一种嵌合型不依赖于CAR的膀胱癌特异性溶瘤腺病毒构建及其抗膀胱癌作用研究

发布时间：2018-01-07 18:03

本文关键词：一种嵌合型不依赖于CAR的膀胱癌特异性溶瘤腺病毒构建及其抗膀胱癌作用研究　出处：《兰州大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:膀胱癌是泌尿系统疾病中最常见的恶性肿瘤之一,且随着空气污染的加重和吸烟人数的增加膀胱癌的发病率逐年增高。膀胱癌的治疗多采用手术、放疗、化疗和灌注疗法,但这些方法尚存在远期治愈率低、复发率高的缺点。近些年膀胱癌的基因治疗成为了一个新兴的治疗手段,其中通过构建携带目的基因的溶瘤腺病毒靶向杀伤膀胱癌细胞是一个新的研究方向。大部分关于膀胱癌溶瘤腺病毒生物治疗的研究中,应用的载体多为5型腺病毒,该病毒主要通过识别膀胱癌细胞表面的柯萨奇-腺病毒受体(coxsackie adenovirus receptor,CAR)进入肿瘤细胞发挥溶瘤作用。但是对于一些分化程度低的肿瘤细胞,其表面CAR的表达量低,从而阻碍了5型腺病毒发挥抗膀胱癌作用。因此,本课题致力于构建一种不依赖于CAR的嵌合型膀胱癌特异性溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,该病毒可以通过膀胱癌广谱表达的CD46分子进入肿瘤细胞发挥溶瘤作用。顺铂是膀胱癌的治疗中较为常用的一种化疗药物,我们通过一系列体外实验研究Ad5/F11p-PSCAE-UPII-E1A联合顺铂是否具有协同抗肿瘤作用。不依赖于CAR的嵌合型溶瘤腺病毒的构建及其与顺铂的联合应用为膀胱癌的基因治疗提供了一个新的思路,拓展了膀胱癌治疗领域。方法:将嵌合5型腺病毒纤毛尾区和11型腺病毒纤毛轴区及旋钮区的骨架质粒Ad5/F11p,与我们实验室前期构建的穿梭质粒Rp-PSCAE-UPII-E1A在大肠杆菌BJ5183中进行同源重组形成重组质粒pAd5/F11p-PSCAE-UPII-E1A,并通过限制性内切酶和聚合酶链式反应(Polymerase chain reaction,PCR)这两种方法对重组质粒进行鉴定。根据pAdEasy-1系统,重组质粒pAd5/F11p-PSCAE-UPII-E1A在HEK293细胞内包装出不依赖于CAR的嵌合型膀胱组织特异性溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,通过PCR的方法对其进行鉴定,对鉴定正确的病毒进行纯化、扩增和滴度测定。将新构建的溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A作用于5637、EJ和T24三种膀胱癌细胞,通过CCK-8、实时荧光定量PCR(qRT-PCR)、Western blot、RNA干扰技术及流失细胞术等方法测定Ad5/F11p-PSCAE-UPII-E1A对不同CAR表达情况的膀胱癌细胞的抗肿瘤作用,以及探讨Ad5/F11p-PSCAE-UPII-E1A对膀胱癌细胞周期的影响。最后通过体外实验研究Ad5/F11p-PSCAE-UPII-E1A联合顺铂对5637、EJ和T24三种膀胱癌细胞的抗肿瘤作用,并通过对相关凋亡基因Fas、Bax、Bcl-2及cleaved Caspase 3的检测初步探讨Ad5/F11p-PSCAE-UPII-E1A联合顺铂抗膀胱癌的作用机制。结果:(1)我们将嵌合5型腺病毒纤毛尾区和11型腺病毒纤毛轴区及旋钮区的骨架质粒Ad5/F11p,和穿梭质粒Rp-PSCAE-UPII-E1A进行同源重组,经鉴定形成了正确的重组质粒pAd5/F11p-PSCAE-UPII-E1A,并根据pAdEasy-1包装系统,包装出了不依赖于CAR的嵌合型溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,通过纯化、扩增及病毒滴度测定,我们得到的病毒滴度为1×1010PFU/ml。(2)实时荧光定量PCR结果显示在5637、EJ及T24细胞中,T24细胞CAR的表达量低,而5637、EJ及T24这三种细胞均高表达CD46分子。(3)Ad5/F11p-PSCAE-UPII-E1A可以进入CAR高表达5637、EJ细胞和CAR低表达的T24进行复制,并发挥抗肿瘤作用,且杀伤作用均优于Ad5-PSCAE-UPII-E1A,而对人正常尿路上皮细胞株SV-HUC-1均无杀伤作用。(4)嵌合型病毒Ad5/F11p-PSCAE-UPII-E1A进入膀胱癌细胞进行复制发挥抗肿瘤作用和细胞表面CAR的表达量无关:该病毒分别感染未经CAR-SiRNA处理的膀胱癌细胞(EJ、5637、T24)和CAR-SiRNA干扰的膀胱癌细胞(EJ-CAR-SiRNA、5637-CAR-Si RNA、T24-CAR-SiRNA)后,E1A蛋白的表达和体外抗增殖作用在在EJ和EJ-CAR-SiRNA、5637和5637-CAR-SiRNA、T24和T24-CAR-Si RNA之间无差异。(5)Ad5/F11p-PSCAE-UPII-E1A感染5637、EJ及T24这三种膀胱癌细胞后可以阻滞大部分膀胱癌细胞周期于G1期。(6)Ad5/F11p-PSCAE-UPII-E1A联合顺铂作用于5637、EJ及T24这三种膀胱癌细胞后具有协同抗膀胱癌作用。(7)Ad5/F11p-PSCAE-UPII-E1A联合顺铂可以诱导膀胱癌细胞发生早期凋亡,且发生早期凋亡的细胞数目多于顺铂单用组和Ad5/F11p-PSCAE-UPII-E1A单用组。(8)Ad5/F11p-PSCAE-UPII-E1A联合顺铂作用于5637、EJ及T24这三种膀胱癌细胞后可以上调Fas、Bax和cleaved Caspase3基因的表达,下调Bcl-2基因的表达。结论:我们构建出了一种不依赖于CAR的嵌合型膀胱癌特异性溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,经纯化、鉴定和滴度测定,该病毒纯度好滴度高可用于后续研究;这种嵌合型病毒Ad5/F11p-PSCAE-UPII-E1A可进入5637、EJ及T24这三种膀胱癌细胞发挥抗肿瘤作用,且效果优于Ad5-PSCAE-UPII-E1A;Ad5/F11p-PSCAE-UPII-E1A可以阻滞膀胱癌细胞周期于G1期;Ad5/F11p-PSCAE-UPII-E1A联合顺铂作用于5637、EJ及T24这三种膀胱癌细胞后具有协同抗肿瘤作用;Ad5/F11p-PSCAE-UPII-E1A联合顺铂可以通过增强内源性和外源性凋亡通路诱导膀胱癌细胞发生凋亡。嵌合型溶瘤腺病毒的构建及其与顺铂的联合应用为膀胱癌的基因治疗提供了一个新的思路,拓展了膀胱癌治疗领域,为下一步的研究提供理论基础。
