腺病毒载体与肿瘤治疗研究进展

基因导入系统是基因治疗的核心技术，可分为病毒载体系统和非病毒载体系统。对于病毒载体而言，大多数野生型病毒对机体都具有致病性，因此需要对其进行改造后才能用于人体。近年来，一些病毒如反转录病毒（包括HIV病毒）、腺病毒、腺病毒伴随病毒、疱疹病毒（包括单纯疱疹病毒、痘苗病毒及EB病毒）、甲病毒等被成功地改造成为基因转移载体并开展了不同程度的应用。对于腺病毒载体，由于它具有①靶细胞种类多，导入效率高；②既可感染分裂期细胞，对增殖停止期细胞也有很高的导入效率，这是逆转录病毒载体所不具备的；③不整合到宿主基因组中从而不会引起插入突变。     

非病毒性基因治疗递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k的构建及性能研究

目的构建非病毒性基因治疗递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k,探讨其细胞毒性及对质粒基因(pDNA)的递送性能。方法通过开环聚合反应制备共聚物mPEG5k-PCL1.2k-OH,将其羟基末端化学转化为N-羟基琥珀酰亚胺(-NHS)生成mPEG5k-PCL1.2k-NHS,随后同枝化PEI10k进行反应生成三元共聚物(mPEG5k-PCL1.2k)1.4-g-PEI10k。应用傅立叶变换红外光谱(FTIR)、核磁共振(NMR)和凝胶渗透色谱对(mPEG5k-PCL1.2k)1.4-g-PEI10k进行结构表征分析;通过MTT分析,比较(mPEG5k-PCL1.2k)1.4-g-PEI10k与PEI10k的细胞毒性;通过复凝聚法制备(mPEG5k-PCL1.2k)1.4-g-PEI10k/pDNA复合物;通过细胞转染实验,观察不同质量比的复合物转染细胞后报告基因的表达效果,考察转染时间和培养基pH值对报告基因表达的影响。结果成功合成了递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k。浓度小于62.5g/ml时,(mPEG5k-PCL1.2k)1.4-g-PEI10k的细胞毒性显著小于PEI10k。(mPEG5k-PCL1.2k)1.4-g-PEI10k载体能够压缩绿色荧光蛋白质粒报告基因(pEGFP)形成(mPEG5k-PCL1.2k)1.4-g-PEI10k/pEGFP复合物,并有效转染细胞获得目的基因表达;当载体与质粒的质量比为10∶1时,绿色荧光蛋白的表达效果最好。转染时间和培养基pH值均可影响(mPEG5k-PCL1.2k)1.4-g-PEI10k/pEGFP的转染效率,转染效率在48h优于24h和72h,pH6.8培养基优于pH7.2培养基。结论递送载体(mPEG5k-PCL1.2k)1.4-g-PEI10k细胞毒性较低,能够递送pDNA进入细胞并表达目的蛋白。     

登革2型病毒PrM基因对不同型登革病毒复制的特异抑制作用

目的 :将我国登革 2型病毒PrM(D2 _PrM)基因导入甲病毒载体系统 ,探讨含正、反义PrM基因的重组病毒对不同血清型登革病毒复制的特异抑制作用。方法 :采用体外转录和电穿孔的方法 ,首先分别将构建的含正、反义PrM基因的重组质粒DNA和辅助载体DNA转录成RNA ,然后将这两种RNA共转染BHK细胞 ,进而包装成重组病毒颗粒。再将经激活的重组病毒感染宿主细胞 ,分别用不同型登革病毒攻击 ,然后通过免疫荧光法 ,观察对不同型登革病毒复制的抑制作用。结果 :含登革 2型正、反义PrM基因的重组甲病毒 ,不仅可完全抑制该型病毒在宿主细胞中的复制 ,而且对其他 3个型病毒的复制也具有不同程度的抑制作用。含反义PrM基因的重组病毒对 4个型登革病毒复制的抑制作用最强。结论 :登革 2型病毒的正、反义PrM基因在宿主细胞中通过重组甲病毒 ,可介导对登革 1～ 4型病毒复制的特异抑制作用     

