【作者】 李湘平；

【导师】 丁彦青；

【作者基本信息】 第一军医大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 鼻咽癌是我国南方及亚洲中国人种高发的头颈部肿瘤，中晚期鼻咽癌患者5-10年生存率仅有40％左右，局部复发和远处转移是影响患者生存率的主要因素。目前所采用的放化疗在治疗鼻咽癌时常发生严重的并发症，影响患者生活质量，这就需要我们更积极地寻找全身综合治疗手段，预防和治疗鼻咽癌的复发和转移，全面提高鼻咽癌的总生存率，改善生存质量。随着肿瘤生物学的发展，肿瘤的生物治疗已逐渐成为治疗恶性肿瘤的第四模式，肿瘤的生物治疗在肿瘤综合治疗中发挥越来越大的作用。干扰素（IFN）作为主要的肿瘤生物治疗制剂之一，以其抑制肿瘤相关病毒的繁殖、抑制肿瘤细胞增生和诱导分化、促进凋亡，免疫调节及抑制肿瘤血管生成等功能，已被广泛应用于治疗14种以上的恶性肿瘤。但干扰素在鼻咽癌的治疗作用了解不多。本研究通过体内外实验，探讨干扰素对EB病毒阳性鼻咽癌细胞生长、转移的作用。目前临床使用干扰素为蛋白制剂，蛋白形式的干扰素在体内半衰期短，需长期给药，使用药量大且血药浓度并不稳定，易出现全身各系统的毒副作用，目前认为干扰素抗肿瘤作用效果有限，这可能与干扰素在体内很难达到有效治疗剂量有关等。针对干扰素治疗肿瘤策略上存在的问题，本实验以重组腺相关病毒（rAAV）作为转基因载体，介导干扰素基因的表达，使机体细胞长期稳定表达基因，产生有生物活性的干扰素蛋白，同时以临床常规外源性干扰素治疗方法为对照，探讨干扰素在鼻咽癌的治疗作用及作用机制，为鼻咽癌生物治疗提供有价值的理论依据。一、重组腺相关病毒（rAAV）载体的包装与鉴定采用分子克隆的方法将IFN-α表达序列插入pAAV质粒的BamHⅠ及EcoRⅠ酶切位点之间，将EGFP表达序列插入pAAV质粒的XhoⅠ及EcoRⅠ酶切位点之间分别成功构建pAAV-IFN-α及pAAV-EGFP；采用无辅助病毒的双质粒共转染方法，pAAV-IFN-α（或pAAV-EGFP）及rAAV包装辅助质粒pDG共同转染HEK-293FT细胞，包装病毒；采用氯仿处理-PEG／NaCL沉淀-氯仿抽提3个步骤分离、浓缩和纯化重组腺相关病毒（rAAV）。通过Real-Time PCR定量检测，确定包装病毒滴度均介于10¹¹v.g.／ml～10¹²v.g.／ml之间。通过不同滴度rAAV-EGFP转染C666-1细胞，其结果显示48小时以5×10⁴v.g.／细胞转染效率最高，达95％以上，纯化后的rAAV仍保持较强的感染性。RT-PCR及Western结果显示IFN-α在转录及翻译水平表达，表明rAAV-IFN-α载体的高效性。二、rAAV-IFN-α对EBV（+）鼻咽癌细胞株的作用研究IFN-α蛋白抑制EBV（+）鼻咽癌细胞株C666-1细胞生长的半数抑制剂量（IC50）实验；rAAV-IFN-α转染C666-1细胞的细胞增殖实验（MTT）；rAAV-IFN-α转染C666-1细胞流式细胞检测；细胞凋亡检测；RT-PCR鉴定rAAV-IFN-α转染C666-1细胞后IFN-α表达及转移相关基因表达变化；高效液相芯片技术（Liquichip）检测rAAV-IFN-α转染C666-1细胞后细胞上清中人IFN-α的含量。数据采用SPSS10.0统计软件包进行统计学处理。结果显示：采用MTT试验，当IFN-α蛋白浓度达到1×10⁵IU／ml，C666-1细胞增殖明显受抑制（达50％），即IC50；MTT试验显示，rAAV-IFN-α转：染C666-1细胞培养48小时至72小时后，细胞平均吸光度与对照组比较无统计学差异（F=1.181，p=0.334）。流式细胞术结果显示，与对照组比较，rAAV-IFN-α转染48小时后能改变C666-1细胞周期，使G1期细胞比例增加（F=39.675，P=0.000），效果优于IFN-α蛋白，但无统计学差异（P=0.059）。运用三种细胞凋亡检测方法结果显示，体外IC50浓度的IFN-α及rAAV-IFN-α均不能引起C666-1细胞明显凋亡。RT-PCR显示rAAV介导IFN-α表达后显著抑制C666-1细胞中EB病毒LMP-1及EBNA-1的表达，并下调肿瘤细胞MMP-9及VEGF的表达，IC50浓度的IFN-α亦能引起相同效应，但效果较弱。高效液相芯片技术（Liquichip）检测结果显示rAAV-IFN-α转染C666-1细胞后培养上清中IFN-α的含量随培养时间延长而增加，但培养至72小时总体浓度仍低于20U／ml。三、rAAV介导IFN-α基因对EBV（+）鼻咽癌肝移植瘤生长和转移的实验研究BALB／c-nu／nu雄性裸鼠64只建立EBV（+）鼻咽癌肝移植瘤动物模型。将64只裸鼠随机分成4组，即A组rAAV-IFN-α组、B组IFN-α组、C组rAAV-EGFP组及D组PBS组，每组16只；其中每组随机取8只用于观察抗肿瘤效果，其余8只用于生存期观察。观察rAAV-IFN-α、IFN-α对裸鼠鼻咽癌肝移植瘤生长和转移抑制的效应。免疫组化检测移植瘤组织中MMP-9的表达和微血管密度（MVD）。原位末端标记（TUNEL法）检测肝脏移植瘤肿瘤细胞凋亡。RT-PCR分析肿瘤组织IFN-α表达及转移相关基因表达变化。高效液相芯片技术检测血清中人IFN-α及鼠IL-12、GM-CSF、IFN-r、IL-2、IL-10、TNF-α的含量。确定不同组动物的生存期。数据采用SPSS10.0统计软件包进行统计学处理。结果显示：肝脏鼻咽癌移植瘤3周后，A、B、C、D组肿瘤平均体积分别为（239.00±44.80）mm³、（340.25±75.96）mm³、（424.75±67.22）mm³、（430.75±112.05）mm³，四组肿瘤体积经方差分析提示有非常显著意义（F=10.397，P=0.000），经多重比较rAAV-IFN-α与其他三组差异均有显著意义（P=0.000），IFN-α组与EGFP及PBS组比较均有显著差异（P=0.029），而EGFP组与PBS组比较差异无显著意义（P=0.880）。A、B、C、D组肝内转移分别为12.5％、37.5％、100％、100％；肺转移为0.0％、50.0％、87.5％、100％；平均生存期分别为（18.25±2.49）天、（15.13±1.13）天、（12.88±1.25）天、（12.63±1.19）天，4组之间有显著统计学差异（F=20.848，P＜0.001），其中rAAV-IFN-α生存时间较IFN-α组（P=0.001）及对照组（P=0.000）显著延长，IFN-α组较EGFP组与PBS组亦显著延长（P＜0.01）。凋亡平均指数分别为（12.20±1.92）、（8.80±1.64）、（4.00±1.58）、（3.40±1.14），经单因素方差分析差异有非常显著意义（F=34.118，P=0.000），多重比较提示A组较其他三组均有显著差异，IFN-α较rAAV-EGFP及PBS组亦有显著差异（P=0.000），而rAAV-EGFP与PBS组间无统计差异（P=0.561）。rAAV-IFN-α及IFN-α组移植鼻咽癌肿瘤组织MMP-9表达较rAAV-EGFP及PBS组显著下降，经秩和检验差异有显著差异（P=0.000）。