【作者】 苗雷英；

【导师】 孙宏晨； 杨柏；

【作者基本信息】 吉林大学 ， 口腔医学， 2007， 硕士

【摘要】 腺样囊性癌（adenoid cystic carcinoma，ACC）是常见的涎腺恶性肿瘤，易复发、早期浸润面神经、术后早期转移且对放疗、化疗不敏感，其治疗一直是临床上比较棘手的问题，众多学者曾作过大量的研究，但目前尚无十分有效的治疗方法。近年来，基因治疗成为肿瘤治疗领域的一新的研究热点。而基因治疗的瓶颈在于寻找无毒副作用且具有靶向性的基因转移载体。传统的基因载体包括病毒载体和非病毒载体。质粒等非病毒载体在水相中传递时，容易被核酸酶水解，而且由于带大量负电荷而不利于靶细胞的胞吞，致使转染效率较低；腺病毒、逆转录病毒等病毒载体制备复杂，费用昂贵，尤其是它的安全性一直来是限制其广泛应用的主要因素。我们将表达增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）的重组腺病毒载体转染正常涎腺细胞，观察其对正常涎腺细胞增殖、功能的影响，以研究其毒副作用。并针对腺病毒载体安全性、靶向性等问题，采用引发原子转移自由基聚合的方法合成超顺磁性Fe₃O₄纳米微粒，经表面修饰后与pACTERT-TRAIL质粒组装。初步尝试制备磁性Fe₃O₄-pACTERT-TRAIL纳米基因复合载体。更多还原

【Abstract】 Salivary tumors are frequent tumor in oromaxillo-facial region.Adenoid cystic carcinoma （ACC） is a common salivary malignant tumor, which infiltrates facial nerve and metastasis earlier and often leads to obvious maxillo-facial malformation after operation. It also reoccurs easily and is not sensitive to radio-therapy or chemo-therapy, so has a deep effect to patient’s life.It is very important to approach it’s reason and find new effective therapy for it.Gene therapy have develop more than 20 years since 1980s.In 1990,NIH used ADA gene cure a 4 years old girl who had severe immune deficiency for ADA gene defect.In 1994, Shillitoe advocate that use gene therapy to treat oral tumors.Gene therapy have marked achievement during last 10years.It has played more and more therapy function for hereditary disease,metabolic disease and malignant tumor.But when deliveried in the aqueous phase,nucleic acid are easy to be hydrolysis by nuclease,and because nucleic acid have a lot of negative charge,it can’t be swallowed by cell easily.For these reason ,we must solve the key problem that find high performance,safe and tissue-specific gene vector.There are two kinds of gene vectors, viral vectors and nonviral vectors.Viral vectors are the most effective up to now such as etroviral vectors, adenoviral vectors, adeno-associated viral vecto rs,herps simplex viral vectors, lentiviral vectors et al.But this kind vector exist side effect and immune reaction to confine it’s widespread application.So people began to pay attention to the safe nonviral vectors,especially the development of nano material.Nanoparticles which size between 0.1-100nm ,also called ultramicroparticle,are smaller than common cell,so it’s possible to be vector to enter cell.Nanoparticle have a lot of physical and chemical character for example large specific surface,surface atomic number,surface energy and surface tension rapid increase with the particle diameter decrease.All these character lead to specific heat,magnetic,photaesthesia character so we can control nano vector target deliver gene by electricity,magnetic and light.We also can modify nanoparticle’s surface structure and condition to raise surface activity,to improve nanoparticle’s compatibility and to form new physical , chemical and mechanism character and new function.Magnetic nanoparticle is a important kind of nanoparticle.It has superparamagntism,so can easily phase separation and localization by magnetic field. So the Magnetofection by the Magnetic nanoparticle may be a valid tumor therapy method.Nanotechnology,biotechnology and bioinformatics have been generally accepted as motive power for medical science development.Hitherto ,there are great stride in vivo and in vitro research.Maybe in recent future, nanobiotechnology will become more effective therapy for genopathy than chemo-therapy, radio-therapy and surgical treatmentThe salivary glands of mammalian （parotid and submandibular gland） are more excellent target of gene therapy due to their anatomy characteristic than other internal organ.It’s location superficial so easily observe the transfection effect.We believe salivary tumors gene therapy have potential application perspective with the research development.In this study, we aim directly at salivary to test the safe problem of recombinate adenovirus vector and prepare the Fe₃O₄@PDMA-pACTERT-TRAIL magnetic nano gene vector.We try to develop new magnetic nano gene vector to target therapy salivary tumor .The field of this research is following: 1 .We used collagenase and trypsinization degested method culture normal Wistar rat submaxillary salivary gland cells.Then transfected the salivary cells with recombinate adenovirus vector which express enhanced green fluorescent protein with CMV as promoter.The results show when we used higher MOI（ more than 200 particle/cell ） recombinate adenovirus vector can inhibited the proliferation of the salivary cells and influented salivary cells secrete amylase. So it is toxiferous for salivary cells.In the other hand,the result also indicate that used proper MOI ,the adenovirus vector expressed EGFP can be used to mark salivary cells for trace labeling and research submaxillary cells’s biological behaviour. 2.We used ARTP reaction prepare Fe₃O₄@PDMA magnetic nano gene vector which surface contain positive charge.Then Fe₃O₄@PDMA combinate with plasmid by electrostatic interation.We used electrophoresis et al methods to detect the complex.The result show they successful combinated.Our research achievements provide a foundation for us to investigate the target transgene ability of magnetic nanoparticle in vivo and in salivary 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 tumors .更多还原