二种方法：首先BALB／c小鼠皮下注射SP2／0-LMP2或SP2／0细胞，10天后肌肉注射rAAV-LMP2／1-hsp或rAAV GFP免疫动物，定期测量肿瘤直径。6．为分析rAAV疫苗的细胞免疫反应，BALB／c小鼠分为3组，分别肌肉注射rAAV-LMP2／1-hsp、rAAVGFP或PBS，3周后杀死动物，取脾细胞。SP2／0-LMP2或SP2／0细胞作为靶细胞，小鼠脾细胞作为效应细胞，以LDH释放法测定细胞毒性T细胞功能。7．为研究rAAV疫苗的免疫机制，我们把BALB／c小鼠分为3组，分别肌肉注射rAAV-LMP2／1-hsp、rAAV GFP或PBS。3周后杀死动物，取脾细胞，分别以LMP2特异性表位肽或非特异性肽干预，培养4天后，以MTT法测定T细胞增殖。结果：1．经过酶切鉴定、PCR、测序法证实重组腺相关病毒质粒pAAV-LMP2／1-hsp构建成功。2．免疫印迹法证实该病毒转染293细胞后可表达融合蛋白。3．先用rAAVLMP2／1-hsp或rAAV GFP免疫BALB／c小鼠，然后接种SP2／0或SP2／0-LMP2细胞，虽然各组都能成瘤，但是rAAV-LMP2／1-hsp组的肿瘤生长速度明显比其他各组缓慢，而其他各组之间无差异。第35天，rAAV LMP2／1-hsp免疫组的肿瘤体积大约是rAAV GFP免疫组的1／5。4．先接种SP2／0或SP2／0-LMP2细胞，然后用rAAVLMP2／1-hsp或rAAV GFP免疫BALB／c小鼠。rAAV-GFP免疫组的小鼠肿瘤进展迅速，在免疫的第20天全部死亡；而rAAV LMP2／1-hsp免疫组的小鼠肿瘤生长缓慢，生存时间明显延长。5．BALB／c小鼠接种rAAV-LMP2／1-hsp，rAAV-GFP或生理盐水，3周后杀死动物，分离培养脾细胞。用SP2／0-LMP2或SP2／0细胞作为靶细胞，小鼠脾细胞作为效应细胞，以LDH释放法测定细胞毒性T细胞功能，结果显示rAAV-LMP2／1-hsp免疫组的脾细胞在LMP2表位肽的刺激下裂解SP2／0-LMP2和SP2／0细胞，而其他各组不发生裂解。6．以MTT法测定T细胞增殖反应，结果显示来源于rAAV-E7CTL-hsp免疫组的小鼠T细胞在LMP2表位肽的刺激作用下发生增值，而其他组T细胞不增值。结论：利用腺相关病毒载体成功构建了一种鼻咽癌疫苗，以EB病毒潜伏期膜蛋白1、2的基因作为表位，并融合了热休克蛋白作为佐剂，该疫苗可能通过诱导细胞毒性T细胞反应，发挥抗肿瘤的效应。该项研究有希望投入临床应用。更多还原

【Abstract】 Background and objective:Nasopharyngeal carcinoma （NPC） is a common cancerin Southern China and Epstein-Barr virus （EBV） is the most important pathogenesis.Hence,there is considerable interest in the possibility of targeting the virus-specific immuneresponse to the viral antigens,which are expressed in these malignancies.These antigensinclude LMP1,and LMP2.Preview studies have shown a low CTL precursor frequency toepitopes within LMP1 in healthy virus carriers,suggesting that reconstitution of bothLMP1- and LMP2- specific CTL responses may be necessary for a long-term therapeuticbenefit of NPC patients.Adeno-associated virus （AAV）,a single-stranded virus,has beenstudied as a vector for gene therapy.AAV has many natural features that are attractive for ahuman gene therapy vector,such as nonpathogenicity,targeted integrating capability,and abroad host range.In this study,we explore a potential that recombinant adeno-associatedvirus （rAAV） carrying a fusing gene containing heat shock protein as an adjuvant,EBVlatent membrane proteins cytotoxic T-lymphocyte epitope DNA as a vaccine preventsNasopharyngeal carcinoma.The tumor vaccine was devised by constructing a chimericgene which contained EBV latent membrane proteins cytotoxic T-lymphocyte epitopeDNA fused with the heat shock protein gene as a tumor vaccine delivered via rAAV.Method:1.First we cloned the coding sequence of EBV LMP1 and LMP2,andconstructed pAAV-LMP2/1-hsp.The rAAV-LMP2/1-hsp was obtained by calciumphosphate-DNA co-transfection methods with the help of packaging-plasmid pXX2 andhelper-plasmid phelper.The control rAAV-GFP was simultaneously packed using the samemethods,followed by purification and tittering of the purified rAAVs with real-time PCRtechniques.2.The SP2/0 cell line was transfected with plasmid pCDNA3.1-LMP2 to allow stable LMP2 expression,and the positive cell clones were selected bv G418 （400ug/ml） andnamed SP 2/0-LMP2 cell line.3.LMP2_228-242 synthetic peptide was injected beneath theskin of two New Zealand white rabbits.After 6 weeks,rabbits were sacrificed,and theirblood was collected and purified to acquire rabbit Anti-LMP2 polyclonal antibodies.4.Todetermine whether the chimerically constructed LMP2/1CTL-hsp gene can be expressedvia rAAV delivered to cells,we infected 293 cells using rAAV-LMP2/1-hsp.Two dayslater,the cellular protein was harvested and the expression of the LMP2/1-hsp