新一代肿瘤条件复制腺病毒构建及其喉癌生物治疗研究

发布时间：2018-07-03 10:42

本文选题：条件复制腺病毒 + 启动子　；参考：《第四军医大学》2010年硕士论文

【摘要】： 凋亡抑制因子(survivin)属凋亡抑制蛋白(Inhibitor of apoptosis protein, IAP)家族成员,由142个氨基酸组成,通过抑制Caspase3、Caspase7活性抑制细胞凋亡,并通过与微管、纺锤体作用调节细胞分裂。hTERT基因是编码人端粒逆转录酶基因,调节端粒酶活性的关键基因。Survivin和hTERT基因选择性地在多种恶性肿瘤细胞中表达而在正常成熟组织中不表达。我们在前期实验中成功克隆了survivin及hTERT启动子基因,经pGL3-Basic荧光素酶报告基因表达载体证实,该两个启动子能高特异性地在肿瘤细胞中调控下游基因表达,其活性甚至高于CMV启动子。survivin及hTERT基因启动子高活性及恶性肿瘤表达谱广泛性,使其更适合于构建肿瘤特异性复制腺病毒(Tumour- Selectively Replicating Oncolytic Adenovirus,T-SROAd)。因此,我们拟通过双启动子(survivin和hTERT启动子)分别调控腺病毒复制必需基因E1A及E1B的表达,并携带蛋白转导域(Protein Transduction Domain,PTD)和野生型p53融合基因,构建新一代的T-SROAd。目的:通过构建和包装双启动子(survivin和hTERT启动子)调控的腺病毒复制必需基因E1A及E1B的表达,并携带蛋白转导域(PTD)和野生型p53融合基因的重组腺病毒,为喉癌乃至所有survivin及hTERT高表达的恶性肿瘤探索出一种新的、疗效更高、毒副作用更低并适用于转移瘤的生物治疗新方法方法:(1) PCR方法分别扩增肿瘤特异性survivin及hTERT启动子,并克隆入腺病毒载体pXC1的两个复制必需基因E1A和E1B序列上游启动子区,hTERT启动子同时调控PTD-P53及IRES连接的E1B基因,即HP-PTDP53-IRES融合基因,构建出双肿瘤特异性启动子调控的条件复制腺病毒载体pXC1-SP-HP53;(2)重组腺病毒载体与腺病毒骨架质粒pBHGE3在脂质体介导下共转染293E细胞进行同源重组,空斑技术获取病毒,并进行病毒扩增、纯化、滴度测定及遗传稳定性鉴定,将病毒命名为Ad-SP-HP53;(3)重组腺病毒Ad-SP-HP53感染人喉癌细胞Hep-2,应用光学显微镜观察细胞病变效应,MTT检测方法观察其对喉癌细胞Hep-2的特异性溶瘤作用,并以正常人的血管内皮细胞ECV304作为对照。流式细胞术及AO/EB染色检测重组腺病毒感染后Hep-2细胞凋亡;(4)裸鼠体内注射Hep-2细胞成瘤后,瘤体注射Ad-SP-HP53,观察其对肿瘤的生长抑制作用。结果: (1)限制性酶切及测序方法鉴定结果证实,成功构建了双肿瘤特异性启动子调控的复制腺病毒载体pXC1-SP-HP53; (2)同源重组获得高滴度重组腺病毒,滴度为3.9×1010TCID50/ml。重组腺病毒遗传稳定性较好,经RT-PCR分析重组病毒感染的Hep-2细胞显示,目的基因在Hep-2细胞中呈阳性表达; (3) MTT结果显示,Ad-SP-HP53可有效抑制喉癌细胞增殖而对正常细胞无增殖抑制作用(p0.05);活细胞计数及细胞形态观察结果显示,重组腺病毒在喉癌细胞中选择性复制并发挥溶细胞作用;流式细胞术及凋亡染色显示,Ad-SP-HP53可有效促进Hep-2的凋亡; (4)裸鼠体内抑瘤实验显示,构建的重组腺病毒Ad-SP-HP53可有效抑制肿瘤生长,延长裸鼠生存时间。结论:成功构建了肿瘤特异性启动子调控的、并携带有野生型P53的重组腺病毒,体内外实验显示该重组腺病毒具有显著的溶瘤作用和促进肿瘤细胞凋亡作用但对正常人血管内皮细胞不发挥溶细胞作用,实验结果为喉癌基因治疗提供了更为良好的条件复制型病毒载体及新的治疗策略。
[Abstract]:Apoptosis suppressor (survivin) is a member of the Inhibitor of apoptosis protein (IAP) family, composed of 142 amino acids, which inhibits apoptosis by inhibiting Caspase3, Caspase7 activity and regulating cell division of the.HTERT gene by the action of microtubules and spindles to encode human telomere reverse transcriptase gene and regulate telomerase activity. The key genes,.Survivin and hTERT, are selectively expressed in a variety of malignant tumor cells and are not expressed in normal mature tissues. We successfully cloned the Survivin and hTERT promoter genes in the early experiments. The two promoters can be highly specific in the tumor by the pGL3-Basic luciferase reporter gene expression vector. The cells regulate the expression of downstream genes, and their activity is even higher than the high activity of the promoter.Survivin and hTERT gene promoter and the broad spectrum of the expression of malignant tumor, making it more suitable for the construction of the tumor specific replicating adenovirus (Tumour- Selectively Replicating Oncolytic Adenovirus, T-SROAd). Therefore, we intend to use the dual promoter (survi) (survi). VIN and hTERT promoter) regulate the expression of E1A and E1B of the essential gene for adenovirus replication, and carry protein transduction domain (Protein Transduction Domain, PTD) and wild type p53 fusion gene to construct a new generation of T-SROAd..
Objective: to construct and package the expression of essential genes E1A and E1B, which are regulated by the double promoter (survivin and hTERT promoter), and carry the recombinant adenovirus of the protein transduction domain (PTD) and the wild type p53 fusion gene, and explore a new, more effective, and toxic pair for the malignant tumors of the larynx and all the Survivin and hTERT. A new biotherapy method that is less effective and suitable for metastatic tumors
