肝癌靶向性溶瘤腺病毒的构建及其对肝癌细胞的特异性杀伤研究
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摘要
1.溶瘤腺病毒Adhaf E1△55的构建及在体外对肝癌细胞的特异杀伤研究
     肿瘤的发生发展往往是多基因协同作用的结果,仅靠恢复1个或几个抑癌基因功能的基因治疗方法一般难以凑效,因此发展了一种使用肿瘤选择复制性腺病毒(亦称特异性溶瘤腺病毒)治疗肿瘤的新策略。
     肝癌细胞中约30~40%不产生AFP,但此类肝癌细胞内仍有痕量的AFP启动子活性;并且肝癌经常发生于肝硬化病人,这类病人的预后在很大程度上受肝储备能力(即由肝脏干细胞导致的肝脏再生能力)的影响。因此抗肿瘤作用的特异性对于肝癌基因治疗的成败是非常重要的,尤其不能误伤了肝脏干细胞。
     血管内皮生长因子VEGF上游的缺氧应答元件HRE与甲胎蛋白AFP核心启动子AF0.3构成的杂合启动子HREAF,已在逆转录病毒载体上被证明可有效介导自杀基因HSVtk在几乎所有各种AFP产量不等的肝癌细胞中特异表达,并且在理论上具有不介导目的基因在肝脏干细胞内表达的可能。因此我们将杂合启动子HREAF用于靶向重组溶瘤腺病毒在肝癌中的特异复制。
     特异性溶瘤腺病毒具有在肿瘤细胞内“复制与裂解的一一对应关系”特点,将HREAF用于靶向溶瘤腺病毒在肝癌细胞内特异复制并裂解之,至少在理论上可避免对肝储备能力的影响。因为肝脏再生过程呈有序适度特征,氧供充足,不同于肝炎、肝硬化等组织中由缺氧条件及多种细胞因子和生长因子而介导VEGF的表达,亦不同于在实体瘤中会因瘤体增长过快及无序新生血供而导致主要由缺氧条件介导VEGF表达。
     在这部分研究工作中,我们构建了肝癌靶向性溶瘤腺病毒Adhaf E1△55,并进行了初步的体外肝癌细胞特异杀伤研究。包括以下工作:
     1)从肝癌基因组DNA克隆了VEGF基因的增强子--缺氧应答元件HRE和AFP基因的核心启动子--AF0.3,将二者重组成杂合启动子HREAF,经测序证明序列
    
     中文摘要
     正确;并将质粒pshuttle-HREAF-EI转染于人肝癌细胞HepGZ、Bel7402。
     SMMC7721和肺癌细胞PLA801C,缺氧诱导培养24h后以RT-PCR方法检测Ela
     的表达状况,对HREAF介导目的基因在人肝癌细胞中特异表达的特性进行了
     验证。在此项工作中,构建了pGEM-HRE、PGEM-AFO.3、PGEM-HREAF、
     pshuttle-HREAF-EI质粒。
     2)克隆己完全剔除了 EIa 5’端转录调控序列的腺病毒 AdE 区基因,构建了
     pGEM-AdEI、pAC-AdEI。pEllRESlneo质粒,并对克隆的此段AdEI基因启
     动腺病毒复制的功能进行了证实。首先将真核表达载体pAC-AdEI转染人肺巨
     细胞癌PLA-801D细胞株,辅以Ad-LacZ复制缺陷型腺病毒感染,结果显示转
     染 pAC-AdE细胞的 p-半乳糖苦酶活性明显高于转染 pAC-GFP的阴性对照细
     胞,初步证明了 m 区启动腺病毒复制的功能;之后再将真核共表达载体
     pAdE-DIESI.neo转染人宫颈癌细胞系Hela细胞,经G418筛选出抗性细胞克
     隆,以低剂量AdLacZ复制缺陷型腺病毒攻击,结果抗性细胞对Ad-LacZ较敏
     感,易被病毒感染裂解死亡,而亲本Hela细胞则末见明显毒性,进一步证实了
     EI区启动腺病毒复制的功能。
     3)将 AdEasy XL adenoviral vector system中的穿梭载体 pshuttle 进订改构,在
     pshuttle结构中克隆入 SV40 polyA片段,改构成适宜于本研究应用的
     pshuttle};将杂合启动子HREAF克隆入pshutleK,构建成肝癌特异的腺病
     毒穿梭载体pshuttle-HREAF;将腺病毒AdEI区基因和己缺失了Elb55基因的
     AdE区基因分别克隆入pshuttle-HREAF,构建了肝癌特异的腺病毒穿梭质粒
     pshuttle-HREAF-EI和 pshuttle-HREAF-EI凸 55。
     4)将腺病毒穿梭质粒 pshuttle-HREAF-EI、pshuttle-HREAF-EIA 55分别与腺病
     毒骨架质粒pAdEasyl共同电转化细菌BJ5183,在细菌内重组出肝癌特异的腺
     病毒质粒 pAdhafEI和 pAdhafEIA 55。
     5)将重组腺病毒质粒 pAdhafEIA 55经 Lipofectamine 2000介导,分别转染 293细
     胞和 HepGZ细胞,分别于正常和缺氧诱导条件下培养,7叶0d后收获细胞,反
     复冻融获病毒上清。提取病毒基因组DNA,经PCR扩增出特异调控序列
     HREAf,证明在两种细胞中均包装出了肝癌特异性重组溶瘤腺病毒 Adhaffi A
     55。快速 CPE法测定重组腺病毒 AdhafEI凸 55滴度均约为 2刁 X 10‘pm/ml。
     4
    ——___
    