高效的红细胞特异性表达系统的建立和优化

目的探讨慢病毒载体中红细胞特异性基因调控元件的优化对小鼠红白血病(murine erythroleu-kemia,MEL)细胞向红系终端分化期间组织特异性表达重组蛋白的影响。方法改变红细胞特异性β-珠蛋白启动子编码长度、改变β-珠蛋白基因位点控制区DNaseⅠ高敏位点(hypersensitive site,HS)的组合、删除β-珠蛋白3’端增强子或β-珠蛋白内含子IVS2,由此构建一系列由不同缺失组合的基因调控元件控制目的基因(如人凝血因子Ⅸ)表达的慢病毒载体,包装成重组慢病毒,测定病毒滴度,并分别感染小鼠MEL细胞。用O6-苄基鸟嘌呤(O6-benzylguanine,BG)-卡莫司汀[1,3-bis(2-chloroethyl)-1-nitrosourea,BCNU]联合筛选以富集感染的阳性细胞,用酶联免疫吸附测定方法检测目的基因的表达,评估不同基因调控元件组合对重组慢病毒载体所携带的目的基因表达的影响。结果经限制性内切酶谱分析和序列测定,构建的慢病毒载体结构正确;三质粒系统瞬时转染人肾胚胎293T细胞包装的重组自灭活慢病毒可通过长时间高速离心有效浓缩;用50μmol/L BG-50μmol/L BCNU联合筛选可有效富集感染的阳性细胞;经环六亚甲基双乙酰胺(N,N’-hexamethyle-nebisacetamide,HMBA)诱导第8天的106个小鼠MEL细胞中表达的重组人凝血因子Ⅸ平均质量浓度达到正常水平的3.8%(191 ng/ml)。结论通过适当减少调控元件优化慢病毒载体不仅有助于提高携带的外源基因片段长度、增加载体制备的有效性、实现温和提高目的基因产物达到治疗水平需求量的目的,而且为开展以红细胞特异性表达载体介导的携带蛋白类药物的红细胞治疗以及非血液病基因治疗奠定临床前的研究基础。     

基因治疗中的动物病毒载体

基因治疗是新近发展起来的一种新治疗技术,有着广阔的应用前景,它的迅猛发展与真核细胞基因转移技术的发展密切相关.目前常用的基因转移技术主要有:①病毒方法:包括RNA病毒和DNA病毒载体;②化学方法:如磷酸钙共沉淀法;③膜融合的方法:即利用包被着DNA分子的膜性载体,如脂质体、红细胞膜、原生质体等;④物理方法     

大鼠B细胞淋巴瘤-2基因的克隆及其慢病毒表达载体的构建

目的 克隆大鼠B细胞淋巴瘤-2(bcl-2)基因全长cDNA,构建及鉴定大鼠bcl-2基因慢病毒表达载体.方法 采用RT-PCR法从大鼠脾脏组织中扩增全长bcl-2 cDNA,将扩增产物克隆至pMD18-T载体中,测序正确后,将基因连接到慢病毒载体pGC-FU(含EGFP 基因)中,构建慢病毒载体表达质粒pGC-FU-bcl-2,将pGC-FU-bcl-2质粒和包装质粒(pHelper1.0、pHclp-er2.0)共同转染人胚胎肾上皮细胞系293T细胞后,细胞即可分泌携带bcl-2基凶的重组慢病毒.将重组慢病毒感染293T细胞,通过Western blot-ring鉴定目的 蛋白bcl-2的表达.结果 经DNA序列分析测定证实大鼠bcl-2基因序列与GenBank中一致;pGC-FU-bcl-2中携有正确的bcl-2基因,并能转染人胚肾上皮细胞;pGC-FU-bcl-2及包装质粒共转染包装细胞293T细胞能产生重组病毒pGC-FU-bcl-2;目的 基因bcl-2能被重组慢病毒高效地转导人人胚肾上皮细胞中,并达到稳定表达,荧光显微镜下能直接观察到荧光蛋白,Westernblotting能榆测到bcl-2蛋白在靶细胞中的表达.结论 成功克隆了大鼠bcl-2基因并构建了重组慢病毒载体,包装出高浓度慢病毒,转染人胚肾上皮细胞后能够稳定表达bcl-2基因,为进一步研究该基因的功能及细胞凋亡相关疾病的治疗奠定了基础.     

