【作者】 周丽君；

【导师】 李秋香； 李冬田；

【作者基本信息】 天津医科大学 ， 病原生物学， 2008， 硕士

【摘要】 由于腺病毒载体在基因治疗方面的诸多优势,现已被广泛应用于人类恶性肿瘤的基因治疗。本研究鉴于以重组复制缺陷型腺病毒治疗恶性肿瘤存在着靶向性差、目的基因表达持续时间短等缺点,将前期构建的含有大肠杆菌胞嘧啶脱氨酶（EC-CD）基因和单纯疱疹病毒胸苷激酶（HSV-TK）基因的重组复制缺陷型腺病毒rAd-CDglyTK进行了基因改造,即将具有抑癌作用的人类腺病毒5型E1A基因和脑/心肌炎病毒的内部核糖体转入位点（internal ribosome entry site,IRES）片段连接后,插入到rAd-CDglyTK中,使其成为一种能够在肿瘤细胞中特异性复制进而裂解肿瘤细胞的新型重组溶瘤腺病毒rAd-E1A-CDglyTK,并探讨这种含有多个治疗基因（包括E1A基因、CD基因和TK基因）以及GFP报告基因的新型重组腺病毒与酶前体药物5-氟胞嘧啶（5-flurocytosine,5-FC）和更昔洛韦（ganciclovir,GCV）联合在体内外对肿瘤细胞的杀伤作用,并与重组复制缺陷型腺病毒rAd-CDglyTK的治疗作用进行比较。本研究首先将成功构建的重组溶瘤腺病毒质粒prAd-E1A-CDglyTK用PacⅠ酶切线性化,经脂质体介导转染293细胞,在293细胞中成功包装的rAd-E1A-CDglyTK能够表达报告基因GFP,在荧光显微镜下可观察到逐日增多的绿色荧光。经多次病毒的传代扩增,待其扩增至较高滴度时采用氯化铯密度梯度超速离心法进行纯化。纯化的重组溶瘤腺病毒经PCR法对其所含的E1A和CDglyTK基因片段进行鉴定,经紫外分光光度法测得纯化病毒的颗粒浓度为9.47×10¹¹VP/ml,用组织培养半数感染剂量(50%tissue culture infectious dose,TCID₅₀)法测得其感染性滴度为3.16×10¹⁰IU/ml,进而计算出该病毒的比活性为3.34%,达到了SFDA规定的重组腺病毒临床级制品的比活性要求。为了探讨重组溶瘤腺病毒rAd-E1A-CDglyTK的体外抑杀瘤作用,选择两种肿瘤细胞系:人卵巢癌细胞系SKOV3和人肺癌细胞系A549。首先,观察rAd-E1A-CDglyTK在肿瘤细胞中的特异性复制能力及其对肿瘤细胞的感染效率,并确定能使细胞感染率达90%以上的最小病毒感染复数（multiplicity ofinfection,MOI）。以一定MOI（单位:IU/cell）的rAd-E1A-CDglyTK感染肿瘤细胞后,用MTT法观察细胞对酶前体药物5-FC和GCV的敏感性,并计算出各药的半数抑制浓度,进而,观察并比较一定MOI的重组溶瘤腺病毒rAd-E1A-CDglyTK或重组复制缺陷型腺病毒rAd-CDglyTK与半数抑制浓度的5-FC和/或GCV联合应用对SKOV3细胞和A549细胞的生长抑制作用。实验结果显示,rAd-E1A-CDglyTK能够在SKOV3细胞和A549细胞中特异性复制。以不同MOI的rAd-E1A-CDglyTK感染SKOV3细胞或A549细胞,发现能使细胞感染率达90%以上的最小MOI为50。因此,以50MOI的rAd-E1A-CDglyTK感染肿瘤细胞,加入不同浓度的5-FC或GCV,发现肿瘤细胞的生长抑制率随药物浓度的增加而逐渐升高,存在明显的剂量依赖效应,经IC₅₀计算器计算得,5-FC和GCV对感染rAd-E1A-CDglyTK的SKOV3细胞的IC₅₀浓度分别为4.56 mmol/L和0.11mmol/L;5-FC和GCV对感染rAd-E1A-CDglyTK的A549细胞的IC₅₀浓度分别为1.38 mmol/L和0.13 mmol/L。两种重组腺病毒/前药系统的抑瘤作用比较发现,rAd-E1A-CDglyTK组细胞生长抑制率为（16.57±1.59）%,与rAd-CDglyTK组（10.44±1.80）%比较,P＜0.01,差异有明显的统计学意义,rAd-E1A-CDglyTK与近似半数抑制浓度的单药,即5mmol/L 5-FC或0.1mmol/L GCV联合,其细胞生长抑制率分别达（51.59±2.31）%和（49.61±2.90）%,而与5 mmol/L 5-FC和0.1mmol/L GCV双药联合应用,其抑制效应达（71.68±2.63）%,与rAd-CDglyTK/5-FC+GCV组的（63.64±2.91）%比较,P＜0.01,差异有明显的统计学意义。从而,证实了含有E1A基因的rAd-E1A-CDglyTK在体外具有一定的抑杀瘤作用;rAd-E1A-CDglyTK/5-FC+GCV系统较rAd-CDglyTK/5-FC+GCV系统具有更好的体外抑杀瘤效果。为了探讨重组溶瘤腺病毒rAd-E1A-CDglyTK的体内抑杀瘤作用,本研究将小鼠Lewis肺癌细胞株接种到C57BL近交纯系小鼠右侧腓肠肌内,按照实验方案分别给予rAd-E1A-CDglyTK、rAd-CDglyTK和/或酶前体药物进行治疗,观察各组荷瘤小鼠的瘤体大小变化,比较各组抑瘤率及组织病理学改变。结果发现,rAd-E1A-CDglyTK/5-FC+GCV组的抑瘤率最高,除rAd-E1A-CDglyTK/5-FC组、rAd-E1A-CDglyTK/GCV组和rAd-CDglyTK/5-FC+GCV组两两间无显著性差异（P＞0.05）外,其余各组间均有明显的统计学差异（P＜0.01）。小鼠肿瘤组织病理学切片HE染色结果显示,rAd-E1A-CDglyTK/5-FC+GCV组除瘤结节包膜边缘见少许瘤细胞外,其余均为坏死区域;rAd-E1A-CDglyTK/5-FC组、rAd-E1A-CDglyTK/GCV组和rAd-CDglyTK/5-FC+GCV组坏死面积相当,均少于rAd-E1A-CDglyTK/5-FC+GCV组;rAd-E1A-CDglyTK组和rAd-CDglyTK组坏死面积更少,其中rAd-E1A-CDglyTK组仍可见成片坏死区域,而rAd-CDglyTK组仅见灶片状坏死,与空白肿瘤对照组差别不大。从而,证实了含有E1A基因的rAd-E1A-CDglyTK在体内发挥了一定的抑杀瘤作用;rAd-E1A-CDglyTK/5-FC+GCV系统较rAd-CDglyTK/5-FC+GCV系统具有更好的体内抑杀瘤效果。本研究在国内外首次成功构建了含有治疗基因,包括人类腺病毒5型E1A基因、大肠杆菌胞嘧啶脱氨酶（EC-CD）基因、单纯疱疹病毒胸苷激酶（HSV-TK）基因,以及报告基因GFP的重组溶瘤腺病毒rAd-E1A-CDglyTK,并通过对重组溶瘤腺病毒rAd-E1A-CDglyTK体内外抑杀瘤作用的实验研究,证实了含有E1A基因的重组溶瘤腺病毒rAd-E1A-CDglyTK在体内外均能发挥一定的抑杀瘤作用;其与酶前体药物5-FC和GCV联合的抑杀瘤作用大大提高,治疗效果优于重组复制缺陷型腺病毒rAd-CDglyTK,此结果为探索更为优化的恶性肿瘤基因治疗腺病毒载体的研究提供了新的思路和可靠的实验依据。更多还原

