溶瘤腺病毒载体系统的构建及其功能研究

发布时间：2018-04-18 04:32

本文选题：溶瘤腺病毒 + 载体构建　；参考：《中国人民解放军军事医学科学院》2008年博士论文

【摘要】： 溶瘤腺病毒载体(Oncolytic adenovectors),又称条件复制型腺病毒载体(Conditionally replicating adenoviruses, CRADs),现已广泛用于肿瘤基因治疗研究中。CRADs名称中条件复制的含义是指这种病毒载体能够在肿瘤细胞中特异性复制,通过病毒扩增达到裂解、杀伤肿瘤细胞的目的,其在正常细胞中不能复制,对正常细胞无伤害或伤害小。传统的肿瘤基因治疗的策略是利用复制缺陷型的载体将治疗基因转导到肿瘤细胞,实践证明这种方法由于基因转移效率低,难以杀灭大多数肿瘤细胞。CRADs由于能够在肿瘤细胞中复制、传播,较好地克服了复制缺陷病毒载体的不足。一般认为,CRADs携带治疗基因后,较不含治疗基因的CRADs能够更有效的杀伤肿瘤细胞。 CRADs的构建通常采用穿梭质粒和骨架质粒在293细胞中同源重组、拯救获得重组病毒的传统方法。真核细胞内重组发生的效率低,需要的时间长;为了排除污染、获得均一的目的病毒,还需要进行噬斑纯化操作。1998年,AdEasy系统的发明有效地克服了在上述传统方法的不足,其通过穿梭质粒和骨架质粒在细菌中重组,方便快速地获得重组病毒基因组DNA,转染包装细胞后能快速产生均一的重组病毒。AdEasy系统原本只能用于构建复制缺陷型腺病毒载体,本研究对其进行了改造,建立了一种携带1-2个目的基因的CRAD构建系统,利用该系统构建了携带治疗基因的CRADs,观察了体内外的肿瘤杀伤效应。首先构建一个携带腺病毒E1区的新质粒pTE-TPE-GM,它具备三个特征:一、携带的5型腺病毒(Ad5)E1区序列,这将赋予溶瘤腺病毒选择性复制的特性,二、其基因组较小,便于对E1区进行改造;三、E1区序列能够方便的切下,并亚克隆到穿梭质粒的合适部位。在这个新质粒的E1区序列中,E1A的启动子由肿瘤或组织特异型启动子(TSP)替代,E1B19K序列由第1个治疗基因替代,在治疗基因和E1B55K之间添加内部核糖体进入位点(IRES)序列。将改造的E1区切下,插入到穿梭质粒pShuttle-CMV的腺苷酸加尾信号和腺病毒右臂序列之间,得到一个新的穿梭质粒,其与AdEasy-1共转化大肠杆菌BJ5183,同源重组生成了携带1个治疗基因转录调控的溶瘤腺病毒载体。如果先在pShuttle-CMV的多克隆序列处插入第2个治疗基因,再将改造的E1区引入这个穿梭质粒,同理就能够获得携带2个治疗基因的溶瘤腺病毒载体。选择端粒酶启动子(TERTp)控制E1A基因的表达,在成功构建pTE-TPE-GM后,利用此系统,设计并构建了两个溶瘤腺病毒,一个是携带粒细胞巨噬细胞集落刺激因子(GM-CSF)和细胞表面共刺激分子B7-1(CD80)的Ad-CD80-TPE-GM,另一个是携带GFP和GM-CSF的Ad-GFP-TPE-GM。本研究还构建了复制缺陷腺病毒Ad-GFP,作为阴性对照。病毒构建后,进行了功能检测。体外实验中,检测了Ad-CD80-TPE-GM和Ad-GFP-TPE-GM的在体外培养肿瘤细胞中的复制和选择性肿瘤杀伤效应。预实验观察到两种溶瘤腺病毒能够杀死端粒酶阳性的PC3M细胞,并证明病毒在其中复制。然后,选用8种端粒酶阳性肿瘤细胞系和2种端粒酶阴性细胞系,进一步评价载体的选择性肿瘤杀伤效应。将病毒感染不同的细胞系,感染后7d,结晶紫染色法检测细胞存活百分数,实验结果表明Ad-CD80-TPE-GM和Ad-GFP-TPE-GM均能够高效特异地杀伤端粒酶阳性肿瘤细胞,以Ad-CD80-TPE-GM的溶瘤效果更为显著。在10MOI感染条件下,Ad-CD80-TPE-GM能够杀死几乎所有端粒酶阳性肿瘤细胞,在1MOI感染条件下,Ad-CD80-TPE-GM能够杀死80%的端粒酶阳性肿瘤细胞,甚至在0.1 MOI感染条件下,Ad-CD80-TPE-GM的溶瘤效果也非常明显。同时两病毒对端粒酶阴性的正常细胞没有明显的杀伤作用。Ad-CD80-TPE-GM能够选择性地在端粒酶阳性肿瘤细胞中复制,每个细胞约可产生375感染单位(Infectious units,IU)的子代病毒。体外实验中,以复制缺陷病毒Ad-p53/GM-CSF/B7-1做对照,我们还检测了Ad-CD80-TPE-GM介导目的基因CD80和GM-CSF的表达。在5MOI感染条件下,Ad-CD80-TPE-GM感染端粒酶阳性人喉癌细胞Hep2,培养48h,培养上清中GM-CSF的表达量达到100ng/ml。与对照相比,GM-CSF的表达增加了9000多倍。同时细胞表面CD80的表达阳性率接近100%,相对平均荧光强度与对照相比,增加了146倍。在体内裸鼠荷瘤实验中,我们检测了溶瘤腺病毒Ad-CD80-TPE-GM的功能。选择BALB/C裸鼠,皮下接种端粒酶阳性的人喉癌细胞Hep2,建立荷瘤裸鼠模型,进行了动物实验。以Ad-GFP做对照,瘤内注射溶瘤腺病毒Ad-CD80-TPE-GM 1×10~9 IU,可明显抑制肿瘤生长,给药后35天,肿瘤抑制率达到了78%,血清转氨酶没有升高。瘤内注射溶瘤腺病毒Ad-CD80-TPE-GM 1×10~9 IU,给药后4天,肿瘤组织中可检测到CD80和GM-CSF的表达,以及病毒的复制。总之,本研究建立了一种携带1-2个外源基因溶瘤腺病毒载体生成系统,在此系统中,组织特异性启动子和治疗基因均可根据需要进行相应替换;利用该系统成功构建了携带CD80和GM-CSF的溶瘤腺病毒载体Ad-CD80-TPE-GM,体内外实验表明其有较好的肿瘤杀伤作用。为利用CRADs进行肿瘤基因治疗研究建立了平台。
