本文選題：條件復(fù)制腺病毒 + 啟動(dòng)子�。� 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 凋亡抑制因子(survivin)屬凋亡抑制蛋白(Inhibitor of apoptosis protein, IAP)家族成員,由142個(gè)氨基酸組成,通過(guò)抑制Caspase3、Caspase7活性抑制細(xì)胞凋亡,并通過(guò)與微管、紡錘體作用調(diào)節(jié)細(xì)胞分裂。hTERT基因是編碼人端粒逆轉(zhuǎn)錄酶基因,調(diào)節(jié)端粒酶活性的關(guān)鍵基因。Survivin和hTERT基因選擇性地在多種惡性腫瘤細(xì)胞中表達(dá)而在正常成熟組織中不表達(dá)。我們?cè)谇捌趯?shí)驗(yàn)中成功克隆了survivin及hTERT啟動(dòng)子基因,經(jīng)pGL3-Basic熒光素酶報(bào)告基因表達(dá)載體證實(shí),該兩個(gè)啟動(dòng)子能高特異性地在腫瘤細(xì)胞中調(diào)控下游基因表達(dá),其活性甚至高于CMV啟動(dòng)子。survivin及hTERT基因啟動(dòng)子高活性及惡性腫瘤表達(dá)譜廣泛性,使其更適合于構(gòu)建腫瘤特異性復(fù)制腺病毒(Tumour- Selectively Replicating Oncolytic Adenovirus,T-SROAd)。因此,我們擬通過(guò)雙啟動(dòng)子(survivin和hTERT啟動(dòng)子)分別調(diào)控腺病毒復(fù)制必需基因E1A及E1B的表達(dá),并攜帶蛋白轉(zhuǎn)導(dǎo)域(Protein Transduction Domain,PTD)和野生型p53融合基因,構(gòu)建新一代的T-SROAd。 目的:通過(guò)構(gòu)建和包裝雙啟動(dòng)子(survivin和hTERT啟動(dòng)子)調(diào)控的腺病毒復(fù)制必需基因E1A及E1B的表達(dá),并攜帶蛋白轉(zhuǎn)導(dǎo)域(PTD)和野生型p53融合基因的重組腺病毒,為喉癌乃至所有survivin及hTERT高表達(dá)的惡性腫瘤探索出一種新的、療效更高、毒副作用更低并適用于轉(zhuǎn)移瘤的生物治療新方法 方法:(1) PCR方法分別擴(kuò)增腫瘤特異性survivin及hTERT啟動(dòng)子,并克隆入腺病毒載體pXC1的兩個(gè)復(fù)制必需基因E1A和E1B序列上游啟動(dòng)子區(qū),hTERT啟動(dòng)子同時(shí)調(diào)控PTD-P53及IRES連接的E1B基因,即HP-PTDP53-IRES融合基因,構(gòu)建出雙腫瘤特異性啟動(dòng)子調(diào)控的條件復(fù)制腺病毒載體pXC1-SP-HP53;(2)重組腺病毒載體與腺病毒骨架質(zhì)粒pBHGE3在脂質(zhì)體介導(dǎo)下共轉(zhuǎn)染293E細(xì)胞進(jìn)行同源重組,空斑技術(shù)獲取病毒,并進(jìn)行病毒擴(kuò)增、純化、滴度測(cè)定及遺傳穩(wěn)定性鑒定,將病毒命名為Ad-SP-HP53;(3)重組腺病毒Ad-SP-HP53感染人喉癌細(xì)胞Hep-2,應(yīng)用光學(xué)顯微鏡觀察細(xì)胞病變效應(yīng),MTT檢測(cè)方法觀察其對(duì)喉癌細(xì)胞Hep-2的特異性溶瘤作用,并以正常人的血管內(nèi)皮細(xì)胞ECV304作為對(duì)照。流式細(xì)胞術(shù)及AO/EB染色檢測(cè)重組腺病毒感染后Hep-2細(xì)胞凋亡;(4)裸鼠體內(nèi)注射Hep-2細(xì)胞成瘤后,瘤體注射Ad-SP-HP53,觀察其對(duì)腫瘤的生長(zhǎng)抑制作用。 結(jié)果: (1)限制性酶切及測(cè)序方法鑒定結(jié)果證實(shí),成功構(gòu)建了雙腫瘤特異性啟動(dòng)子調(diào)控的復(fù)制腺病毒載體pXC1-SP-HP53; (2)同源重組獲得高滴度重組腺病毒,滴度為3.9×1010TCID50/ml。重組腺病毒遺傳穩(wěn)定性較好,經(jīng)RT-PCR分析重組病毒感染的Hep-2細(xì)胞顯示,目的基因在Hep-2細(xì)胞中呈陽(yáng)性表達(dá); (3) MTT結(jié)果顯示,Ad-SP-HP53可有效抑制喉癌細(xì)胞增殖而對(duì)正常細(xì)胞無(wú)增殖抑制作用(p0.05);活細(xì)胞計(jì)數(shù)及細(xì)胞形態(tài)觀察結(jié)果顯示,重組腺病毒在喉癌細(xì)胞中選擇性復(fù)制并發(fā)揮溶細(xì)胞作用;流式細(xì)胞術(shù)及凋亡染色顯示,Ad-SP-HP53可有效促進(jìn)Hep-2的凋亡; (4)裸鼠體內(nèi)抑瘤實(shí)驗(yàn)顯示,構(gòu)建的重組腺病毒Ad-SP-HP53可有效抑制腫瘤生長(zhǎng),延長(zhǎng)裸鼠生存時(shí)間。 結(jié)論:成功構(gòu)建了腫瘤特異性啟動(dòng)子調(diào)控的、并攜帶有野生型P53的重組腺病毒,體內(nèi)外實(shí)驗(yàn)顯示該重組腺病毒具有顯著的溶瘤作用和促進(jìn)腫瘤細(xì)胞凋亡作用但對(duì)正常人血管內(nèi)皮細(xì)胞不發(fā)揮溶細(xì)胞作用,實(shí)驗(yàn)結(jié)果為喉癌基因治療提供了更為良好的條件復(fù)制型病毒載體及新的治療策略。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張曉晴,王力紅,劉世喜,歐陽(yáng)雪松,梁傳余;p53基因突變與喉癌生物學(xué)行為的關(guān)系[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2002年01期

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本文編號(hào)：2093370