【Abstract】 Adenovirus as gene tranfer vectors with a great number of advantages,including its wide host range,high titers and high transduction efficiency has been used extensively in the context of cancer gene therapy.However,due to its non-target transduction and transient exogenous gene expression,recombinant replication defective adenovirus-based gene therapy in clinical trials has thus far yielded dissatisfactory results.In order to find a better adenovirus-based cancer gene therapeutic modality,we inserted the connecting fragment E1A-IRES,deriving from adenovirus 5 type E1A gene and human EMCV internal ribosome entry site fragment, into recombinant replication defective adenovirus,rAd-CDglyTK,and constructed a novel recombinant oncolytis adenovirus,rAd-E1A-CDglyTK,which contains adenovirus E1A gene,fusion suicide gene CDglyTK fragment and GFP reporter gene and can specificly replicate in the tumor cells.Firstly,we had the successfully constructed recombinant plasmid, prAd-E1A-CDglyTK,digested by PmeⅠenzyme,then transfected into 293 cells with liposome.The successful package was verified by observing the expression of green fluorescence protein under the fluorescent microscope.After packed and proliferated in 293 cells,rAd-E1A-CDglyTK was purified through the method of cesium chloride density gradient ultracentrifugation.The existence of E1A gene and CDglyTK fragment was identified by PCR assay.The result of viral particle concentration tested by ultraviolet spectrophotometry was 9.47×10¹¹VP/ml and its infectious titre was 3.16×10¹⁰IU/ml tested through 50%tissue culture infectious dose assay.The virual ratio-activity,3.34%,was gained by the ratio of infectious titre and viral particle concentration,having achieve the SFDA rated ratio-activity requirement of the recombinant adenovirus clinical grade products.To investigate the antitumor effect of the novel recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,we chose two kind of tumor cell lines,human ovarian cancer SKOV3 cell line and human lung cancer A549 cell line.Firstly,we verified the specific replicating ability of rAd-E1A-CDglyTK in the two kind of tumor cells,observed its infectious efficiency to SKOV3 or A549 cells and determine the minimal multiplicity of infection that can lead to the infectious efficiency up to above 90%.With the minimal MOI virus dose to infect tumor cells,SKOV3 or A549 cells,and to observe the drug sensitivity to enzyme prodrug 5-FC or GCV by MTT assay,and then to determine the 50%inhibiting concentration of 5-FC