• 1.第四軍醫(yī)大學西京醫(yī)院燒傷與皮膚外科（陜西西安 710032）;;
  • 2.第四軍醫(yī)大學醫(yī)學教育研究室（陜西西安 710032）;;
  • 3.第四軍醫(yī)大學西京醫(yī)院肝膽外科（陜西西安 710032）;

目的構(gòu)建含木薯linamarase (lis)基因的穩(wěn)定轉(zhuǎn)染肝癌細胞系，并對其生物學特性進行研究。方法 應(yīng)用PCR法從木薯的DNA中擴增出lis基因，將其克隆至pcDNA3.1(+)質(zhì)粒中，構(gòu)建重組質(zhì)粒pcDNA3.1/lis。 通過脂質(zhì)體法將其轉(zhuǎn)染人肝癌細胞系HepG2，進行G418篩選，獲得穩(wěn)定轉(zhuǎn)染的肝癌細胞系HepG2/lis，通過免疫熒光染色、RT-PCR和Western blot法鑒定lis在細胞中的表達; 采用Lambert法對細胞內(nèi)lis酶的活性進行分析; 采用MTT法觀察穩(wěn)定轉(zhuǎn)染細胞系生長曲線，流式細胞儀分析細胞周期，裸鼠成瘤實驗分析其體內(nèi)成瘤特性。結(jié)果 lis基因能穩(wěn)定整合于真核細胞中，并包裝出具備β-葡萄糖苷酶活性的蛋白。lis在細胞中的穩(wěn)定表達對細胞形態(tài)、生長特性、細胞周期、體內(nèi)成瘤性等生物學特征并無明顯影響。 結(jié)論成功構(gòu)建含lis基因的穩(wěn)定轉(zhuǎn)染肝癌細胞系，為將lis自殺基因系統(tǒng)應(yīng)用于肝癌的治療提供了實驗基礎(chǔ)。

引用本文： 李軍,朱靚,鄭瑞國,李海民,陳明浩,馬俊,竇科峰. linamarase穩(wěn)定轉(zhuǎn)染肝癌細胞系的建立及研究. 中國普外基礎(chǔ)與臨床雜志, 2011, 18(8): 829-834. doi: 復制

1. Santana MA, Vásquez V, Matehus J, et al. Linamarase expression in cassava cultivars with roots of low and highcyanide content [J]. Plant Physiol, 2002, 129(4): 16861694.
2. ItohNashida T, Hiraiwa M, Uda Y. Purification and properties of betaDglucosidase (linamarase) from the butter bean, Phaseolus lunatus [J]. J Biochem, 1987, 101(4): 847854.
3. Link N, Aubel C, Kelm JM, et al. Therapeutic protein transduction of mammalian cells and mice by nucleic acidfree lentiviral nanoparticles [J]. Nucleic Acids Res, 2006, 34(2): e16.
4. GarcíaEscudero V, Gargini R, Izquierdo M. Glioma regression in vitro and in vivo by a suicide combined treatment [J]. Mol Cancer Res, 2008, 6(3): 407417.
5. Lambert JL, Ramasamy J, Paukstelis JV. Stable reagents for the colorimetric determination of cyanide by modified Koenig reactions [J]. Anal Chem, 1975, 47 (6): 916918.
6. Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002 [J]. CA Cancer J Clin, 2005, 55(2): 74108.
7. 沈志勇, 劉驊, 曹暉. 胃癌基因診斷和治療的研究進展 [J]. 中國普外基礎(chǔ)與臨床雜志, 2009, 16(6): 500502.
8. 任濤, 李枚娟, 顏江華. 自殺基因治療惡性腫瘤的研究現(xiàn)狀及展望 [J]. 現(xiàn)代腫瘤醫(yī)學, 2009, 17(12): 24352437.
9. 李越. 肝癌的自殺基因治療的研究進展 [J]. 醫(yī)學理論與實踐, 2008, 21(1): 3940.
10. Sung MW, Yeh HC, Thung SN, et al. Intratumoral adenovirusmediated suicide gene transfer for hepatic metastases from colorectal adenocarcinoma: results of a phase I clinical trial [J]. Mol Ther, 2001, 4(3): 182191.
