• 1.第三軍醫(yī)大學(xué)西南醫(yī)院全軍肝膽外科研究所 西南醫(yī)院肝膽外科(重慶400038);;
  • 2.成都軍區(qū)昆明總醫(yī)院肝膽外科(昆明650032);

目的　 構(gòu)建包含IL-13基因片段的慢病毒載體，培養(yǎng)IL-13基因工程大鼠肝干細(xì)胞(肝卵圓細(xì)胞,HOC,WB-F344細(xì)胞)，將IL-13基因重組至WB-F344細(xì)胞中，使之可分泌大鼠IL-13。
方法 　人工合成大鼠IL-13基因，利用pWPXL-MOD(TG-005)載體構(gòu)建包含大鼠IL-13基因片段的質(zhì)粒； 制備慢病毒穿梭質(zhì)粒及其輔助包裝元件載體質(zhì)粒，四質(zhì)粒載體(組成為pRsv-REV、pMDlg-pRRE、pMD2G及目的穿梭質(zhì)粒)分別進(jìn)行高純度無(wú)內(nèi)毒素抽提，共轉(zhuǎn)染293T細(xì)胞，培養(yǎng)48 h后，收集富含慢病毒顆粒的細(xì)胞上清液，對(duì)其濃縮后得到高滴度的慢病毒濃縮液，定量PCR檢測(cè)病毒滴度。將構(gòu)建好的慢病毒感染W(wǎng)B-F344細(xì)胞，5 d后采用real-time PCR檢測(cè)WB-F344細(xì)胞中IL-13的表達(dá)水平，Western blot檢測(cè)重組蛋白表達(dá)水平。
結(jié)果 　成功構(gòu)建了IL-13過表達(dá)慢病毒載體，并獲得IL-13基因工程大鼠肝干細(xì)胞； 將IL-13基因重組至WB-F344細(xì)胞基因組中，并可在細(xì)胞中進(jìn)行轉(zhuǎn)錄，慢病毒感染W(wǎng)B-F344細(xì)胞存在IL-13 mRNA表達(dá)，可分泌大鼠IL-13。
結(jié)論 　構(gòu)建的基因工程WB-F344細(xì)胞能顯著分泌大鼠IL-13，感染后的WB-F344細(xì)胞的IL-13表達(dá)水平顯著高于感染前，滿足動(dòng)物實(shí)驗(yàn)要求。

引用本文： 陳剛,韓江,劉永康,艾軍華,董家鴻. 攜帶IL-13基因大鼠肝干細(xì)胞系的建立. 中國(guó)普外基礎(chǔ)與臨床雜志, 2009, 16(4): 276-280. 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

