【作者】 马树东；

【导师】 罗荣城；

【作者基本信息】 第一军医大学 ， 肿瘤内科学， 2004， 博士

【摘要】 恶性肿瘤已成为危害人类健康的常见病，占据人类死亡的第一或第二位原因，在全世界范围内每年大约有700万人死于恶性肿瘤。近年来，恶性肿瘤的发病率不仅逐渐上升，且发病年龄也趋于年轻化。世界卫生组织预测，恶性肿瘤将是21世纪人类的“头号杀手”。其治疗一直是人们关注的热点。恶性肿瘤治疗除传统的手术、放射、化学治疗外，免疫治疗在现阶段占据越来越重要的位置。抗肿瘤疫苗，特别是近年掀起的基因工程载体疫苗向人们展示了诱人的前景。作为肿瘤疫苗的相关抗原，Her2／neu在许多上皮组织起源的恶性肿瘤中过量表达，如：乳腺癌，卵巢癌，胃癌和肺癌等，显示了作为疫苗目标抗原的潜在价值。5型腺病毒载体具有安全，高效等特点，在目前的病毒载体疫苗中占主要地位。为此我们以研制载体疫苗为目的，构建表达Her2／neu基因膜外第一受体区（Her2-ECDs），全长膜外区（Her2-ECD）和膜外跨膜区（Her2-TM）蛋白的重组腺病毒（rAd）载体，观察并比较rAdHer2-ECDs，rAdHer2-ECD和rAdHer2-TM作为DNA疫苗在哺乳动物体内诱导的免疫效果，并探讨了其用于转基因树突状细胞（DC）所发挥的抗肿瘤效应。 采用RT-PCR法从高表达Her2／neu蛋白的SK-BR-3细胞总RNA中扩增出Her2／neu基因膜外第一受体区（Her2-ECDs），全长膜外区（Her2-ECD）和膜外跨膜区（Her2-TM）基因：将PCR产物加A后克隆进PMD-18T载体中，经M13引物测序，序列完全正确。还发现我们钓出的TM片段基因在跨膜区第一和二个氨基酸均为缬氨酸（val），这是Her2／neu基因多态性在这个位点出现频率最低（1.2％）的一种排列。二次克隆将目的基因插入穿梭质粒pAdTrack-CMV中，在大肠杆菌BJ5183中与5型腺病毒骨架质粒Adeasy^TM进行同源重组，获得rAdHer2-ECDs，rAdHer2-ECD和rAdHer2-TM三个质粒；用脂质体polyfect转染线性化博士论文:HerZ/neu膜外及跨膜区蛋白基因重组腺病毒的构建和诱导的免疚效应的重组质粒到293包装细胞系中，筛选得到rAdHerZ一ECDs，rAdHerZ一ECD和rAdHerZ一TM;大量扩增，氯化艳梯度密度超速离心纯化后，采用物理方法测定病毒滴度，证明分别得到5.46xl0’ZvP/ml，2.95xl0‘ZvP/m1，8.40X10’Zvp加l的高滴度病毒。Hela细胞感染传代三次未出现细胞病变。感染293细胞后，经Westem Blot检测证明其在细胞内获得了表达，并且表达量随着感染复数的增加而升高。提示我们成功地获得了高纯度和高滴度的，能有效感染包装细胞的rAdHerZ一ECDs，rAdHerZ一ECD和rAdHerZ一TM。 将rAdHerZ一ECDs，rAdHerZ一ECD和rAdHerZ一TM以1 x 10”VP通过胃饲、肌肉注射、腹腔注射和尾静脉注射途径免疫Balb/。小鼠，观察rAdHerZ一ECDs，rAdHerZ一ECD和rAdHerZ一TM在小鼠体内表达后诱导免疫应答情况。结果显示，不同病毒同一途径之间抗HerZ抗体滴度无明显差异。在不同的途径同一病毒之间抗HerZ抗体滴度以尾静脉注射组为最高，其次为肌肉注射组和腹腔注射组，两者无明显差异（P>0.05），但均明显高于胃饲组（P<0.05）。在不同途径中，以静脉注射诱发的CTL活性为最强，肌肉注射和腹腔注射次之，胃饲最低（P<0.05）。在三个重组腺病毒中，以rAd HerZ一TM诱发的cTL活性为最高。用1 xlo”vP的rAdHerZ一ECDS肌肉注射小鼠，注射后2周外周血中即出现HerZ抗体，其后滴度逐渐升到高，第三周达到高峰，然后缓慢有所下降。免疫后第2周诱发了小鼠出现特异性的CTL活性，于第3周亦达到最高峰，并在高水平上维持较长时间;以不同的病毒颗粒数的rAdHerZ一TM肌肉注射小鼠，只有在病毒量达到1 xlo，”vP时才‘产生可测定的抗HerZ抗体，随着病毒量的升高，HerZ抗体滴度亦随之升高。但只在病毒量达到sx10’“vP，较高效靶细胞之比时，才能检测到cTL活性，随着感染的病毒数量的升高，CTL活性有所升高。提示，将rAdHerZ一ECDs，rAdHerZ一ECD和rAdHerZ一TM作为载体疫苗，在小鼠体内成功地诱导了HerZ/neu特异性的免疫应答，具有明显的应用价值，值得进一步深入研究。 其后，我们将认dHerZ一ECDs，rAdHerZ一ECD和rAdHerZ^TM应用于转基因DC观察其对DC免疫功能的影响，结果显示利用El、E3缺陷型的叭dHerZ一TM感染未成熟De，通过x一gal染色，在Mol为3.75 x 104时，有91%以上的DC被感染，胞浆被染成蓝色。westem blot检测表明HerZ一ECDs， 2博士论文:HerZ/neu膜外及跨膜区蛋白基因重组腺病毒的构建和诱导的免疫效应ECD，TM蛋白在DC中获得表达，但在培养上清中未检测到。在培养的第5天，转染rAdHerZ一ECDS，ECD和TM的DC培养上清中IL一12的含量比未转染DC含量为高（P<0.05），但三个不同重组病毒之间无明显差异（P>.05）。检测转染和未转染的DC刺激淋巴细胞增殖后培养上清中IFN-Y的含量，显示随着时间的延长，培养上清中的IFN一丫的含量逐步增高，但病毒感染DC明显高于非感染DC（P<0.05）。在混合淋巴细胞反应中，DC明显介导淋巴细胞增殖反应，但转染和非转染DC之间无差异。在DC诱导的CTL反应中，病毒感染DC诱导的杀伤率明显高于SK一OV一3修饰和非修饰DC，而SK一OV一3修饰又高于更多还原