11. Huang X, Zhang W, Wakimoto H, et al. Adenovirusmediated tissuespecific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression levels [J]. J Exp Ther Oncol, 2002, 2(2): 100106.
12. 李鋒, 張萍, 王楚, 等. PNP/fludarabine自殺基因系統(tǒng)對人肝癌細胞HepG2體外殺傷效應(yīng) [J]. 中國腫瘤, 2004, 13(12): 800805.
13. Huber BE, Richards CA. Regulated expression of artificial chimeric genes contained in retroviral vectors: implications for virusdirected enzyme prodrug therapy (VDEPT) and other gene therapy applications [J]. J Drug Target, 1996, 3(5): 349356.
14. 陳耿臻， 韓慧， 許銘炎， 等. ADVtk重組腺病毒的構(gòu)建及檢測 [J]. 中國普外基礎(chǔ)與臨床雜志， 2010， 17（7）： 703706.
15. Cool V, Pirotte B, Gérard C, et al. Curative potential of herpes simplex virus thymidine kinase gene transfer in rats with 9L gliosarcoma [J]. Hum Gene Ther, 1996, 7(5): 627635.
16. Cortes ML, de Felipe P, Martin V, et al. Successful use of a plant gene in the treatment of cancer in vivo [J]. Gene Ther, 1998, 5(11): 14991507.
17. Kousparou CA, Epenetos AA, Deonarain MP. Antibodyguided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells [J]. Int J Cancer, 2002, 99(1): 138148.
18. Birchall JC, Kellaway IW, Gumbleton M. Physical stability and in vitro gene expression efficiency of nebulised lipidpeptideDNA complexes [J]. Int J Pharm, 2000, 197(12): 221231.
19. 盧銀平, 王寶菊, 黃紅平, 等. 中國旱獺干擾素α家族基因在真核細胞和原核細胞中的表達 [J]. 中華肝臟病雜志, 2006, 14(2): 124128.
20. Thompson L. Gene therapy. Monkey tests spark safety review [J]. Science, 1992, 257(5078): 1854.
21. 代文杰, 姜洪池. 肝細胞基因轉(zhuǎn)導載體系統(tǒng)應(yīng)用進展 [J]. 中國普外基礎(chǔ)與臨床雜志, 2002, 9(1): 5860.
22. Hernández T, Lundquist P, Oliveira L, et al. Fate in humans of dietary intake of cyanogenic glycosides from roots of sweet cassava consumed in Cuba [J]. Nat Toxins, 1995, 3(2): 114117.
23. Frakes RA, Sharma RP, Willhite CC. Comparative metabolism of linamarin and amygdalin in hamsters [J]. Food Chem Toxicol, 1986, 24(5): 417420.
24. Cortés ML, GarcíaEscudero V, Hughes M, et al. Cyanide bystander effect of the linamarase/linamarin killersuicide gene therapy system [J]. J Gene Med, 2002, 4(4): 407414.
25. 胡春松, 洪均言, Losordo DW. 基因治療的安全性、有效性和穩(wěn)定性原則 [J]. 中國實驗血液學雜志, 2004, 12(3): 392396.
  1. 1. Santana MA, Vásquez V, Matehus J, et al. Linamarase expression in cassava cultivars with roots of low and highcyanide content [J]. Plant Physiol, 2002, 129(4): 16861694.
  2. 2. ItohNashida T, Hiraiwa M, Uda Y. Purification and properties of betaDglucosidase (linamarase) from the butter bean, Phaseolus lunatus [J]. J Biochem, 1987, 101(4): 847854.
  3. 3. Link N, Aubel C, Kelm JM, et al. Therapeutic protein transduction of mammalian cells and mice by nucleic acidfree lentiviral nanoparticles [J]. Nucleic Acids Res, 2006, 34(2): e16.
  4. 4. GarcíaEscudero V, Gargini R, Izquierdo M. Glioma regression in vitro and in vivo by a suicide combined treatment [J]. Mol Cancer Res, 2008, 6(3): 407417.
  5. 5. Lambert JL, Ramasamy J, Paukstelis JV. Stable reagents for the colorimetric determination of cyanide by modified Koenig reactions [J]. Anal Chem, 1975, 47 (6): 916918.
