• 汕頭大學(xué)醫(yī)學(xué)院第二附屬醫(yī)院外三科（廣東汕頭 515041）;

目的 應(yīng)用基因重組技術(shù)，構(gòu)建含胸苷激酶（tk）自殺基因的復(fù)制缺陷型腺病毒（ADV-tk），為進(jìn)一步研究tk自殺基因?qū)δ[瘤的抑制作用奠定基礎(chǔ)。
方法 采用細(xì)胞內(nèi)同源重組法構(gòu)建攜帶tk基因的ADV-tk，經(jīng)PCR鑒定正確后進(jìn)行擴(kuò)增、純化和滴度測(cè)定。重組腺病毒ADV-tk體外轉(zhuǎn)染人肝癌SMMC-7721細(xì)胞，RT-PCR檢測(cè)tk基因的整合和表達(dá)。建立裸鼠人肝癌動(dòng)物模型，腹腔注射ADV-tk，熒光顯微技術(shù)觀察ADV-tk基因在肝臟的轉(zhuǎn)染情況和對(duì)腫瘤細(xì)胞的抑制作用。
結(jié)果 構(gòu)建的重組腺病毒中帶有tk基因，經(jīng)擴(kuò)增、純化后測(cè)得重組腺病毒滴度為1.4×1010 pfu/ml。ADV-tk體外轉(zhuǎn)染SMMC-7721細(xì)胞后，RT-PCR檢測(cè)顯示tk基因在細(xì)胞中整合并且有效表達(dá)。體內(nèi)檢測(cè)結(jié)果發(fā)現(xiàn)ADV-tk在肝臟有明顯轉(zhuǎn)染，腫瘤組織中凋亡細(xì)胞明顯增多。
結(jié)論 本研究構(gòu)建的攜帶tk基因的復(fù)制缺陷型腺病毒獲得成功，可用于下一步研究。

引用本文： 陳耿臻,韓慧,許銘炎,鄧小玲. ADV-tk重組腺病毒的構(gòu)建及檢測(cè). 中國(guó)普外基礎(chǔ)與臨床雜志, 2010, 17(7): 703-706. doi: 復(fù)制

版權(quán)信息： ?四川大學(xué)華西醫(yī)院華西期刊社《中國(guó)普外基礎(chǔ)與臨床雜志》版權(quán)所有，未經(jīng)授權(quán)不得轉(zhuǎn)載、改編

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2. Portsmouth D, Hlavaty J, Renner M. Suicide genes for cancer therapy [J]. Mol Aspects Med, 2007; 28(1): 441.
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8. Li YM, Song ST, Jiang ZF, et al. Telomerasespecific oncolytic virotherapy for human hepatocellular carcinoma [J]. World J Gastroenterol, 2008; 14(8): 12741279.
9. Zhou J, Gao Q, Chen G, et al. Novel oncolytic adenovirus selectively targets tumorassociated pololike kinase 1 and tumor cell viability [J]. Clin Cancer Res, 2005; 11(23): 84318440.
10. Majumdar AS, Hughes DE, Lichtsteiner SP, et al. The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters [J]. Gene Ther, 2001; 8(7): 568578.
11. Aguilar LK, The BS, Vaachaki MT, et al. AdVtk/valacyclovir gene therapy in combination with radiotherapy for prostate cancer: interim results of a phase Ⅰ/Ⅱ clinical trial [C]. Proc Am Soc Clin Oncol, 21: 2002 (abstr 25).
12. Ling CQ, Li B, Zhang C, et al. Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma [J]. Ann Oncol, 2005; 16(1): 109115.
13. Gentry BG, Boucher PD, Shewach DS. Hydroxyurea induces bystander cytotoxicity in cocultures of herpes simplex virus thymidine kinaseexpressing and nonexpressing HeLa cells incubated with ganciclovir [J]. Cancer Res, 2006; 66(7): 38453851.
14. Li N, Zhou J, Weng D, et al. Adjuvant adenovirusmediated delivery of herpes simplex virus thymidine kinase administration improves outcome of liver transplantation in patients with advanced hepatocellular carcinoma [J]. Clin Cancer Res, 2007; 13(19): 58475854.
15. Palmer DH, Mautner V, Mirza D, et al. Virusdirected enzyme prodrug therapy: intratumoral administration of a replicationdeficient adenovirus encoding nitroreductase to patients with resectable liver cancer [J]. J Clin Oncol, 2004; 22(9): 15461552.