小鼠RelB基因RNAi慢病毒载体的构建与鉴定

目的 构建小鼠RelB基因RNA干扰(RNAi)慢病毒载体,有效沉默骨髓树突细胞(DC)的RelB基因表达,为构建骨髓致耐受DC用于自身免疫病的防治提供研究基础.方法 利用Invitrogen公司在线软件设计小鼠RelB基因(NM_009046)shRNA序列,合成、退火形成dsoligo后克隆到pENTRTM/U6载体的黏性末端,测序,得到的阳性重组子再与慢病毒载体进行重组,转化stbl3感受态细胞,测序鉴定,在脂质体的介导下将慢病毒的包装混合物和RelB基因重组慢病毒载体导入293FT细胞,包装成病毒后,收集病毒上清,采用系列稀释法测定病毒滴度.结果 测序结果显示pENTRTM/U6-RelB-shRNA为阳性克隆,将该阳性重组载体与慢病毒载体重组,转化,氨苄青霉素抗性筛选阳性克隆,U6前引物测序鉴定,测序结果 显示该重组慢病毒载体也为阳性克隆,在293FT细胞中进行病毒包装,收集上清并在-80℃贮存.系列稀释法检测病毒悬液的滴度为6×105TU/ml.结论 成功构建出小鼠RelB基因shRNA慢病毒RNAi表达载体,为制备致耐受DC和研究DC在自身免疫病中的应用提供了稳定的转染细胞载体.     

骨肉瘤基因治疗及载体系统研究现状

近年来,有关骨肉瘤基因治疗的研究在基因治疗方法、基因载体、载体导入途径等方面取得了较大进展。骨肉瘤的基因治疗主要有免疫基因治疗、反义基因治疗、抑基因治疗、自杀基因治疗及联合基因治疗等。基因载体包括病毒载体和非病毒载体。     

肿瘤基因治疗载体的选择及使用安全性的研究进展

随着对肿瘤发生发展分子机制认识的不断深入,肿瘤的基因治疗已成为攻克和治愈肿瘤最具希望和挑战的研究领域。如何建立安全有效的治疗基因导人系统成为研究者们首要解决的问题。目前,常用的载体主要有病毒载体和非病毒载体两类：病毒载体的转移效率高,在基础研究和临床试验中应用广泛,但近年来因存在安全性的问题,其使用受到了一定的制约;非病毒载基因导入的效率相对较低,但具有安全性好且容易制备的特点,日益受到人们的重视。现就近年来国内外对于肿瘤基因治疗载体的使用选择及安全性研究作一综述。     