rAAV-IFN-α及IFN-α组肝脏移植鼻咽癌肿瘤组织MVD（1.60±1.14）、（1.80±0.84）较对照组（13.00±2.74）、（13.20±2.59），明显下降，经单因素方差分析差异有非常显著意义（F=53.498，P=0.000）。RT-PCR结果显示rAAV-IFN-α组肝脏移植鼻咽癌细胞内有IFN-αmRNA表达，在IFN-α组及对照组无IFN-αmRNA表达。在rAAV-IFN-α组及IFN-α组鼻咽癌细胞MMP-9mRNA、VEGFmRNA表达与对照组比较表达下降。用rAAV-IFN-α及IFN-α治疗荷瘤裸鼠后3周，A、B、C、D组裸鼠血清中IFN-α含量（U／ml，（?）±s）分别为（7.01±2.68）（5.59±1.42）（1.81±0.48）、（1.30±0.34），rAAV-IFN-α组血清中IFN-α的含量较IFN-α组含量高，但无统计学差异（P=0.126），但较对照组显著升高（P=0.000）；检测四组裸鼠血清中IL-12含量分别为（pg／ml，（?）±s）（105.02±43.43）（56.99±13.93）（0.00±0.00）（0.00±0.00），rAAV-IFN-α组较IFN-α组（P=0.002）及对照组比较均有显著统计学差异；四组裸鼠血清中GM-CSF含量（pg／ml，（?）±s）分别为（105.23±21.05）、（100.52±14.59）、（72.42±22.76）、（63.08±26.100），rAAV-IFN-α组比较IFN-α组无统计学差异（P=0.687），较对照组有显著增长（P＜0.01）。IFN-r、IL-2、IL-10、TNF-α四种细胞因子含量治疗组与对照组之间无统计学差异。结论：1、本研究采用的rAAV-IFN-α治疗策略能有效抑制鼻咽癌的生长和转移；从抑制移植瘤生长、转移抑制效果和生存时间比较，rAAV-IFN-α基因治疗优于外源性IFN-α蛋白治疗。2、IFN-α主要通过抑制肿瘤血管增生，免疫调节作用发挥其抗EBV（+）鼻咽癌生长和转移作用，IFN-α对EBV（+）鼻咽癌细胞的直接抑制作用有限。3、IFN-α可能通过抑制EBV LMP-1及EBNA-1基因表达发挥其抗EBV（+）鼻咽癌生长和转移作用。更多还原

【Abstract】 Nasopharyngeal carcinoma （NPC）, a type of head and neck cancer, shows adistinct racial distribution among Chinese living in South China and and SoutheastAsia.The 5-10 years survival rate of advanced NPC patients is only 40%. The majorfactors affecting the survival of NPC patients include local recurrence andmetastases. Besides this, a serious side-effect was observed in patients with currentionizing-radiation therapy and chemotherapy, which affect the living quality ofNPC patients. It is critical to develop some novel therapies, which can enhanceoverall survival rate and improve living quality by preventing and antagonizingmetastases in NPC patients.Biotherapy has been becoming progressively the fourth model in controlingmaligmant tumor with development of tumor biology. Biotherapy of tumor playsthe key role in general treatment, especialy in targeting recurrence and metastases.Interferon（IFN）, one of major biotherapic medicine, has been used worldwidely forthe treatment of more than 14 types of cancer, including some hematogenousmalignancies and some solid tumors.IFN has diverse biological functions, includingsurppressing tumor related virus, inducing differentiation of tumor cells, promotingapoptosis of tumor cells, inhibiting proliferating of tumor cells, immunomodulatingimmune systems of patients and inhibiting angiogenesis of tumor. However, there is a poor understanding about effects of IFN on NPC. In the present study, we willinvestigate the effects of IFN on proliferative activity and metastases formation inEBV positive NPC tumor cells in vitro and in vivo.Pharmacokinetics studies indicated that IFNs exhibit an extremely shorthalf-life in the blood system after parenteral adminstration. Injections of high dosesof cytokines repeately, apparently needed to achieve effective antitumor response,often cause the adverse effects on the human body. In some clinical trials, somepoor response with cancer tissue were observed on patients due to an insuficientdelivery of the cytokine to the correct target site.Thus, some alternative strategies ofstable, effective