[Abstract]:Objective: bladder cancer is one of the most common malignant tumor of urinary system diseases, and with the increasing rate of air pollution and smoking the incidence of bladder cancer increased year by year. The treatment of bladder cancer with surgery, radiotherapy, chemotherapy and infusion therapy, but these methods are low cure rate in the long term, high recurrence rate disadvantages. Gene therapy of bladder cancer in recent years has become a new means of treatment, the oncolytic adenovirus carrying target gene into killer cells of bladder cancer is a new research direction. Most researches about adenovirus biological treatment solution of bladder cancer, the application of type 5 adenovirus vector the virus, mainly through the surface recognition of bladder cancer cells of coxsackie adenovirus receptor (coxsackie adenovirus, receptor, CAR) into tumor cells play an oncolytic effect. But for some low degree of differentiation The surface of tumor cells, expression of CAR is low, which hinders the adenovirus type 5 inhibit bladder cancer cells. Therefore, the aim of this research is to construct chimeric bladder cancer specific oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1A which does not rely on CAR, CD46 molecules of the virus through bladder cancer spectrum expression into tumor cells play oncolysis. Cisplatin is a chemotherapeutic drug commonly used in the treatment of bladder cancer, we are through a series of in vitro experimental study of Ad5/F11p-PSCAE-UPII-E1A combined with cisplatin have synergistic anticancer effect. Do not depend on the construction of CAR chimeric oncolytic adenovirus combined with cisplatin and its application for gene therapy of bladder cancer provides a new the way to expand the field of the treatment of bladder cancer. Methods: the chimeric adenovirus type 5 pili tail region and adenovirus type 11 axonemal region and knob area backbone plasmid Ad5/ F1 1p, our laboratory and the pre constructed shuttle plasmid Rp-PSCAE-UPII-E1A to form recombinant plasmid pAd5/F11p-PSCAE-UPII-E1A homologous recombination in Escherichia coli BJ5183, and by restriction endonuclease and polymerase chain reaction (Polymerase chain reaction, PCR) identification of the recombinant plasmid was identified by the two methods. According to the pAdEasy-1 system, the recombinant plasmid pAd5/F11p-PSCAE-UPII-E1A packaging chimeric bladder tissue specific oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1A is not dependent on CAR in HEK293 cells, identified by the PCR method, the correct identification of virus was purified and amplified. The titer of the newly constructed oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1A in 5637, EJ and T24 three kinds of bladder cancer cells by real-time fluorescent CCK-8 quantitative PCR (qRT-PCR), Western blot, RNA interference technique and flow cytometry were used to measure the Ad5/F 11p-PSCAE-UPII-E1A on the expression of CAR in bladder cancer cells and the antitumor effect, to explore the effects of Ad5/F11p-PSCAE-UPII-E1A on the cell cycle of bladder cancer. Finally by in vitro experimental study of Ad5/F11p-PSCAE-UPII-E1A combined with cisplatin on 5637, EJ and T24 in three bladder cancer cells and the antitumor effect of apoptosis related gene Fas, Bax, detection mechanism 3 the Bcl-2 and cleaved Caspase of Ad5/F11p-PSCAE-UPII-E1A and cisplatin against bladder cancer. Results: (1) we will plasmid Ad5/F11p type 5 adenovirus cilia tail and adenovirus type 11 axonemal region and knob area and fitted the shuttle plasmid Rp-PSCAE-UPII-E1A for homologous recombination, the