【Abstract】 Malignant tumor, a common disease to threaten public health, is the 1 st/2nd cause of human death that is about seven million worldwide every year. The incidence of malignant tumor goes up gradually in recent years and patients tend to become younger and younger. World Health Organization foresees that it will be "No.l killer to human " in 21st century. Its therapy has always been the hot spot that people show solicitude for. Immunotherapy, besides traditional surgical therapy, radiotherapy and chemotherapy, plays more and more important role in the treatment of malignant tumor at the present. Anti-tumor vaccine has presented tempting prospect for people, specially the carrier vaccine of genetic engineering raised in recent years. Her2/neu, one of the tumor-associated antigens, over-expressed in many epithelial tumors, such as breast cancer, ovary cancer, gastric cancer and lung cancer, has show potential as targeting antigen of vaccine. Type 5 adenovirus, which is safe and high-efficacy, takes dominant position in vector for gene delivery. In this study we developed a Her2/neu vaccine and then investigated its immunogenicity by rAdenovirus vector expressing Her2/neu proteins of extracellular and transmembrane domains. Meanwhile, we also have estimated the feasibility that rAdHer2 was introduced to dendritic cell-based immunotherapy of anti-tumor.Genes of Her2/neu extracellular first-receptor domain(Her2-ECDs), full-length extracellular domain(Her2-ECD), extracellular and transmembrane domain(Her2-TM) proteins were amplified from total RNA resulted from SK-BR-3 cells over-expressing Her2/neu by RT-PCR, and then cloned into the PMD-18T vector after adding A. The right sequences were identified by sequencing with Ml3 primers and a lowest frequency (1.2%) of4occurrence of Her2/neu gene polymorphism was detected that both of the first and the second amino acids of transmembrane domain protein were Valines. During the second cloning, objective genes cut off from PMD-18 vectors were inserted into shuttle pAdTrack-CMV plasmids and then rAdHer2-ECDs, rAdHer2-ECD and rAdHer2-TM plasmids were obtained from homologous recombination in E coli. BJ5183, into which the Adeasy?plasmid of adenoviral framework and the recombined pAdTrack-CMV plasmid were co-transfected. rAdHer2-ECDs , rAdHer2-ECD and rAdHer2-TM were isolated by screening after linearized rAdHer2-ECDs, rAdHer2-ECD and rAdHer2-TM plasmids were transfected into 293 package cell line with polyfect. Virus with 5.4VP/ml, 2.95 10l2VP/ml and 8.40 1012VP/ml titre, respectively, measured by physical method, were generated after large scale virus amplification in 293 cell and purified by Cesium Chloride Gradient. The CPE didn’t achieve in Hela cell undergone 3 passages, after infected by purified recombined adenovirus. The express of proteins was probed by Western Blot Check in infected 293 cell, and the amounts of Her2/neu elevated with the increase of MO. These results indicated that generated recombined adenovirus with high purification and titre can transfer package cell line efficiently in vitro.To estimat the transduction efficiencies of rAdHer2-ECDs , rAdHer2-ECD and rAdHer2-TM in vivo and their ability of eliciting immune response, The Balb/c mice were infected by recombined adenovirus via intravenous injection(IV), intrauscular injection(IM), intraperitoneal injection(IP) and gastric gavage(gg). The results showed that there is no significant difference in Her2 antibody titres among via the same administration route. The titre level of Her2 antibody induced via intravenous injection was the highest in same recombined adenovirus among the different administration routes, following with intramuscular injection and intraperitoneal injection route in turn, but no difference was detected between them, and that of anyi-Her2 antibody via gastric gavage was the lowest. The CTL responses induced by recombined adenovirus were same as the administration of rAdHer2-ECDs , rAdHer2-ECD or rAdHer2-TM via different routes. rAdHer2-TM can induce the strongest CTL response via更多还原