and targeted delivery systems of INFs are urgently needed to bedeveloped. In this study, we constructed a recombinant adeno-associated viralvector harboring interferon alpha gene （rAAV-IFN-α）,demonstrating efficient andcontinous expression of active IFN-αprotein.The antitumor effects of IFN-αgenedelivered by this new strategy are much better compared to that by conventionalclinical use of IFN-αprotein.Our results may suggest a novel therapeutic approachfor treatment of patients with advanced NPCConstruction of the recombinant adeno-associated virus（rAAV） and AssayPlasmid pAAV-IFN-αwas constructed by inserting IFN-αgene into BamH I andEcoRI sites of plasmid pAAV using BglⅡⅠ/EcoRI enzymes.Plasmid pAAV-EGFPwas similarly constructed by inserting EGFP gene between the XhoI and EcoRI siteof pAAV. Recombinant AVV particles were produced by using a helper virus-freesystem.rAAV vectors and helper plasmid pDG were co-transfected into HEK 293-FTcells by calcium phosphate precipitation method.The supernatant fraction containingrAAV-IFN-αor rAAV-EGFP particles was decanted.rAAV particles were purified byHiTrap Heparin column chromatography.The final titer of the purified viral vectors was quantified by real-time PCR.The concentration of viral particles ranged from 10¹¹ to 10¹² v.g./ml. After C666-1cells, a kind of EBV-positive NPC cell line, were infected with rAAV-EGFP atdifferent concentration at 48 hr, the highest transfective efficiency （95%） was obtained with 5×10⁴ v.g./cell multiplicity of infection （MOI）.Transfective efficiencyof the purified viral vectors was maintained.rAAV-IFN-αinfected C666-1 cellsshowed the stable expression of IFN-αby RT-PCR and Western blot assay.The resultssuggested the higher transfective efficiency of rAAV-IFN-α.Effect of rAAV-IFN-αon EBV-positive NPC cell lineA series of experiments were performed to study effects of IFN-αonEBV-positive NPC cell line C-666-1.These methods included half-percent cell growthsuppressed(IC₅₀) dose test of IFN-α, MTT assay for cell proliferation, flow Cytometryassay and apoptosis assay, detection of IFN-αand metastases-related genes inC-666-1 cell infected rAAV-IFN-αby RT-PCR and Western bloting, measurement ofIFN-αlevel in cell culture supematant by liquichip technique.The results showed that C-666-1 were relatively resistant to antiproliferativeeffects of IFN-α,whose IC₅₀ was 1×10⁵IU/ml. To determine the effect ofrAAV-IFN-α-infected C666-1 cells on growth,antiproliferative activity of withHighest transduction efficiency （95%） had not revealed for 48 hr, which opticaldensity（OD） was no statistically significant compared to control （F=1.181,p=0.334）.Until 72hr rAAV-IFN-α-infected C666-1 cells growth had not beensuppressed. Flow Cytometry assay showed a significant increasing percentage ofrAAV-IFN-α-infected C666-1 cells in the G₁ phase compared to therAAV-EGFP-infected C666-1 cells in the G₁ phase （F=39.675,P=0.000）.The effectof rAAV-IFN-αon cell cycle was better than that of IFN-αprotein bymulti-comparison, but no statistically significant. In vitro, no