發(fā)布時(shí)間：2018-01-07 18:03

  本文關(guān)鍵詞：一種嵌合型不依賴于CAR的膀胱癌特異性溶瘤腺病毒構(gòu)建及其抗膀胱癌作用研究　出處：《蘭州大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 膀胱癌 嵌合型溶瘤腺病毒 基因治療 CAR 順鉑 凋亡

【摘要】：目的:膀胱癌是泌尿系統(tǒng)疾病中最常見的惡性腫瘤之一,且隨著空氣污染的加重和吸煙人數(shù)的增加膀胱癌的發(fā)病率逐年增高。膀胱癌的治療多采用手術(shù)、放療、化療和灌注療法,但這些方法尚存在遠(yuǎn)期治愈率低、復(fù)發(fā)率高的缺點(diǎn)。近些年膀胱癌的基因治療成為了一個(gè)新興的治療手段,其中通過構(gòu)建攜帶目的基因的溶瘤腺病毒靶向殺傷膀胱癌細(xì)胞是一個(gè)新的研究方向。大部分關(guān)于膀胱癌溶瘤腺病毒生物治療的研究中,應(yīng)用的載體多為5型腺病毒,該病毒主要通過識(shí)別膀胱癌細(xì)胞表面的柯薩奇-腺病毒受體(coxsackie adenovirus receptor,CAR)進(jìn)入腫瘤細(xì)胞發(fā)揮溶瘤作用。但是對(duì)于一些分化程度低的腫瘤細(xì)胞,其表面CAR的表達(dá)量低,從而阻礙了5型腺病毒發(fā)揮抗膀胱癌作用。因此,本課題致力于構(gòu)建一種不依賴于CAR的嵌合型膀胱癌特異性溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,該病毒可以通過膀胱癌廣譜表達(dá)的CD46分子進(jìn)入腫瘤細(xì)胞發(fā)揮溶瘤作用。順鉑是膀胱癌的治療中較為常用的一種化療藥物,我們通過一系列體外實(shí)驗(yàn)研究Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑是否具有協(xié)同抗腫瘤作用。不依賴于CAR的嵌合型溶瘤腺病毒的構(gòu)建及其與順鉑的聯(lián)合應(yīng)用為膀胱癌的基因治療提供了一個(gè)新的思路,拓展了膀胱癌治療領(lǐng)域。方法:將嵌合5型腺病毒纖毛尾區(qū)和11型腺病毒纖毛軸區(qū)及旋鈕區(qū)的骨架質(zhì)粒Ad5/F11p,與我們實(shí)驗(yàn)室前期構(gòu)建的穿梭質(zhì)粒Rp-PSCAE-UPII-E1A在大腸桿菌BJ5183中進(jìn)行同源重組形成重組質(zhì)粒pAd5/F11p-PSCAE-UPII-E1A,并通過限制性內(nèi)切酶和聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase chain reaction,PCR)這兩種方法對(duì)重組質(zhì)粒進(jìn)行鑒定。根據(jù)pAdEasy-1系統(tǒng),重組質(zhì)粒pAd5/F11p-PSCAE-UPII-E1A在HEK293細(xì)胞內(nèi)包裝出不依賴于CAR的嵌合型膀胱組織特異性溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,通過PCR的方法對(duì)其進(jìn)行鑒定,對(duì)鑒定正確的病毒進(jìn)行純化、擴(kuò)增和滴度測(cè)定。將新構(gòu)建的溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A作用于5637、EJ和T24三種膀胱癌細(xì)胞,通過CCK-8、實(shí)時(shí)熒光定量PCR(qRT-PCR)、Western blot、RNA干擾技術(shù)及流失細(xì)胞術(shù)等方法測(cè)定Ad5/F11p-PSCAE-UPII-E1A對(duì)不同CAR表達(dá)情況的膀胱癌細(xì)胞的抗腫瘤作用,以及探討Ad5/F11p-PSCAE-UPII-E1A對(duì)膀胱癌細(xì)胞周期的影響。最后通過體外實(shí)驗(yàn)研究Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑對(duì)5637、EJ和T24三種膀胱癌細(xì)胞的抗腫瘤作用,并通過對(duì)相關(guān)凋亡基因Fas、Bax、Bcl-2及cleaved Caspase 3的檢測(cè)初步探討Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑抗膀胱癌的作用機(jī)制。