携带自杀基因的重组AAV载体的构建及其在乳腺癌基因治疗中的研究
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摘要
乳腺癌是人类常见的一种恶性肿瘤，也是女性主要恶性肿瘤之一。手术、放疗、化疗构成了肿瘤治疗的三大模式。在现代分子生物学和基因工程技术飞速发展的推动下，肿瘤的生物学治疗日益受到人们的重视，显示出良好的应用前景，成为肿瘤治疗的第四模式。肿瘤的自杀基因治疗是近年来基因治疗中研究的热点，也是目前常用的基因治疗方法。
本实验将处于Tet基因调控系统调控下的HSVtk基因插入腺相关病毒载体中，构建两个重组腺相关病毒：AAV／TRE／HSVtk／Tet-On和AAV／TRE／HSVtk／Tet-Off，并研究它们在乳腺癌基因治疗中的作用。首先，使用PCR技术从pRevTet-On／pRevTet-Off扩增Tet-On／Tet-Off基因片段，回收Tet-On／Tet-Off基因片段，以ClaⅠ和HpaⅠ消化后定向克隆插入质粒pRevTRE／HSVtk中相应的酶切位点，通过酶切、PCR鉴定筛选阳性重组质粒，再以XhoⅠ和ClaⅠ酶切该质粒，将含有HSVtk基因和Tet-On／Tet-Off调控序列的片段插入pAAV-MCS中后，经部分酶切除去不受Tet-On／Tet-Off调控序列调控的P_(CMV)启动子，然后经过Klenow DNA聚合酶末端补平、自身环化形成相应的重组腺相关病毒载体。重组腺相关病毒载体后，将所构建的重组载体与其辅助质粒以磷酸钙共沉淀的方法转染HEK293包装细胞，转染后的细胞经反复冻融收集病毒颗粒。以CsCl_2密度梯度离心法纯化所得的重组腺相关病毒，最后得到两株重组腺相关病毒AAV／TRE／HSVtk／Tet-On和AAV／TRE／HSVtk／Tet-Off，病毒的物理滴度分别为2.37×10~(11)／ml和3.86×10~(11)／ml。以病毒感染宿主细胞MCF-7后，提取细胞基因组DNA，进行斑点杂交检测，发现该重组病毒能够将目的基因整合到MCF-7细胞基因组中。
硕士学位论文 中文摘要
此外，还将所构建的pRevTRE／HSVtlUltton质粒以脂质体方法
转染 MCFj细胞，经过 Hygromycin B筛选，建立了一株稳定的受四
环素衍生物强力霉素（Doxcycline，Dox）调控HSVtk基因表达的乳
腺癌细胞株MCF／TRE／HSVtklTeton。用RTICR的方法检测不同Dox
浓度下HSVtk基因的表达；以MTT法检测不同DOX浓度下两氧鸟苦
（Ganciclovir，GCV）对乳腺癌细胞的杀伤作用。结果表明HSVtk基
因产物可以将无毒性的GCV转变成一种有毒的代谢产物，从而杀死
乳腺癌细胞。该基因的表达受到Tet－On基因调控系统的调控，由强
力霉素调节HSVtk基因表达。
Human breast cancer is a common malignancy in women all over the world. Operation, radiotherapy and chemotherapy are three patterns in the therapy of human tumor. With the development of molecular biology and biotechnology, biotherapy become more and more important and become the forth pattern in tumor gene therapy. Suicide gene therapy is one of the choices and a highlight in human gene therapy recently.
In this research, the HSVtk gene, which is controlled by Tet gene regulation system, was inserted into AAV vector. Two viral vectors: AAV/TRE/HSVtk/Tet-On and AAV/TRE/HSVtk/Tet-Off were constructed. PCR amplification was performed using primers based on Tet-On/ Tet-Off gene sequence from pRevTet-On/pRevTet-Off. Tet-On/Tet-Off gene was inserted into pRevTRE/HSVtk and then the recombinant was digested by Xho I and Cla I. The fragment, which contains HSVtk gene and Tet-On/ Tet-Off gene, was inserted into pAAV-MCS. The PCA/K promoter, which is not controlled by Tet-On/ Tet-Off gene, was got rid of by partially digested. The end was flushed by Klenow DNA polymerase and then connected by T4 DNA ligase. pAAV/TRE/HSVtk/Tet-On or pAAV/TRE/HSVtk/Tet-On, pAAV-RC, pHelper were co-transfected into incasing cells HEK293 by using modified calcium phosphate co-precipitation method. Cells transfected with those vectors were harvested by scraping at 3 or 4 days after the initial transfection and lysed through three freeze/thaw cycles. Virus was purified by CsCl2 density gradient purification method. The liter of those viruses are 2.37X 10n/ml and 3.86X 10!Vml . The two recombinant AAV viruses were used to infect MCF-7 cell line. The HSVtk gene in MCF-7 genome was detected by dot-blot after the cells was infected by rAAV.