or GCV.With the minimal MOI virus dose and the 50%inhibiting concentration of of 5-FC and/or GCV to observe the cell growth inhibiting ratio,and to compare the antitumor effect of the recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,with the recombinant replication defecient adenovirus,rAd-CDglyTK,as well as the combination effect with prodrug,5-FC and/or GCV.As a result,the data indicated that rAd-E1A-CDglyTK itself had a significant cytocidal effect on SKOV3 cells,and combined with the 50%inhibiting concentration of 5-FC or GCV,the growth inhibition ratio could be respectively up to（51.59±2.31）%and（49.61±2.90）%. rAd-E1A-CDglyTK combined with the 50%inhibiting concentration of 5-FC and GCV,the growth inhibition ratio could reach（71.68±2.63）%,which comparied with rAd-CDglyTK/5-FC+GCV（63.64±2.91）%,P＜0.01.The resemble data attained from the experiment of A549 cells.Therefore,we could easily have a conclusion that the recombinant oncolytic adenovirus combined with enzyme prodrugs,5-FC and GCV,can result in a better ant^tumor effect in vitro in comparison with the replication defecient adenovirus.To investigate the antitumor effect in vivo of the novel recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,we prepared Lewis lung cancer-bearing C57BL mouse model.The tumor-bearing mice were divided into seven groups with random and subsequently accepted different administration,including recombinant adenovirus,rAd-CDglyTK or rAd-E1A-CDglyTK,and enzyme prodrug 5-FC and/or GCV.From the tendency of average tumor weight changes and the comparision of tumor inhibition rate as well as the tumor pathological changes of different groups,to observe the antitumor effect of the recombinant oncolytic adenovirus combined with enzyme prodrugs,5-FC and GCV.The experimental data presented that rAd-E1A-CDglyTK could have a considerable antitumor effect on Lewis lung cancer-bearing C57BL mouse,rAd-E1A-CDglyTK combined with enzyme prodrugs, 5-FC and GCV,could achieve a siginificant antitumor effect,better than the replication defecient adenovirus,rAd-CDglyTK.In short,our study successfully constructed a novel recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,which contains adenovirus E1A gene,fusion suicide gene CDglyTK fragment and GFP reporter gene and can specificly replicate in the tumor cells.And we verified that the insertion of E1A gene rendered rAd-E1A-CDglyTK a considerable antitumor effect in vitro and in vivo and combined with enzyme prodrugs,5-FC and GCV,the antitumor effect could be significantly increased,which was better than the antitumor effect of replication defecient adenovirus,rAd-CDglyTK.These results set up a reliable experimental basis for the research of adenovirus vectors for malignant tumor gene therapy.更多还原