16. van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSVtk/GCV gene therapy. A review [J]. Curr Gene Ther, 2002; 2(3): 307322.
17. Unger MM, Wahl J, Ushmorov A, et al. Enriching suicide gene bearing tumor cells for an increased bystander effect [J]. Cancer Gene Ther, 2007; 14(1): 3038.
18. Zhang Q, Chen G, Peng L, et al. Increased safety with preserved antitumoral efficacy on hepatocellular carcinoma with dualregulated oncolytic adenovirus [J]. Clin Cancer Res, 2006; 12(21): 65236531.
19. Hsieh JL, Lee CH, Teo ML, et al. Transthyretindriven oncolytic adenovirus suppresses tumor growth in orthotopic and ascites models of hepatocellular carcinoma [J]. Cancer Sci, 2009; 100(3): 537545.
20. Li G, Sham J, Yang J, et al. Potent antitumor efficacy of an E1B 55kDadeficient adenovirus carrying murine endostatin in hepatocellular carcinoma [J]. Int J Cancer, 2005; 113(4): 640648.
21. He LF, Gu JF, Tang WH, et al. Significant antitumor activity of oncolytic adenovirus expressing human interferonβ for hepatocellular carcinoma [J]. J Gene Med, 2008; 10(9): 983992.
22. Grimm D, Kay MA. From virus evolution to vector revolution: use of naturally occurring serotypes of adenoassociated virus (AAV) as novel vectors for human gene therapy [J]. Curr Gene Ther, 2003; 3(4): 281304.
23. Coura Rdos S, Nardi NB. The state of the art of adenoassociated virusbased vectors in gene therapy [J]. Virol J, 2007; 4: 99105.
24. Liu H, Peng CH, Liu YB, et al. Inhibitory effect of adenoassociated virusmediated gene transfer of human endostatin on hepatocellular carcinoma [J]. World J Gastroenterol, 2005; 11(22): 33313334.
25. Blechacz B, Splinter PL, Greiner S, et al. Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma [J]. Hepatology, 2006; 44(6): 14651477.
26. 劉自明, 嚴(yán)律南, 李馨筱. 包載攜有HSVTK基因腺病毒PLG微球的制備 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2007; 14(4): 399402.
27. 羅濤, 張強(qiáng), 張建, 等. 腺病毒介導(dǎo)胸苷激酶基因?qū)o脈平滑肌細(xì)胞增殖及內(nèi)膜增生抑制作用的研究 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006; 13(3): 291294.
28. 李梅生, 梁力建, 黃潔夫, 等. HSVtk/CD融合基因殺傷人膽管癌細(xì)胞QBC939的體內(nèi)實(shí)驗(yàn) [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2005; 12(6): 581584.
  1. 1. Vassaux G, MartinDuque P. Use of suicide genes for cancer gene therapy: study of the different approaches [J]. Expert Opin Biol Ther, 2004; 4(4): 519530.
  2. 2. Portsmouth D, Hlavaty J, Renner M. Suicide genes for cancer therapy [J]. Mol Aspects Med, 2007; 28(1): 441.
  3. 3. Altaner C. Prodrug cancer gene therapy [J]. Cancer Lett, 2008; 270(2): 191201.
  4. 4. Si S, Sun Y, Li Z, et al. Gene therapy by membraneexpressed superantigen for αfetoproteinproducing hepatocellular carcinoma [J]. Gene Ther, 2006; 13(22): 16031610.
  5. 5. Stefani AL, Barzon L, Castagliuolo I, et al. Systemic efficacy of combined suicide/cytokine gene therapy in a murine model of hepatocellular carcinoma [J]. Hepatol, 2005; 42(5): 728735.
  6. 6. Song TJ, Eisenberg DP, Adusumilli PS, et al. Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines [J]. J Gastrointest Surg, 2006; 10(4): 532542.
  7. 7. Cai X, Zhou J, Chang Y, et al. Targeting gene therapy for hepatocarcinoma cells with the E.coli purine nucleoside phosphorylase suicide gene system directed by a chimeric αfetoprotein promoter [J]. Cancer Lett, 2008; 264(1): 7182.
  8. 8. Li YM, Song ST, Jiang ZF, et al. Telomerasespecific oncolytic virotherapy for human hepatocellular carcinoma [J]. World J Gastroenterol, 2008; 14(8): 12741279.
  9. 9. Zhou J, Gao Q, Chen G, et al. Novel oncolytic adenovirus selectively targets tumorassociated pololike kinase 1 and tumor cell viability [J]. Clin Cancer Res, 2005; 11(23): 84318440.