apoptosis wasobserved in rAAV-IFN-α-infected C666-1 cells or IFN-α- incubated C666-1 cells.By RT-PCR.assay, we found that the mRNA expression of LMP-1 and EBNA-1 inEBV-positive C666-1 cells were suppressed, and the mRNA expression MMP-9 andVEGF were down-regulated by rAAV-IFN-α.. IFN-αprotein of IC50 had the sameefffect on C666-1 cells but it was weak. The results of Liquichip test showed theamounts of IFN-αin culture supematant of rAAV-IFN-αinfected C666-1 cell wereelevated with increasing incubated cell time,but the total concentration of IFN-α measured at 72 hr was less than 20U/ml.Effects of rAAV-IFN-αand IFN-αon growth and metastases of establishedxenograft EBV-positive NPC tumor in vovo64 male nude mice （BALB/c nu/nu） were used to establish liver xenograftEBV-positive NPC matastases model by direct injection of 1×10⁶/ml C666-1 cellsinto the liver.The 64 nude mice were divided into 4 groups of 16,A group withrAAV-IFN-α, B group with IFN-α, C group with rAAV-EGFP and D group with PBs.8 mice from each group were randomly chosen to evaluate the therapeutic effects andwere sacrificed at the third weekend of C666-1 cell injection.The remainder were leftto evaluate the long-term survival time until die naturelly.The survival time of theremainder mice were recorded, and a survival curve was constructed and analyzed bya log-rank test.Effect of rAAV-IFN-αand IFN-αon growth and metastases ofxenograft NPC were observed in vovo.Immunohistochemical assay was used to detectthe expression of MMP9 and MVD.The apoptosis of xenograft NPC tissue wasanalyzed by TUNEL staining. RT-PCR was performed to test the mRNA level ofIFN-α,MMP9 and VEGF in transplanted tumor.Liquichip technique was applied tomeasure the amount of human IFN-αand murine IL-12、GM-CSF、IFN-r、IL-2、IL-10、TNF-αin the mice serum. The data were Statistically analyzed by SPSS10.0.The results showed the volume of the transplanted tumor in 4 groupsrespectively were （239.00±44.80）mm³、（340.25±75.96）mm³、（424.75±67.22）mm³、（430.75±112.05）mm³ after implanting directly C666-1 cells into the liver for 3weeks.The volumes of the transplanted tumors in 4 groups differed significantly（F=10.397,P=0.000）.rAAV-IFN-αgroup had a significant differences with othergroups by mul-comparesion （P=0.000）.Satistical difference were significant, whichwere found between IFN-αand the controls （P=0.029） but not between group C andgroup D. Metastases rate in liver were 12.5%、37.5%、100%、100% and metastasesrate in lung were 0.0%、50.0%、87.5%、100% in group A,B,C,D respectively. Theaverage survival time in 4 groups were respectively （18.25±2.49）days、 （15.13±1.13）)days、（12.88±1.25）)days、（12.63±1.19）)days. Survival analysisdemonstrated satistical significance among 4 groups （F=20.848,P＜0.001）.Survivaltime of rAAV-IFN-αwas much longer than IFN-αgroup （P=0.001） and 2 controlsgroups （P=0.000）. Furthermore, survival time of IFN-αgroup was longer than twocontrol groups （P＜0.01）.The apoptotic indices for rAAV-IFN-α, IFN-α,rAAV-EGFP,PBS groups were calculated to be （12.20±1.92）、（8.80±1.64）、（4.00±1.58）、（3.40±1.14）, There