In addition, transfection of pRevTRE/HSVtk/Tet-On was performed for MCF-7 cell line by using lipofectin. After selected by Hygromycin B, MCF-7 cell line stably expressing Tet-regulated HSVtk gene was established. mRNA level of MCF/RevTRE/HSVtk/Tet-On at different Dox concentration was detected by RT-PCR and MTT method was performed to detect the cell survival rate of MCF/TRE/HSVtk/Tet-On at different Dox concentration when GCV
IV
presented. The product of HSVtk gene can convert the nontoxic GCV into toxic product and kill the breast cancer cells. The expression of HSVtk gene was controlled by Tet-On regulation system under the inducement of Dox.
本实验将处于Tet基因调控系统调控下的HSVtk基因插入腺相关病毒载体中，构建两个重组腺相关病毒：AAV／TRE／HSVtk／Tet-On和AAV／TRE／HSVtk／Tet-Off，并研究它们在乳腺癌基因治疗中的作用。首先，使用PCR技术从pRevTet-On／pRevTet-Off扩增Tet-On／Tet-Off基因片段，回收Tet-On／Tet-Off基因片段，以ClaⅠ和HpaⅠ消化后定向克隆插入质粒pRevTRE／HSVtk中相应的酶切位点，通过酶切、PCR鉴定筛选阳性重组质粒，再以XhoⅠ和ClaⅠ酶切该质粒，将含有HSVtk基因和Tet-On／Tet-Off调控序列的片段插入pAAV-MCS中后，经部分酶切除去不受Tet-On／Tet-Off调控序列调控的P_(CMV)启动子，然后经过Klenow DNA聚合酶末端补平、自身环化形成相应的重组腺相关病毒载体。重组腺相关病毒载体后，将所构建的重组载体与其辅助质粒以磷酸钙共沉淀的方法转染HEK293包装细胞，转染后的细胞经反复冻融收集病毒颗粒。以CsCl_2密度梯度离心法纯化所得的重组腺相关病毒，最后得到两株重组腺相关病毒AAV／TRE／HSVtk／Tet-On和AAV／TRE／HSVtk／Tet-Off，病毒的物理滴度分别为2.37×10~(11)／ml和3.86×10~(11)／ml。以病毒感染宿主细胞MCF-7后，提取细胞基因组DNA，进行斑点杂交检测，发现该重组病毒能够将目的基因整合到MCF-7细胞基因组中。
硕士学位论文 中文摘要
此外，还将所构建的pRevTRE／HSVtlUltton质粒以脂质体方法
转染 MCFj细胞，经过 Hygromycin B筛选，建立了一株稳定的受四
环素衍生物强力霉素（Doxcycline，Dox）调控HSVtk基因表达的乳
腺癌细胞株MCF／TRE／HSVtklTeton。用RTICR的方法检测不同Dox
浓度下HSVtk基因的表达；以MTT法检测不同DOX浓度下两氧鸟苦
（Ganciclovir，GCV）对乳腺癌细胞的杀伤作用。结果表明HSVtk基
因产物可以将无毒性的GCV转变成一种有毒的代谢产物，从而杀死
乳腺癌细胞。该基因的表达受到Tet－On基因调控系统的调控，由强
力霉素调节HSVtk基因表达。
Human breast cancer is a common malignancy in women all over the world. Operation, radiotherapy and chemotherapy are three patterns in the therapy of human tumor. With the development of molecular biology and biotechnology, biotherapy become more and more important and become the forth pattern in tumor gene therapy. Suicide gene therapy is one of the choices and a highlight in human gene therapy recently.
In this research, the HSVtk gene, which is controlled by Tet gene regulation system, was inserted into AAV vector. Two viral vectors: AAV/TRE/HSVtk/Tet-On and AAV/TRE/HSVtk/Tet-Off were constructed. PCR amplification was performed using primers based on Tet-On/ Tet-Off gene sequence from pRevTet-On/pRevTet-Off. Tet-On/Tet-Off gene was inserted into pRevTRE/HSVtk and then the recombinant was digested by Xho I and Cla I. The fragment, which contains HSVtk gene and Tet-On/ Tet-Off gene, was inserted into pAAV-MCS. The PCA/K promoter, which is not controlled by Tet-On/ Tet-Off gene, was got rid of by partially digested. The end was flushed by Klenow DNA polymerase and then connected by T4 DNA ligase. pAAV/TRE/HSVtk/Tet-On or pAAV/TRE/HSVtk/Tet-On, pAAV-RC, pHelper were co-transfected into incasing cells HEK293 by using modified calcium phosphate co-precipitation method. Cells transfected with those vectors were harvested by scraping at 3 or 4 days after the initial transfection and lysed through three freeze/thaw cycles. Virus was purified by CsCl2 density gradient purification method. The liter of those viruses are 2.37X 10n/ml and 3.86X 10!Vml . The two recombinant AAV viruses were used to infect MCF-7 cell line. The HSVtk gene in MCF-7 genome was detected by dot-blot after the cells was infected by rAAV.