結(jié)果:(1)我們將嵌合5型腺病毒纖毛尾區(qū)和11型腺病毒纖毛軸區(qū)及旋鈕區(qū)的骨架質(zhì)粒Ad5/F11p,和穿梭質(zhì)粒Rp-PSCAE-UPII-E1A進(jìn)行同源重組,經(jīng)鑒定形成了正確的重組質(zhì)粒pAd5/F11p-PSCAE-UPII-E1A,并根據(jù)pAdEasy-1包裝系統(tǒng),包裝出了不依賴于CAR的嵌合型溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,通過純化、擴(kuò)增及病毒滴度測(cè)定,我們得到的病毒滴度為1×1010PFU/ml。(2)實(shí)時(shí)熒光定量PCR結(jié)果顯示在5637、EJ及T24細(xì)胞中,T24細(xì)胞CAR的表達(dá)量低,而5637、EJ及T24這三種細(xì)胞均高表達(dá)CD46分子。(3)Ad5/F11p-PSCAE-UPII-E1A可以進(jìn)入CAR高表達(dá)5637、EJ細(xì)胞和CAR低表達(dá)的T24進(jìn)行復(fù)制,并發(fā)揮抗腫瘤作用,且殺傷作用均優(yōu)于Ad5-PSCAE-UPII-E1A,而對(duì)人正常尿路上皮細(xì)胞株SV-HUC-1均無殺傷作用。(4)嵌合型病毒Ad5/F11p-PSCAE-UPII-E1A進(jìn)入膀胱癌細(xì)胞進(jìn)行復(fù)制發(fā)揮抗腫瘤作用和細(xì)胞表面CAR的表達(dá)量無關(guān):該病毒分別感染未經(jīng)CAR-SiRNA處理的膀胱癌細(xì)胞(EJ、5637、T24)和CAR-SiRNA干擾的膀胱癌細(xì)胞(EJ-CAR-SiRNA、5637-CAR-Si RNA、T24-CAR-SiRNA)后,E1A蛋白的表達(dá)和體外抗增殖作用在在EJ和EJ-CAR-SiRNA、5637和5637-CAR-SiRNA、T24和T24-CAR-Si RNA之間無差異。(5)Ad5/F11p-PSCAE-UPII-E1A感染5637、EJ及T24這三種膀胱癌細(xì)胞后可以阻滯大部分膀胱癌細(xì)胞周期于G1期。(6)Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑作用于5637、EJ及T24這三種膀胱癌細(xì)胞后具有協(xié)同抗膀胱癌作用。(7)Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑可以誘導(dǎo)膀胱癌細(xì)胞發(fā)生早期凋亡,且發(fā)生早期凋亡的細(xì)胞數(shù)目多于順鉑單用組和Ad5/F11p-PSCAE-UPII-E1A單用組。(8)Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑作用于5637、EJ及T24這三種膀胱癌細(xì)胞后可以上調(diào)Fas、Bax和cleaved Caspase3基因的表達(dá),下調(diào)Bcl-2基因的表達(dá)。結(jié)論:我們構(gòu)建出了一種不依賴于CAR的嵌合型膀胱癌特異性溶瘤腺病毒Ad5/F11p-PSCAE-UPII-E1A,經(jīng)純化、鑒定和滴度測(cè)定,該病毒純度好滴度高可用于后續(xù)研究;這種嵌合型病毒Ad5/F11p-PSCAE-UPII-E1A可進(jìn)入5637、EJ及T24這三種膀胱癌細(xì)胞發(fā)揮抗腫瘤作用,且效果優(yōu)于Ad5-PSCAE-UPII-E1A;Ad5/F11p-PSCAE-UPII-E1A可以阻滯膀胱癌細(xì)胞周期于G1期;Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑作用于5637、EJ及T24這三種膀胱癌細(xì)胞后具有協(xié)同抗腫瘤作用;Ad5/F11p-PSCAE-UPII-E1A聯(lián)合順鉑可以通過增強(qiáng)內(nèi)源性和外源性凋亡通路誘導(dǎo)膀胱癌細(xì)胞發(fā)生凋亡。嵌合型溶瘤腺病毒的構(gòu)建及其與順鉑的聯(lián)合應(yīng)用為膀胱癌的基因治療提供了一個(gè)新的思路,拓展了膀胱癌治療領(lǐng)域,為下一步的研究提供理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.14

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