【作者】 韩志强；

【导师】 马丁；

【作者基本信息】 华中科技大学 ， 妇产科， 2007， 博士

【摘要】 第一部分选择性封闭肿瘤STAT3的新型溶瘤腺病毒M3的构建目的本实验室的第一代腺病毒载体(Ad5/dE1/TK)不能复制,体内存在时间短;第二代腺病毒载体(Ad5/dE1A/6.7K/gp19K)能复制,有一定的选择性,病毒裂解肿瘤细胞释放的肿瘤抗原可激发机体的特异性免役反应,对正常细胞有一定的毒性;针对以上两种腺病毒载体缺点,本课题构建一种具有更高肿瘤特异性的新型溶瘤腺病毒基因治疗载体(Ad5/dE1A/ADP),并将该载体和癌症治疗的一个理想靶点相结合-命名为M3,使之具有更高的选择性复制能力、病毒扩增量大、高效表达靶基因、有一定程度的免疫逃避能力和对正常细胞几无毒性的特点,具有强大的肿瘤特异性杀伤效应,为肿瘤的治疗提供了一个强有力的工具。方法应用层析技术获取腺病毒的TP-DNA,二次PCR技术对Ad5基因组E3区的ADP基因进行定点缺失,通过限制酶消化、连接酶连接、转化、克隆、体外同源重组、钙磷转染、细胞病毒内包装、腺病毒单克隆纯化等技术获得重组腺病毒,通过PCR扩增、测序技术对该重组腺病毒进行鉴定。CsCl梯度液超速离心获得纯化的重组腺病毒。结果成功的获得了具有高包装效率的腺病毒TP-DNA,通过该TP-DNA和穿梭载体的同源重组获得了正确的重组腺病毒。结论获得的重组腺病毒基因治疗载体M3具有预想的各种技术特征,为研究该载体的应用价值奠定了基础。第二部分高度肿瘤特异性新型溶瘤腺病毒载体M3基本生物学特性的鉴定目的对已构建成功的这种具有更高肿瘤特异性的新型溶瘤腺病毒载体M3的基本生物学特性进行鉴定,判断该载体是否满足本课题最初的设想。方法应用PCR检测该载体携带的外源基因(反义STAT3 cDNA片段)的表达模式,外源基因的表达是否模拟了被替代的腺病毒基因(ADP)的表达模式;real-time PCR检测ADP基因的缺失或反义STAT3 cDNA片段的插入对E3区其它基因表达的影响;CPE实验检测该载体对不同肿瘤细胞和正常细胞的裂解能力;病毒爆发实验及TCID50检测ADP基因的缺失或反义STAT3 cDNA片段的插入对子代病毒产量的影响。结果M3所携带外源基因主要在病毒复制的晚期表达,且其表达依赖于病毒DNA的复制,与被替代的腺病毒基因(ADP)的表达模式相一致;ADP基因的缺失或反义STAT3 cDNA片段的插入对其周围E3区的其它基因没有产生显著的影响;M3在肿瘤细胞种引起的CPE效应迟于相应的野生型病毒,5000倍于野生型腺病毒感染量时亦未在正常的细胞中观察到明显的CPE效应;ADP基因的缺失使得肿瘤细胞内子代病毒的量显著增加。结论获得的重组腺病毒M3具有预想的各种基本生物学特性,为该载体的应用奠定了基础。第三部分高度肿瘤特异性新型溶瘤腺病毒载体M3治疗效应的检测目的验证M3对靶蛋白的封闭效应、体外对肿瘤细胞表型的影响和体内的溶瘤效应。方法Western blot免疫印迹和real-time PCR检测M3对靶蛋白的封闭及其下游效应蛋白的调节;应用流式细胞分析技术检测M3在体外对不同组织来源的肿瘤细胞的杀伤能力;耐药细胞株的应用检测M3的化疗增敏作用;Transwell体外侵袭实验观察M3对不同肿瘤细胞侵袭能力的影响;异位及原位肿瘤移植模型检测M3的体内治疗效应。结果Western blot结果表明M3可有效抑制肿瘤细胞内靶蛋白STAT3的表达并下调其下游蛋白c-myc、Bcl-xL、survivin、VEGF、MMP-9和Tim3的表达,但对正常细胞HUVEC内的STAT3的表达没有影响;流式分析结果显示与Ad5/dE1A及Ad5/dE1A/ADP相比,M3对不同组织来源的肿瘤细胞具有强大的杀伤力,肿瘤细胞的最大凋亡率可达80%以上,显著增强了卵巢癌耐药细胞系C13K对药物的敏感性;体外侵袭实验表明M3可显著抑制肿瘤细胞的侵袭能力;前列腺癌及胃癌皮下移植瘤动物模型的实验结果表明瘤内直接注射M3可显著抑制肿瘤的生长,胃癌原位移植模型的实验结果表明,经静脉注射病毒不仅可显著抑制肿瘤的生长,同时亦显著的抑制了胃癌细胞的腹腔种植性转移。结论重组腺病毒M3可有效的封闭STAT3蛋白并影响其下游蛋白的表达,对肿瘤的杀伤效应强大,具有潜在的临床应用价值。更多还原