chimericgene was analyzed by Western blotting with rabbit Anti-LMP2 polyclonal antibodies.5.Two different vaccination strategies were used to assess the efficacy of the rAAV vaccine.In the first set of experiments,BALB/c mice were immunized with eitherrAAV-LMP2/1-hsp or rAAV GFP.Three weeks after the immunization,these mice werechallenged s.c.with live SP2/0-LMP2 or SP2/0 cells.After challenge,these animals wereregularly monitored for 30 days,and the tumor size was measured by calipers.In thesecond set of experiments,BALB/c mice were first challenged with SP2/0-LMP2 or SP2/0tumor cells.Ten days after the challenge,mice were immunized i.m.with either rAAVLMP2/1-hsp or rAAV GFP.The therapeutic efficacy of the rAAV vaccine was assessed byregular monitoring of tumor regression.6.To assess the Cellular immune response of therAAV vaccine,BALB/c mice were vaccinated with recombinant virus encoding LMP2/1,rAAV GFP,or normal saline.After 3 weeks,mice were sacrificed,and Spleen cells wereretained.Target cells were SP2/0-LMP2 or WT SP2/0,and Effector cells were T cells.LDH release was measured using the Cytotox 96 cytotoxicity assay.7.To analyse theimmune mechanism of the rAAV vaccine,BALB/c mice were vaccinated withrrAAV-LMP2/1-hsp,rAAV-GFP,or normal saline.After 3 weeks,mice were sacrificed,and T cells were pulsed in vitro with or without LMP2 synthetic peptide （PYLFWLAAI）,or pulsed with nonspecific peptide for a 4-day proliferation assay.Proliferation wasquantified by MTT Assays.Results:1.Restriction enzyme analysis,PCR and sequencing results showed that therecombinant plasmid pAAV-LMP2/1-hsp was successfully constructed.2.Western blotsshowed that recombinant protein was detected by anti-LMP2 antibodies in rAAV-LMP2/1-hsp infected cells but not in the control cells infected with rAAV-GFP.3.BALB/c mice （6 mice in each group） were first immunized with rAAV LMP2/1-hsp orrAAV GFP,and then challenged with SP2/0 or SP2/0-LMP2 cells.Although both groups ofanimals developed tumors,the tumor outgrowth in rAAV-GFP was highly aggressive.Inthe contrast,these tumors grew much less aggressively in animals immunized withrAAV-LMP2/1-hsp,and outgrowth was completely resolved in 90% of the animals by theend of the observation period in the mice with load of SP2/0-LMP2 cells.By day 35,theaverage tumor load in rAAV LMP2/1-hsp immunized mice was 5 fold lower whencompared with rAAV GFP immunized mice.4.In the second set of experiments,followingimmunization,the tumor size progressively increased in almost all animals injected withrAAV-GFP,and by day 20 after immunization,100% mice were dead.In contrast,adramatic reduction in the tumor load was observed in mice immunized with rAAVLMP2/1-hsp,and showed long-term protection.5.T cells from BALB/c mice immunizedwith rAAV-LMP2/1-hsp,rAAV-GFP,or mock were isolated and stimulated in vitro withLMP2 synthetic peptide.The results showed that SP2/0-LMP2 and WT SP2/0 cells pulsedwith LMP2 peptide 131 to 139 were lysed,whereas WT SP2/0 cells alone or WT SP2/0cells pulsed with nonspecific peptide were not lysed.6.T cell proliferation assay showedthat T cells from mice vaccinated with rAAV-LMP2/1-hsp could be stimulated mice byLMP2 peptide_131-139.Conclusions:we successfully developed rAAV encoding EBV LMP2/1 CTL peptideDNA fused with hsp DNA as a tumor vaccine,which may induce CTL response to retardtumor growth.It is a potential vaccine for nasopharyngeal carcinoma treatment using hsp asa carrier protein and delivery by rAAV vector.更多还原