In addition, transfection of pRevTRE/HSVtk/Tet-On was performed for MCF-7 cell line by using lipofectin. After selected by Hygromycin B, MCF-7 cell line stably expressing Tet-regulated HSVtk gene was established. mRNA level of MCF/RevTRE/HSVtk/Tet-On at different Dox concentration was detected by RT-PCR and MTT method was performed to detect the cell survival rate of MCF/TRE/HSVtk/Tet-On at different Dox concentration when GCV
IV
presented. The product of HSVtk gene can convert the nontoxic GCV into toxic product and kill the breast cancer cells. The expression of HSVtk gene was controlled by Tet-On regulation system under the inducement of Dox.
引文
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9. Tomicic MT, Thust R, Kaina B. Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation Oncogene 2002; 21(14): 2141-53
10. Asklund T, Appelskog IB, Ammerpohl O, Langmoen IA, Dilber MS, Aints A, Ekstrom TJ, Almqvist PM. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene. Exp Cell Res. 2003; 284(2): 183-93
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20．胡舜英，冯茹 腺相关病毒在基因治疗中的研究进展《国外医学输血及血液学分册》2000，23 (3)； 19-23
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2. Rosenberg SA. Prin ciples of Cancer management: biologic therapy In Devita VT Jr, Hellman S, Rosenberg SA .eds Cancer: Principles & Practice of oncology. 5th ed. Philadephia: Lippincott-Raven, 1997. 349-373
3. Xu G, McLeod HL. Strategies for enzyme/prodrug cancer therapy. Clin Cancer Res. 2001 Nov; 7(11): 3314-24.
4. Grignet-Debrus C, Cool V, Baudson N The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy. Cancer Gene Ther. 2000;7(11): 1456-68.
5. Brade AM, Szmitko P, Ngo D, Liu FF, Klamut HJ. Heat-directed suicide gene therapy for breast cancer. Cancer Gene Ther. 2003; 10(4): 294-301
6. Moolten FL Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 1986; 46(10): 5276-81
7. Fillat C, Carrio M, Cascante A, Sangro B.Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclovir system: fifteen years of application. Curr Gene Ther. 2003; 3(1): 13-26
8. Kanazawa T, Mizukami H, Okada T, Hanazono Y, Kume A, Nishino H, Takeuchi K. Kitamura K, Ichimura K, Ozawa K. Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Ther. 2003; 10(1): 51-8
9. Tomicic MT, Thust R, Kaina B. Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation Oncogene 2002; 21(14): 2141-53
10. Asklund T, Appelskog IB, Ammerpohl O, Langmoen IA, Dilber MS, Aints A, Ekstrom TJ, Almqvist PM. Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene. Exp Cell Res. 2003; 284(2): 183-93
11. Sturtz FG, Waddell K, Shulok etal. Variable efficiency of the thymidine kinaseganciclovir system in human glioblastoma cell lines:Implications for gene therapy〔J〕.HumanGeneTherapy, 1997,8:1945
12. VrionisFD, WuTK, QiP, etal. The bystander effect exerted by tumor cells expressing the HSV-tk gene is dependent on connexin expression and cell communication via gapjunctions. GeneTherapy, 1997,4:577
13. Ishii-MoritaH,Agbaria R, MullenCA, etal. Mechanism of bystander effect killing in the HSV-tk gene therapy model of cancer treatment (J) .GeneTherapy, 1997,4:244
14. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline responsive promoters. Proc Natl Acad Sci USA, 1992,89(12); 5547-5551
15. Gossen M, Freundlied S , Bender G , Muller G , Hillen W, Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science, 1995, 268(5218): 1766-1769
16. Block A, Puls F, Muller J, Milasinovic D, Igelmann D, Schafer P, Kupfermann N, Schmoldt A, Ameis D, Greten H. Highly suppressible expression of single-chain interleukin-12 by doxycycline following adenoviral infection with a single-vector Tet-regulatory system. J Gene Med 2003; 5(3): 190-200
17. Perez N, Plence P, Millet V Tetracycline transcriptional silencer tightly controls transgene expression after in vivo intramuscular electrotransfer: application to interleukin 10 therapy in experimental arthritis. Hum Gene Ther. 2002: 10; 13(18): 2161-72
18. Rendahl KG, Quiroz D, Ladner M, Tightly regulated long-term erythropoietin expression in vivo using tet-inducible recombinant adeno-associated viral vectors. Hum Gene Ther. 2002 Jan 20; 13(2): 335-42.
19. Jung—Joo H, Zorica S, et al. Novel Retroviral Vector Transferring a Suicide Gene and a Selectable Marker Gene with Enhanced Gene Expression by Using a Tetracycline-Responsive Expression System. J Virol 1996,70(11): 8138-41
20．胡舜英，冯茹 腺相关病毒在基因治疗中的研究进展《国外医学输血及血液学分册》2000，23 (3)； 19-23
21. Wu P, Philips M, John B, et al. Adeno-Associated Virus vector-mediated transgene integration into Neurons and other nondividing cell targets, J Virol, 72(7): 1998; 5919
22. Wright JF, Qu G, Tang C, Sommer JM.Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector. Curr Opin Drug Discov Devel. 2003;6(2): 174-8.
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