[Abstract]:Oncolytic adenovirus vector (Oncolytic adenovectors), also called conditionally replicative adenovirus (Conditionally replicating, adenoviruses, CRADs), which has been widely used in the study of meaning of.CRADs conditional replication in cancer gene therapy in the name refers to the virus vector specific replication in tumor cells by virus amplification to lysis, killing tumor cells the purpose, it cannot replicate in normal cells to normal cells, no injury or damage. The traditional gene therapy strategy is the use of vector replication defective gene therapy will be transferred to tumor cells, it is proved that this method is due to low gene transfer efficiency, it is difficult to kill most tumor cells due to.CRADs replication in tumor cell spreading, overcomes the defects of replication defective viral vectors. It is generally considered that CRADs gene carrying therapy, is free CRADs, a therapeutic gene, can kill tumor cells more effectively.
The construction of CRADs usually adopts the shuttle plasmid and plasmid homologous recombination in 293 cells, save the traditional method to obtain the recombinant virus. The recombination in eukaryotic cells is low efficiency, the need for a long time; in order to eliminate the pollution, to obtain a uniform objective virus, but also the need for plaque purification operation in.1998, the invention of AdEasy system to effectively overcome the shortcomings in the traditional method, the shuttle plasmid and the recombinant plasmid in bacteria, fast and convenient to obtain recombinant virus genomic DNA, recombinant virus.AdEasy transfected into the packaging cell can quickly produce uniform originally can only be used to construct replication defective adenovirus vector, this study was carried out on the transformation. A carry 1-2 CRAD gene to construct the system, construct carry the therapeutic gene CRADs by using this system, to observe the in vivo tumor killing effect.