【Abstract】 PART IConstruction and Application of a Newly Oncolytic Adenovirus Vector M3 Selectively Blocking STAT3 of TumorObjectives The first generation of adenovirus vevtor , Ad5/dE1/TK, vector in our laboratory couldn’t replicate and stayed in vivo for a short time. The second generation of adenovirus vector, Ad5/dE1A/6.7K/gp19K, which could replicate and was selective at some degree, was toxical to normal cells because it could lysis tumor cells and released tumor antigens that could stimulate specifically immune response. To aim directly at the shortcomings of two adenovirus vectors above, we planed to construct a newly oncolytic adenovirus vector, Ad5/dE1A/ADP, with more tumor specifity and then combined with an ideal target of cancer therapy.That would be the third genetion of aenovirus vector named M3. The vector would had powerful tumor-specifically effect and provide a potent approach of cancer therapy because of its more selectively replicative ability, high expression of target gene, some ability of immune evasion and minimal toxicity to normal cells. Methods To obtain TP-DNA of adenovirus by chromotography techniques; to delete ADP gene at fix-point in E3 domain of adenovirus genome by two rounds PCR; to acquire recombinant adenovirus by restriction endonuclease digesting, ligase ligating, transformation, cloning, homologous recombination in vitro, Ca-P transfection, packing adenovirus in cells, adenovirus monoclone purification and so on; to verify the recombinant adenovirus by PCR and sequencing; to gain purified recombinant adenovirus by CsCl gradient centrifuge. Results we obtained TP-DNA with high package effect successfully and gained right recombinant adenovirus through homologous recombination of the TP-DNA and shuttle vector. Conclusion Obtained recombinant adenovirus vector M3 for gene therapy that had a variety of anticipated technological characters established fundament for application of the vector. PARTⅡBiological character verification of a more tumor-specific and newly oncolytic adenovirus vector M3Objectives We verified the biological characters of the more tumor-specific and newly oncolytic adenovirus vector that had constructed successfully to judge whether the vector satisfied the first expectation of our study. Methods We detected the expression model of exogenous gene (antisense STAT3 cDNA fragment) taken by the vector and detected if the expression of exogenous gene imitated the expression of substituted ADP by PCR. After deletion of ADP or insertion of antisense STAT3 cDNA fragment, effects on other gene expression in E3 domain was determined by real-time PCR. CPE assay detected the ability of the vector that lysised different tumor cells and normal cells. We detected whether deletion of ADP or insertion of antisense STAT3 cDNA fragment would affect on progeny virus production by virus burst assay and TCID50 assay. Results Exogenous gene taken by recombinant adenovirus was expressed in the late stage of the virus replication and the expression, which depended on virus DNA replication, was identified with expression models of substituted ADP. There was no effect on surrounding other gene in E3 domain after deletion of ADP or insertion of antisense STAT3 cDNA fragment. The occurrence of CPE effect of tumor cells by the virus was later than it by wild type adenovirus. And we didn’t observed the apparent CPE effect in normal cells when the amount of the recombinant adenovirus was 5000 times as many as that of wild type adenovirus. The production of the recombinant adenovirus was marked increased because of deletion of ADP. Conclusion Obtained recombinant adenovirus vector for gene therapy that had a variety of anticipated biological characters established fundament for application of the vector. PARTⅢAnalysis of Therapeutic Effects of the More Tumor-specific and Newly Oncolytic Adenovirus Vector M3Objectives To verify the effects of M3 on its target protein, affects of M3 on tumor cells phenotype in vitro and oncolytic effect of M3 in vivo. Methods The blocking effects of M3 on its target protein and the regulations of M3 on its downstream effect proteins were determined by western blot assay and real-time PCR. Lethal effect of M3 on a variety of tumor cells in vitro and sensitization effect by chemotherapy of M3 on drug resistant cell lines were determined by flow cytometry. We observed affects of invasion ability of M3 on different tumor cells by transwell invasion assay in vitro. To detect therapeutic effect of M3 in vivo by dystopic and orthotopic tumor transplanted model. Results Western blot results showed that M3 could effectively inhibit expression of STAT3 protein and down-regulate expression of downstream proteins such as c-myc、Bcl-xL、survivin、VEGF、MMP-9 and Tim3,but had no effect on STAT3 expression of mormal cells HUVEC. Analysis of flow cytometry demonstrated that M3 had potent lethal effect on tumor cells derived from different tissues or organs compared to Ad5/dE1A and Ad5/dE1A/ADP and the maximum apoptosis rate of tumor cells induced by M3 reached to more than 80%. And what’s more, M3 still enhanced significantly the sensibility of ovarian cancer cell line C13K to chemotherapeutic drugs. Transwell assay displayed M3 inhibited effectively invasive ability of tumor cells. In subcutaneouly transplant mice model of prostatic carcinoma and gastric cancer, M3 that was intratumorally injected could markedly restrain the growth of tumor cells. In orthotopic mice model of gastric cancer, M3 by intravenous injection not only markedly inhibited the growth of gastric cancer cells, but also prevented enterocoelia implantation metastasis of them. Conclusion Recombinant adenovirus M3 could block validly the expression of STAT3 protein and affect the expression of downstream protein in the mean time. With the potent lethal effect on tumor cells, M3 possessed the potential value of clinical application.更多还原