siRNA抑制Smad4或Runx2基因表达治疗大鼠异位骨化效果比较

目的:研究siRNA抑制Smad4基因表达治疗异位骨化大鼠模型的效果,并与抑制Runx2的疗效进行比较。方法:1、设计6条分别针对大鼠Smad4和Runx2的mRNA模板,用体外转录合成法分别合成6条siRNA,采用RT-PCR从mRNA水平在培养的原代成骨前体细胞中筛选有明显抑制作用的siRNA。2、设计合成针对Smad4基因和Runx2基因编码的发卡样siRNA的双链DNA,与腺病毒的穿梭质粒连接后,取其启动子和终止子连接于非病毒载体上。在培养的大鼠原代成骨细胞中,采用RT-PCR和western blot测定Smad4特异性siRNA非病毒对Smad4的抑制作用和Runx2特异性siRNA非病毒对Runx2的抑制作用。3、采用体内实验测定Smad4或Runx2特异性siRNA非病毒载体对切断大鼠跟腱诱导异位骨化动物模型的影响:实验组行跟腱切断术和植入100μg的Smad4-siRNA或Runx2-siRNA重组非病毒载体,对照组行跟腱切断术和植入100μg的pcDNA4/HisA非病毒载体。10周后,行CT三位重建检测形成的异位骨大小,HE染色观察其组织形态。结果:1、在合成的3条Smad4-siRNA中,siRNA139-159对Smad4的抑制效果最明显,在合成的3条Runx2-siRNA中,siRNA1057-1077对Runx2的抑制效果最明显。2、成功构建重组非病毒载体,转染原代成骨细胞可明显抑制Smad4和Runx2的表达。3、CT三位重建显示,Smad4-siRNA实验组形成的异位骨体积较对照组减小92%,Runx2-siRNA实验组形成的异位骨体积较对照组减小93%,差异均有统计学意义(P<0.05)。组织学观察显示非病毒载体介导的特异性Smad4-siRNA和Runx2-siRNA在体内可以抑制异位骨形成,两者之间的抑制效果差异无统计学意义(P>0.05)。结论:Smad4和Runx2两种信号通路的特异性siRNA可以明显抑制切断跟腱诱导的异位骨化,但两种不同siRNA的抑制效果无差别。     

Molecular Imaging of Biological Gene Delivery Vehicles for Targeted Cancer Therapy: Beyond Viral Vectors

Cancer persists as one of the most devastating diseases in the world. Problems including metastasis and tumor resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of present clinical treatments. To overcome these limitations, cancer gene therapy has been developed over the last two decades for a broad spectrum of applications, from gene replacement and knockdown to vaccination, each with different requirements for gene delivery. So far, a number of genes and delivery vectors have been investigated, and significant progress has been made with several gene therapy modalities in clinical trials. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications. However, both have limitations and risks that restrict gene therapy applications, including the complexity of production, limited packaging capacity, and unfavorable immunological features. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents such as bacteria, bacteriophages, and bacteria-like particles. Recently, many molecular imaging techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been employed to assess vector-mediated gene expression in living subjects. In this review, molecular imaging techniques for monitoring biological gene delivery vehicles are described, and the specific use of these methods at different steps is illustrated. Linking molecular imaging to gene therapy will eventually help to develop novel gene delivery vehicles for preclinical study and support the development of future human applications.     

Intraarterial gene delivery in rabbit hepatic tumors: transfection with nonviral vector by using iodized oil emulsion

PURPOSE: To evaluate the feasibility of an iodized oil emulsion that is used for the chemoembolization of hepatocellular carcinoma as a modifier of a nonviral gene transfer system for intraarterial gene delivery in experimentally induced hepatic tumors. MATERIALS AND METHODS: Experiments were performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the animal research committee at Seoul National University Hospital. VX2 carcinoma was implanted into the liver of 26 rabbits. Four nonviral gene transfer systems were prepared by using pCMV-luc+ as a reporter gene. The first system consisted of a DNA and polyethylenimine (PEI) complex (n = 7); the second, of a DNA and PEI complex mixed with iopamidol and iodized oil (n = 7); the third, of a DNA and PEI complex mixed with iopamidol (n = 7); and the fourth, of a DNA and PEI complex mixed with iodized oil (n = 5). For the DNA and PEI complex that was mixed with iopamidol and iodized oil, iopamidol was used to stabilize the emulsion. Twenty days after tumor implantation, intraarterial gene delivery was performed by selective catheterization of the hepatic artery. Rabbits were euthanized 24 hours after gene delivery. Luciferase activity was assayed in the tumor, left hepatic lobe, right hepatic lobe, and other organs and was statistically analyzed for comparison between complexes by using the Kruskal-Wallis test. RESULTS: Luciferase activity in the tumor was significantly higher for the group that received DNA, PEI, iopamidol, and iodized oil than for any other group (Kruskal-Wallis test, P < .05). Luciferase activity in the left hepatic lobe, right hepatic lobe, and other organs was not significantly different between complexes. Selective gene expression in tumor cells was confirmed by means of immunohistochemical analysis for luciferase. CONCLUSION: It is feasible to use an iodized oil emulsion system for the intratumoral transfection of nonviral vectors in experimentally induced hypervascular hepatic tumors.     