Methods: (1) the tumor specific survivin and hTERT promoter were amplified by the PCR method, and the two replicating essential genes of the adenovirus vector pXC1 were cloned into the upstream promoter region of E1A and E1B sequences. The hTERT promoter simultaneously regulates the E1B gene of PTD-P53 and IRES connection, namely the HP-PTDP53-IRES fusion gene, and constructs the regulation of the dual tumor specific promoter. Conditional replication of adenovirus vector pXC1-SP-HP53; (2) recombinant adenovirus vector and adenoviral skeleton plasmid pBHGE3 co transfected 293E cells under liposome mediated homologous recombination, plaque technology to obtain virus, virus amplification, purification, titer determination and genetic stability identification, the virus named Ad-SP-HP53; (3) recombinant adenovirus Ad-SP-HP 53 human larynx cancer cell Hep-2 was infected with the optical microscope to observe the cytopathic effect. The MTT detection method was used to observe the specific tumor effect on the Hep-2 of the laryngeal cancer cells, and the normal human vascular endothelial cells ECV304 was used as the control. The flow cytometry and AO/EB staining were used to detect the apoptosis of the Hep-2 cells after the recombinant adenovirus infection; (4) the nude mice were injected with He. After P-2 cells were tumor, Ad-SP-HP53 was injected into the tumor to observe its inhibitory effect on tumor growth.
Result:
(1) restriction enzyme digestion and sequencing confirmed that the recombinant adenovirus vector pXC1-SP-HP53 was successfully constructed with double tumor specific promoter.
(2) the recombinant adenovirus with high titer was obtained by homologous recombination, and the genetic stability of the recombinant adenovirus with a titer of 3.9 x 1010TCID50/ml. was better. The Hep-2 cells infected by the recombinant virus by RT-PCR showed that the target gene was expressed in Hep-2 cells.
(3) the results of MTT showed that Ad-SP-HP53 could effectively inhibit the proliferation of laryngeal cancer cells and have no proliferation inhibition to normal cells (P0.05). The results of living cell count and cell morphology showed that the recombinant adenovirus was selectively replicated in the larynx cells and played the role of lysis cells. Flow cytometry and apoptosis staining showed that Ad-SP-HP53 could effectively promote Hep-2 Apoptosis;
(4) tumor inhibition experiments in nude mice showed that the recombinant adenovirus Ad-SP-HP53 could effectively inhibit tumor growth and prolong the survival time of nude mice.
Conclusion: the tumor specific promoter was successfully constructed and the recombinant adenovirus carrying the wild type P53 was carried. The experiment in vitro and in vivo showed that the recombinant adenovirus had significant hemolytic effect and promoted the apoptosis of tumor cells, but did not play the role of cells in the normal human vascular endothelial cells. The experimental results provided the gene therapy for larynx cancer. More favorable conditions for replicating viral vectors and new therapeutic strategies.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.65

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