were significant differences among 4groups（F=34.118, P=0.000）.Group A had a significant differences compared to theother 3 groups by mul-comparison.Satistical difference were also found betweenIFN-αand the controls （P=0.000）,but not between rAAV-EGFP and PBS groups.Immunohistochemical analyses on rAAV-IFN-αand IFN-αgroups showed thatthe MMP9 expression was down-regulated compared to rAAV-EGFP and PBS groupsin the transplanted tumor of liver tissue,which had satistical significance （P=0.000）.Immunohistochemical detection of CD31 revealed significantly a lower microvesseldensity in rAAV-IFN-α（1.60±1.14） and IFN-αgroup （1.80±0.84） than that in theEGFP （13.00±2.74） or PBS （13.20±2.59） groups respectively; which had asignificant difference （F=53.498, P=0.000）.Additionally, the expression of IFN-αmRNA was confirmed in the transplanted tumor of rAAV-IFN-αgroup, but not foundin the other groups.The expression of MMP9 mRNA and VEGF mRNA intransplanted NPC were decreased in group A and B compared to controls.We examined the amounts of IFN-αin the serum produced from intravenousrAAV-IFN-αbefore 3 weeks or from subcutaneous injection of IFN-αfor 3 weeksThe results showed that the serum IFN-αlevel（U/ml, （?）x±s） in rAAV-IFN-αgroup（7.01±2.68） was higher than IFN-αgroup （5.59±1.42）,which had no satisticaldifference （P=0.126）.There was a significant difference in the amounts of IFN-αofserum between treatment groups and controls （1.81±0.48）、（1.30±0.34） （p=0.000）.To confirm the cytokine induction by rAAV-IFN-αand IFN-αused in this study, wefound the level of IL-12,GM-CSF were increased after treatment.The serum IL-12 level（pg/ml,（?）±s） were respectively （105.02±43.43）、（56.99±13.93）、（0.00±0.00）、（0.00、0.00）,which had the satistical difference Between treatment groups andcontrol groups （P=0.002）. The serum GM-CSF level （pg/ml,（?）±s） were respectively（105.23±21.05）、（100.52±14.59）、（72.42±22.76）、（63.08±26.100）, which satisticaldifference was not found between group A and group B but between treatment groupsand control groups.The levels of IFN-r、IL-2、IL-10、TNF-αin serurn were alsomeasured and found no significant differences between treatment groups andcontrols.Conclusion1、Our results suggested that the growth and metastases of liver transplantedNPC were suppressed efficiently and that survival time was prolongedremarkblely after treatment by intravenous rAAV-IFN-αor subcutaneousinjection of IFN-α, The effects on antiprolification,metastasticsuppression,survival time and apoptosis analysis with rAAV-IFN-αdeliverywas more powerful than that of IFN-αprotein delivery, rAAV-mediatedIFN-αgene therapy is a promising strategy in suppressing growth andmetastases of EBV-positive NPC tumor.2、The effects of IFN-αon EBV-positive NPC may be attributed toantiangiogenesis and immunomodulatary role in this experiment.But it islimited that antitumor direct response of IFN-αhad been observed.3、The effects of IFN-αon suppressing growth and metastases of EBV-positiveNPC tumor.may be obtained by inhibiting expression of LMP-1 andEBNA-1 in EB virus.更多还原