报告基因显像监测基因治疗研究进展

为评价基因治疗,需要随时对治疗基因的定位和表达进行监测。放射性核素报告基因技术是检测基因表达的最好方法。用基因融合、双顺反子、双启动子及双向转录等重组技术,构建表达报告基因的腺病毒载体,导入靶细胞或组织内,然后注射与报告基因偶合的核素标记的探针,进行PET、SPECT或γ-照相,可无创伤地、重复地定量显示报告基因表达。目前,用于基因治疗的报告基因和报告探针系统有:HSV1-tk(单纯疱疹病毒胸腺嘧啶核苷激酶基因)和碘、氟同位素标记的尿嘧啶、鸟嘌呤的衍生物;突变的多巴胺D2R(多巴胺2型受体)基因和(18F-FESP(18F-氟乙基螺环哌丁苯);SSTr2(生长抑素2型受体基因)和生长抑素类似物等。其中,部分已用于临床试验治疗。     

甲病毒表达系统研究进展

介绍甲毒表达系统，包括载体和用于包装的辅助系统的构建，以及该系统在异源基因表达和核酸疫苗研究中的应用。     

Gene transfer to the tendon-bone insertion site

This study investigated whether gene transfer to the tendon-bone insertion site is possible during early tendon-transplant healing using viral vectors. In addition, we evaluated the optimal gene delivery technique for an in vivo adenoviral gene transfer to a tendon-bone insertion site in a bone tunnel. Twenty-six rabbits underwent a bilateral transfer of the flexor digitorum longus tendon into a bone canal in the calcaneus. The animals were divided into two groups. The first group (n=18) received a direct injection of an adenoviral vector carrying the luciferase marker gene into the tendon on the left side, while on the right side the adenoviral vector was first injected into the bone trough and the tendon was later inserted into the trough. The analysis of this experiment showed that over a 4-week period a higher luciferase activity was achieved using the bone trough immersion technique. In the second group (n=8) we therefore used the qualitative marker virus (Ad/-LacZ) with the bone trough immersion technique in order to show the site of gene expression. The histological analysis of this experiment demonstrated the presence of -galactosidase positive cells within the tendon-bone interface over a 4-week period. Therefore we showed in the first part of this study that the bone canal provides a more efficient target for direct adenoviral gene delivery than the tendon. In the second part of the study we demonstrated the feasibility of the bone trough immersion technique since sustained gene expression within the tendon-bone interface was obtained for up to 4 weeks. This study has shown the feasibility of gene delivery to the tendon-bone interface and provides the basis for the application adenoviral delivery of growth factor genes to the tendon-bone insertion site.     

基因转移技术与基因靶向性核素治疗

随着在细胞分子水平上对疾病发病机制认识的深入,基因治疗已成为目前医学分子生物学最重要的研究领域之一。基因治疗过程中基因转移技术起着关键作用,携带目的基因的载体有待进一步开发和改进。在基因治疗过程中,需要进行基因监测,基因显像是其最有效的方法。将基因治疗与靶向核素治疗相结合,即"基因靶向核素治疗"为肿瘤基因治疗开辟了一条崭新的途径。     