First, build a new area of adenovirus carrying E1 plasmid pTE-TPE-GM, it has three characteristics: first, type 5 adenovirus carrying the E1 sequence (Ad5), which will give the characteristics of selective replication oncolytic adenovirus two, its genome is small, easy to modify E1; three, E1 sequence easy to cut, and subcloned into the shuttle plasmid. The appropriate parts of the E1 sequences in this new plasmid, E1A promoter by tumor or tissue specific promoter (TSP), E1B19K sequence from first for gene replacement, add in the treatment of internal ribosome entry site between gene and E1B55K (IRES) sequence. E1 will transform the region, into the polyadenylation signals and Adenovirus Shuttle Plasmid pShuttle-CMV sequence of the right arm, get a new shuttle plasmid and AdEasy-1 were transformed into Escherichia coli BJ5183, homologous recombination was generated carrying 1 treatment base Because of the oncolytic adenovirus vector of transcriptional regulation. If the first into the second gene therapy in the cloning sequence at pShuttle-CMV, then the E1 transformation of the region into the shuttle plasmid, oncolytic adenovirus vector can be obtained similarly carrying 2 gene therapy. Telomerase promoter (TERTp) to control the expression of E1A gene in. The successful construction of the pTE-TPE-GM, using this system, the design and construction of two oncolytic adenovirus carrying a granulocyte macrophage colony stimulating factor (GM-CSF) and cell surface costimulatory molecule B7-1 (CD80) Ad-CD80-TPE-GM, the other one is carrying GFP and GM-CSF Ad-GFP-TPE-GM. this study also constructed a replication defective adenovirus Ad-GFP virus, virus as negative control. After construction, the function of detection.
In vitro experiments, the detection of Ad-CD80-TPE-GM and Ad-GFP-TPE-GM in cultured tumor cells in tumor selective replication and cytotoxicity in vitro. The pre experiment observed two kinds of oncolytic adenovirus can kill telomerase positive PC3M cells, and prove that the virus replicates in them. Then, selected 8 kinds of telomerase positive tumor cell lines and 2 telomerase negative cell line, tumor selective killing effect. Further evaluation vector to cell lines of different virus infection after infection, 7d, crystal violet staining method to detect cell survival percentage, the experimental results show that Ad-CD80-TPE-GM and Ad-GFP-TPE-GM can effectively and specifically kill telomerase positive tumor cells by oncolytic Ad-CD80-TPE-GM effect is more significant in the 10MOI infection condition. Ad-CD80-TPE-GM, can kill almost all telomerase positive tumor cells in the 1MOI infection condition, Ad-CD80-TPE-GM can kill Telomerase positive tumor cells die 80%, even at 0.1 MOI under the condition of Ad-CD80-TPE-GM infection, the oncolytic effect is very obvious. At the same time two virus of normal cells telomerase negative no obvious cytotoxicity of.Ad-CD80-TPE-GM can selectively replicate in telomerase positive tumor cells, each cell can produce about 375 infectious units (Infectious units, IU) the progeny virus.
In vitro, the replication defective virus Ad-p53/GM-CSF/B7-1 control, we also examined the expression of Ad-CD80-TPE-GM gene mediated by CD80 and GM-CSF. In the 5MOI infection condition, cultured 48h telomerase positive human laryngeal carcinoma cell Hep2, Ad-CD80-TPE-GM infection and the expression level of culture supernatant reached GM-CSF 100ng/ml. compared with the control group, the expression of GM-CSF increased by 9000 at the same time times. The positive expression rate of cell surface CD80 close to 100%, the relative mean fluorescence intensity compared with the control, increased by 146 times.
In vivo nude mice, we tested the oncolytic adenovirus Ad-CD80-TPE-GM function. BALB/C nude mice subcutaneous inoculation of telomerase positive Hep2 human laryngeal carcinoma cells, the establishment of nude mice model, animal experiments were carried out. In order to control Ad-GFP, intratumoral injection of oncolytic adenovirus Ad-CD80-TPE-GM 1 * 10~9 IU, can inhibit tumor growth, 35 days after the administration, the tumor inhibition rate reached 78%, serum transaminase increased. No intratumoral injection of oncolytic adenovirus Ad-CD80-TPE-GM 1 * 10~9 IU, 4 days after administration, detected CD80 and GM-CSF expression in tumor tissues, and the replication of the virus.
In conclusion, this study established a 1-2 carrying exogenous gene oncolytic adenovirus vector generation system, in this system, tissue specific promoter and gene therapy can be carried out according to the needs of the corresponding replacement; successfully constructed oncolytic adenovirus vector carrying Ad-CD80-TPE-GM CD80 and GM-CSF by using this system, in vitro and in vivo experiments show that it has better the tumor killing effect. For the use of CRADs to establish a platform for the research of tumor gene therapy.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346;R73-3

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