  6. 6. Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002 [J]. CA Cancer J Clin, 2005, 55(2): 74108.
  7. 7. 沈志勇, 劉驊, 曹暉. 胃癌基因診斷和治療的研究進展 [J]. 中國普外基礎(chǔ)與臨床雜志, 2009, 16(6): 500502.
  8. 8. 任濤, 李枚娟, 顏江華. 自殺基因治療惡性腫瘤的研究現(xiàn)狀及展望 [J]. 現(xiàn)代腫瘤醫(yī)學, 2009, 17(12): 24352437.
  9. 9. 李越. 肝癌的自殺基因治療的研究進展 [J]. 醫(yī)學理論與實踐, 2008, 21(1): 3940.
  10. 10. Sung MW, Yeh HC, Thung SN, et al. Intratumoral adenovirusmediated suicide gene transfer for hepatic metastases from colorectal adenocarcinoma: results of a phase I clinical trial [J]. Mol Ther, 2001, 4(3): 182191.
  11. 11. Huang X, Zhang W, Wakimoto H, et al. Adenovirusmediated tissuespecific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression levels [J]. J Exp Ther Oncol, 2002, 2(2): 100106.
  12. 12. 李鋒, 張萍, 王楚, 等. PNP/fludarabine自殺基因系統(tǒng)對人肝癌細胞HepG2體外殺傷效應(yīng) [J]. 中國腫瘤, 2004, 13(12): 800805.
  13. 13. Huber BE, Richards CA. Regulated expression of artificial chimeric genes contained in retroviral vectors: implications for virusdirected enzyme prodrug therapy (VDEPT) and other gene therapy applications [J]. J Drug Target, 1996, 3(5): 349356.
  14. 14. 陳耿臻， 韓慧， 許銘炎， 等. ADVtk重組腺病毒的構(gòu)建及檢測 [J]. 中國普外基礎(chǔ)與臨床雜志， 2010， 17（7）： 703706.
  15. 15. Cool V, Pirotte B, Gérard C, et al. Curative potential of herpes simplex virus thymidine kinase gene transfer in rats with 9L gliosarcoma [J]. Hum Gene Ther, 1996, 7(5): 627635.
  16. 16. Cortes ML, de Felipe P, Martin V, et al. Successful use of a plant gene in the treatment of cancer in vivo [J]. Gene Ther, 1998, 5(11): 14991507.
  17. 17. Kousparou CA, Epenetos AA, Deonarain MP. Antibodyguided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells [J]. Int J Cancer, 2002, 99(1): 138148.
  18. 18. Birchall JC, Kellaway IW, Gumbleton M. Physical stability and in vitro gene expression efficiency of nebulised lipidpeptideDNA complexes [J]. Int J Pharm, 2000, 197(12): 221231.
  19. 19. 盧銀平, 王寶菊, 黃紅平, 等. 中國旱獺干擾素α家族基因在真核細胞和原核細胞中的表達 [J]. 中華肝臟病雜志, 2006, 14(2): 124128.
  20. 20. Thompson L. Gene therapy. Monkey tests spark safety review [J]. Science, 1992, 257(5078): 1854.
  21. 21. 代文杰, 姜洪池. 肝細胞基因轉(zhuǎn)導載體系統(tǒng)應(yīng)用進展 [J]. 中國普外基礎(chǔ)與臨床雜志, 2002, 9(1): 5860.
  22. 22. Hernández T, Lundquist P, Oliveira L, et al. Fate in humans of dietary intake of cyanogenic glycosides from roots of sweet cassava consumed in Cuba [J]. Nat Toxins, 1995, 3(2): 114117.
  23. 23. Frakes RA, Sharma RP, Willhite CC. Comparative metabolism of linamarin and amygdalin in hamsters [J]. Food Chem Toxicol, 1986, 24(5): 417420.
  24. 24. Cortés ML, GarcíaEscudero V, Hughes M, et al. Cyanide bystander effect of the linamarase/linamarin killersuicide gene therapy system [J]. J Gene Med, 2002, 4(4): 407414.
  25. 25. 胡春松, 洪均言, Losordo DW. 基因治療的安全性、有效性和穩(wěn)定性原則 [J]. 中國實驗血液學雜志, 2004, 12(3): 392396.