  10. 10. Majumdar AS, Hughes DE, Lichtsteiner SP, et al. The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters [J]. Gene Ther, 2001; 8(7): 568578.
  11. 11. Aguilar LK, The BS, Vaachaki MT, et al. AdVtk/valacyclovir gene therapy in combination with radiotherapy for prostate cancer: interim results of a phase Ⅰ/Ⅱ clinical trial [C]. Proc Am Soc Clin Oncol, 21: 2002 (abstr 25).
  12. 12. Ling CQ, Li B, Zhang C, et al. Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma [J]. Ann Oncol, 2005; 16(1): 109115.
  13. 13. Gentry BG, Boucher PD, Shewach DS. Hydroxyurea induces bystander cytotoxicity in cocultures of herpes simplex virus thymidine kinaseexpressing and nonexpressing HeLa cells incubated with ganciclovir [J]. Cancer Res, 2006; 66(7): 38453851.
  14. 14. Li N, Zhou J, Weng D, et al. Adjuvant adenovirusmediated delivery of herpes simplex virus thymidine kinase administration improves outcome of liver transplantation in patients with advanced hepatocellular carcinoma [J]. Clin Cancer Res, 2007; 13(19): 58475854.
  15. 15. Palmer DH, Mautner V, Mirza D, et al. Virusdirected enzyme prodrug therapy: intratumoral administration of a replicationdeficient adenovirus encoding nitroreductase to patients with resectable liver cancer [J]. J Clin Oncol, 2004; 22(9): 15461552.
  16. 16. van Dillen IJ, Mulder NH, Vaalburg W, et al. Influence of the bystander effect on HSVtk/GCV gene therapy. A review [J]. Curr Gene Ther, 2002; 2(3): 307322.
  17. 17. Unger MM, Wahl J, Ushmorov A, et al. Enriching suicide gene bearing tumor cells for an increased bystander effect [J]. Cancer Gene Ther, 2007; 14(1): 3038.
  18. 18. Zhang Q, Chen G, Peng L, et al. Increased safety with preserved antitumoral efficacy on hepatocellular carcinoma with dualregulated oncolytic adenovirus [J]. Clin Cancer Res, 2006; 12(21): 65236531.
  19. 19. Hsieh JL, Lee CH, Teo ML, et al. Transthyretindriven oncolytic adenovirus suppresses tumor growth in orthotopic and ascites models of hepatocellular carcinoma [J]. Cancer Sci, 2009; 100(3): 537545.
  20. 20. Li G, Sham J, Yang J, et al. Potent antitumor efficacy of an E1B 55kDadeficient adenovirus carrying murine endostatin in hepatocellular carcinoma [J]. Int J Cancer, 2005; 113(4): 640648.
  21. 21. He LF, Gu JF, Tang WH, et al. Significant antitumor activity of oncolytic adenovirus expressing human interferonβ for hepatocellular carcinoma [J]. J Gene Med, 2008; 10(9): 983992.
  22. 22. Grimm D, Kay MA. From virus evolution to vector revolution: use of naturally occurring serotypes of adenoassociated virus (AAV) as novel vectors for human gene therapy [J]. Curr Gene Ther, 2003; 3(4): 281304.
  23. 23. Coura Rdos S, Nardi NB. The state of the art of adenoassociated virusbased vectors in gene therapy [J]. Virol J, 2007; 4: 99105.
  24. 24. Liu H, Peng CH, Liu YB, et al. Inhibitory effect of adenoassociated virusmediated gene transfer of human endostatin on hepatocellular carcinoma [J]. World J Gastroenterol, 2005; 11(22): 33313334.
  25. 25. Blechacz B, Splinter PL, Greiner S, et al. Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma [J]. Hepatology, 2006; 44(6): 14651477.
  26. 26. 劉自明, 嚴(yán)律南, 李馨筱. 包載攜有HSVTK基因腺病毒PLG微球的制備 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2007; 14(4): 399402.
  27. 27. 羅濤, 張強(qiáng), 張建, 等. 腺病毒介導(dǎo)胸苷激酶基因?qū)o脈平滑肌細(xì)胞增殖及內(nèi)膜增生抑制作用的研究 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006; 13(3): 291294.
  28. 28. 李梅生, 梁力建, 黃潔夫, 等. HSVtk/CD融合基因殺傷人膽管癌細(xì)胞QBC939的體內(nèi)實(shí)驗(yàn) [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2005; 12(6): 581584.