recombinant plasmid pAd5/F11p-PSCAE-UPII-E1A was identified to form the correct. According to the pAdEasy-1 packaging system, the packaging does not depend on the CAR chimeric oncolytic adenovirus Ad5/ F11p-PSCAE-UPI I-E1A, through purification, amplification and determination of virus titer of virus titer, we get 1 * 1010PFU/ml. (2) by real-time quantitative PCR showed that in 5637, EJ and T24 cells, the expression of T24 CAR cells was low, while the 5637, EJ and T24 these three kinds of cells were high expression of CD46 (3. Ad5/F11p-PSCAE-UPII-E1A) can enter the high expression of CAR 5637, the low expression of EJ cells and CAR T24 replication, and exert anti-tumor effect, and the killing effect is better than that of Ad5-PSCAE-UPII-E1A, and on normal human urothelial cell line SV-HUC-1 had no killing effect. (4) chimeric virus Ad5/F11p-PSCAE-UPII-E1A in bladder cancer cell replication independent play expression the amount of anti tumor effect and cell surface CAR: the virus infection were not treated with CAR-SiRNA bladder cancer cells (EJ, 5637, T24) of bladder cancer cells and CAR-SiRNA interference (EJ-CAR-SiRNA, 5637-CAR-Si RNA, T24-CAR-SiRNA) After the expression of E1A protein and in vitro anti proliferation effect in EJ and EJ-CAR-SiRNA, 5637 and 5637-CAR-SiRNA, the difference between T24 and T24-CAR-Si. RNA (5) Ad5/F11p-PSCAE-UPII-E1A infection in 5637, EJ and T24 of the three types of bladder cancer cells can block most bladder cancer cell cycle in G1 phase. (6) Ad5/F11p-PSCAE-UPII-E1A combined with cisplatin in 5637, EJ and T24 of the three types of bladder cancer cells has a synergistic antitumor effect on bladder cancer. (7) Ad5/F11p-PSCAE-UPII-E1A combined with cisplatin can induce bladder cancer cell apoptosis, cell number and apoptosis than cisplatin alone group and Ad5/F11p-PSCAE-UPII-E1A group. (8) Ad5/F11p-PSCAE-UPII-E1A combined with cisplatin for 5637 EJ. T24 and the three types of bladder cancer cells can upregulate the Fas expression of Bax, cleaved and Caspase3 genes, the expression level of Bcl-2 gene. Conclusion: We constructed a Bladder cancer specific chimeric oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1A, CAR dependent after purification, identification and determination of the titer of virus titer, the purity can be used for further research; this chimeric virus can enter the 5637 Ad5/F11p-PSCAE-UPII-E1A, EJ and T24 these three kinds of bladder cancer cells exert anti-tumor effect, and the effect is better than that of Ad5-PSCAE-UPII-E1A; Ad5/F11p-PSCAE-UPII-E1A can block bladder cancer cell cycle in G1 phase; Ad5/F11p-PSCAE-UPII-E1A combined with cisplatin for 5637, EJ and T24 these three kinds of bladder cancer cells has a synergistic antitumor effect; Ad5 /F11p-PSCAE-UPII-E1A combined with cisplatin can enhance bladder cancer cell apoptosis induced by exogenous and endogenous apoptosis pathway. Construction of chimeric oncolytic adenovirus combined with cisplatin and its application provides a new way for gene therapy of bladder cancer, expand the treatment of bladder cancer The field provides a theoretical basis for the next step of research.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.14

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