     中文摘要
     在 HepGZ细胞内包装出重
1. Preparation of the oncolytic adenovirus AdhafE1△55 selectively killing hepatocarcinoma cells in vitro
    The occurrence and progression of malignant tumors is involved in the accumulation of multiple genes, so the effect of gene therapy will be no use if only be relied on the recovery of one or several tumor suppressor genes. One new strategy for treatment of malignant tumors was recently developed by using replication- selective adenovirus (or called selectively oncolytic adenovirus).
    About 30%-40% of hepatocellular carcinoma (HCC) cells don't secret AFP, but the AFP promoter in these cells still have a trace of activity. And HCC frequently occurs in patients with liver cirrhosis and the prognosis of patients is influenced by the hepatic reserve( the liver regeneration resulted from hepatic stem cells ). Hence the selectivity of antitumor effects for gene therapy is vital for HCC, especially not to hurt the hepatic stem cell.
    A hybrid promoter, HREAF, in which a 0.4-kb fragment of human vascular endothelial growth factor (VEGF) 5'-flanking sequences containing hypoxia-responsive element (HRE) was fused to the human AFP core promoter AF0.3,was constructed. It can mediate hypoxia-inducible transcriptions of the herpes simplex virus-thymidine kinase (HSV-tk) which was controlled by it in a retroviral vector in high-,low- and non-AFP-producing human HCC cells, but not in non-HCC cells. And HREAF also will prevent the expression of therapeutic genes in hepatic stem cells that also express AFP. So this hybrid promoter HREAF was introduced into the construction of the recombinant oncolytic adenovirus Adhaf El △ 55 to direct its selectively replication in HCC cells.
    
    
    
    One of the characteristic of the selectively oncolytic adenovirus is that if it replicates in this tumor cell and it will only lyse this cell. The promoter HREAF as controller can direct the replication of the oncolytic adenovirus to lyse the HCC cells, and at least to prevent theoretically the damage of the hepatic reserve. Because the regeneration of liver is in order and proper and supplied with sufficient oxygen, it differs from the VEGF expression stimulated not only by hypoxia but also by various cytokines and growth factors in the tissues of hepatitis or cirrhotic liver. And it also differs from the VEGF expression stimulated mainly by hypoxia which is resulted from the excessive growth and unorderly angiogenesis in solid tumor.
    Basis on the conception above, we constructed HCC selectively oncolytic adenovirus AdhafEl A55 and assess preliminarily its selectively killing ability in HCC cells in vitro. The main results of this part are as follows:
    1) Cloned the enhancer of VEGF gene, HRE, and the core promoter AFP, AF0.3, from HCC cell genome DNA, then the hybrid promoter HREAF was recombined and verified by sequencing. Transfected the plasmid pShuttle-HREAF-El into HCC cells HepG2, Bel7402, SMMC7721 and lung cancer cells PLA801C,the expression of AdEla gene controlled by HREAF was detectable by RT-PCR in HepG2, Bel7402, SMMC7721 but not in PLA801C after 24h culture under hypoxia-inducible condition, and verified the expression specifity of genes controlled by HREAF in human HCC cells. In this study, plasmids pGEM-HRE, pGEM-AF0.3, pGEM-HREAF and pShuttle-HREAF-El were constructed.
    2) Cloned adenovirus Adhaf El fragment that has been completely deleted El a 5' transcriptional regulation sequence, constructed the plasmids pGEM-AdEl, pAC-AdE1, pE1-IRES1neo, and identified the El function for starting adenovirus replication. At first, human lung cancer PLA801D cells was transfected with pAC-AdEl mediated by Lipofectamine, then infected with Ad-LacZ, a replication-defective adenovirus. The results suggest that the activity of p-galactosidase in cells transfected with pAC-AdE1 preliminarily proving El function for starting adenovirus replication is significantly higher than the negative contrast cells with pAC-GFP. Following it, subcloned El to the
    
    
    pIRESl.neo to constructed a co-expression vector pAdE1-IRES1.neo. Then pA
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