微量注射不同载体转基因至损伤肌腱的效率、分布和组织反应

目的 探索微量注射法以不同转基因载体转基因至损伤肌腱的转基因效率、分布和组织反应.方法 用微量注射器将10μl携带增强绿色荧光蛋白(enhanced green fluorescent pro-tein,EGFP)报告基因的pCMV-EGFP、pCAGGS-EGFP、AAV2-EGFP和Ad5-EGFP载体直接注射入鸡的损伤并缝合的趾深屈肌腱.在术后第3,7,14,21天分别取肌腱,进行冰冻切片后在荧光显微镜下观察肌腱内的EGFP表达量及分布,并将肌腱切片作HE染色后观察各不同载体在肌腱中引起的炎症反应.结果 荧光显微镜下观测发现,注射4种转基因载体的肌腱,转染3 d后即可见肌腱内有增强绿色荧光蛋白表达;在第7天时表达最明显;第14天时,各组表达增强绿色荧光蛋白表达量下降;21 d时很少有表达增强绿色荧光蛋白的细胞.用微量注射器在腱段两点注射能保证近损伤肌腱内转染细胞均匀分布.注射AAV2-EGFP和Ad5-EGFP的肌腱的增强绿色荧光蛋白明显多于质粒载体的增强绿色荧光蛋白,而AAV2-EGFP和Ad5-EGFP两组间无明显差异.HE染色发现质粒载体和Ad5-EGFP对肌腱的组织反应较重,可见较多炎症细胞浸润,以淋巴细胞和中性粒细胞为主.注射AAV2-EGFP的肌腱炎症反应较轻.结论 用微量注射器在腱段两点注射转基因载体能转染近损伤处的整个肌腱段的细胞.在研究的4种基因治疗方法中,AAV2和Ad5在损伤肌腱中的转基因效率最强,在转基因后第7天表达最明显,而且AAV2引起的肌腱组织反应最轻.提示AAV2载体比Ad5和质粒载体更有利于作为转基因治疗肌腱损伤的载体,微量注射载体是转基因至损伤肌腱的合适方法.     

放射技术与基因治疗

基因治疗是一个融合了多学科、多种技术的全新医学领域 ,已成为世界医学界的研究热点。放射学技术可在以下方面中发挥独特作用 :选择靶向治疗位点 ,发展和完善载体导入途径 ,建立监测治疗的影象学方法 ,评估基因表达 ,以及放疗与基因治疗的结合等 ,从而在这一迅速发展的诊断和治疗领域作出特有的贡献     

基因治疗在周围神经损伤修复中的应用

随着分子生物学研究的飞速发展，基因治疗在基础和临床得到了广泛的应用，在周围神经损伤修复领域表现出尤为广阔的前景。目前，基因治疗修复周围神经损伤的基本策略是利用载体工具将编码神经营养因子的基因转入靶细胞，使之在体内持续表达，进而达到治疗效果。本文着重就神经营养因子、靶细胞、载体工具3个方面介绍当前国际上基因治疗修复周围神经损伤的研究情况。     

In vitro design and characterization of the nonviral gene delivery vector iopamidol, protamine, ethiodized oil reagent

Purpose

To demonstrate cellular selectivity toward hepatoma cells and compare the efficiency of gene delivery of a novel nonviral vector of iopamidol, protamine, and ethiodized oil reagents (VIPER).

Materials and Methods

Rat hepatocellular carcinoma (HCC) cells were transfected in triplicate under varying conditions by using firefly luciferase as a reporter gene. Conditions included variations of a protamine:DNA (P:D) complex (20:1, 50:1, 100:1, 200:1 mass ratios), iopamidol (0%, 10%, 33%), and ethiodized oil (0%, 1%, 2%, 4%, 8%, and 16%). The conditions affording efficient gene transfer and ease of translation to in vivo studies were selected for cell line comparison (HCC cells vs hepatocytes). Adenoviral transduction was compared with nonviral vector transfection.

Results

At low concentrations, ethiodized oil increased transfection efficiency regardless of P:D mass ratio. However, high concentrations resulted in significant attenuation. Unexpectedly, the addition of iopamidol to P:D complexes markedly improved transfection efficiency. When using an optimal P:D, iopamidol, and ethiodized oil solution, DNA transfection of normal liver and tumor cells showed significant selectivity for tumor cells. In the context of hepatoma cells, transfection efficiency with the nonviral vector was better than 10⁴ pfu adenovirus.

Conclusions

The development and characterization of the VIPER system provides a possible alternative to viral gene therapy of HCC.