1. Alison MR, Vig P, Russo F, et al. Hepatic stem cells: from inside and outside the liver? [Ｊ]. Cell Prolif, 2004; 37(1): 1-21.
2. Kafri T, van Praag H, Gage FH, et al. Lentiviral vectors: regulated gene expression [Ｊ]. Mol Ther, 2000; 1(6): 516-521.
3. Xu K, Ma H, McCown TJ, et al. Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors [Ｊ]. Mol Ther, 2001; 3(1): 97-104.
4. Charrier S, Stockholm D, Seye K, et al. A lentiviral vector encoding the human Wiskott-Aldrich syndrome protein corrects immune and cytoskeletal defects in WASP knockout mice [Ｊ]. Gene Ther, 2005; 12(7): 597-606.
5. Trono D. Lentiviral vectors: turning a deadly foe into a therapeutic agent [Ｊ]. Gene Ther, 2000; 7(1): 20-23.
6. Sinn PL, Sauter SL, McCray PB Jr. Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors—design, biosafety, and production [Ｊ]. Gene Ther， 2005; 12(14): 1089-1098.
7. Romano G. Current development of lentiviral-mediated gene transfer [Ｊ]. Drug News Perspect， 2005; 18(2): 128-134.
8. Zufferey R, Nagy D, Mandel RJ, et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo [Ｊ]. Nat Biotechnol， 1997; 15(9): 871-875.
9. Naldini L, Blmer U, Gage FH, et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector [Ｊ]. Proc Natl Acad Sci USA， 1996; 93(21): 11382-11388.
10. Miyoshi H, Smith KA, Mosier DE, et al. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors [Ｊ]. Science， 1999; 283(5402): 682-686.
11. Kafri T, Blmer U, Peterson DA, et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors [Ｊ]. Nat Genet， 1997; 17(3): 314-317.
12. Chomarat P, Banchereau J. Interleukin-4 and interleukin-13: their similarities and discrepancies [Ｊ]. Int Rev Immunol, 1998; 17(1-4): 1-52.
13. Gorczynski RM, Cohen Z, Fu XM, et al. Interleukin-13, in combination with anti-interleukin-12, increases graft prolongation after portal venous immunization with cultured allogeneic bone marrow-derived dendritic cells [Ｊ]. Transplantation, 1996; 62(11): 1592-1600.
14. 陳剛, 傅志仁, 王梁華, 等. 人白介素-13對(duì)牛主動(dòng)脈內(nèi)皮細(xì)胞表達(dá)黏附分子的影響 [Ｊ]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2005；　12(5): 469-472.
15. 陳剛, 晉云, 李雪松, 等. 保護(hù)性基因A20在血管內(nèi)皮細(xì)胞中的誘導(dǎo)表達(dá) [Ｊ]. 細(xì)胞與分子免疫學(xué)雜志, 2007; 23(12): 1200-1202.
16. Qin AL, Zhou XQ, Zhang W, et al. Characterization and enrichment of hepatic progenitor cells in adult rat liver [Ｊ]. World J Gastroenterol, 2004; 10(10): 1480-1486.
17. Strain AJ, Crosby HA, Nijjar S, et al. Human liver-derived stem cells [Ｊ]. Semin Liver Dis, 2003; 23(4): 373-384.
18. Wang X, Foster M, Al-Dhalimy M, et al. The origin and liver repopulating capacity of murine oval cells [Ｊ]. Proc Natl Acad Sci USA, 2003; 100 Suppl 1: 11881-11888.
19. Sell S. Heterogeneity and plasticity of hepatocyte lineage cells [Ｊ]. Hepatology, 2001; 33(3): 738-750.
20. Oh SH, Hatch HM, Petersen BE. Hepatic oval ‘stem’ cell in liver regeneration [Ｊ]. Semin Cell Dev Biol, 2002; 13(6): 405-409.
21. Muller-Borer BJ, Cascio WE, Anderson PA, et al. Adult-derived liver stem cells acquire a cardiomyocyte structural and functional phenotype ex vivo [Ｊ]. Am J Pathol, 2004; 165(1): 135-145.
22. Chramostová K, Vondrácek J, Sindlerová L, et al. Polycyclic aromatic hydrocarbons modulate cell proliferation in rat hepatic epithelial stem-like WB-F344 cells [Ｊ]. Toxicol Appl Pharmacol, 2004; 196(1): 136-148.
23. Suzuki A, Nakauchi H, Taniguchi H. In vitro production of functionally mature hepatocytes from prospectively isolated hepatic stem cells [Ｊ]. Cell Transplant, 2003; 12(5): 469-473.
24. Poulsom R, Alison MR, Forbes SJ, et al. Adult stem cell plasticity [Ｊ]. J Pathol, 2002; 197(4): 441-456.
25. Yin L, Sun M, Ilic Z, et al. Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats [Ｊ]. Hepatology, 2002; 35(2): 315-324.
26. 〖ＪＰ３〗Zheng YW, Taniguchi H. Diversity of hepatic stem cells in the fetal and adult liver [Ｊ]. Semin Liver Dis, 2003; 23(4): 337-348.
27. Weber A. Immortalization of hepatic progenitor cells [Ｊ]. Pathol Biol (Paris), 2004; 52(2): 93-96.
  1. 1. Alison MR, Vig P, Russo F, et al. Hepatic stem cells: from inside and outside the liver? [Ｊ]. Cell Prolif, 2004; 37(1): 1-21.
  2. 2. Kafri T, van Praag H, Gage FH, et al. Lentiviral vectors: regulated gene expression [Ｊ]. Mol Ther, 2000; 1(6): 516-521.
  3. 3. Xu K, Ma H, McCown TJ, et al. Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors [Ｊ]. Mol Ther, 2001; 3(1): 97-104.
  4. 4. Charrier S, Stockholm D, Seye K, et al. A lentiviral vector encoding the human Wiskott-Aldrich syndrome protein corrects immune and cytoskeletal defects in WASP knockout mice [Ｊ]. Gene Ther, 2005; 12(7): 597-606.
  5. 5. Trono D. Lentiviral vectors: turning a deadly foe into a therapeutic agent [Ｊ]. Gene Ther, 2000; 7(1): 20-23.
  6. 6. Sinn PL, Sauter SL, McCray PB Jr. Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors—design, biosafety, and production [Ｊ]. Gene Ther， 2005; 12(14): 1089-1098.
  7. 7. Romano G. Current development of lentiviral-mediated gene transfer [Ｊ]. Drug News Perspect， 2005; 18(2): 128-134.
  8. 8. Zufferey R, Nagy D, Mandel RJ, et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo [Ｊ]. Nat Biotechnol， 1997; 15(9): 871-875.
  9. 9. Naldini L, Blmer U, Gage FH, et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector [Ｊ]. Proc Natl Acad Sci USA， 1996; 93(21): 11382-11388.
  10. 10. Miyoshi H, Smith KA, Mosier DE, et al. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors [Ｊ]. Science， 1999; 283(5402): 682-686.
  11. 11. Kafri T, Blmer U, Peterson DA, et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors [Ｊ]. Nat Genet， 1997; 17(3): 314-317.
  12. 12. Chomarat P, Banchereau J. Interleukin-4 and interleukin-13: their similarities and discrepancies [Ｊ]. Int Rev Immunol, 1998; 17(1-4): 1-52.
  13. 13. Gorczynski RM, Cohen Z, Fu XM, et al. Interleukin-13, in combination with anti-interleukin-12, increases graft prolongation after portal venous immunization with cultured allogeneic bone marrow-derived dendritic cells [Ｊ]. Transplantation, 1996; 62(11): 1592-1600.
  14. 14. 陳剛, 傅志仁, 王梁華, 等. 人白介素-13對(duì)牛主動(dòng)脈內(nèi)皮細(xì)胞表達(dá)黏附分子的影響 [Ｊ]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2005；　12(5): 469-472.
  15. 15. 陳剛, 晉云, 李雪松, 等. 保護(hù)性基因A20在血管內(nèi)皮細(xì)胞中的誘導(dǎo)表達(dá) [Ｊ]. 細(xì)胞與分子免疫學(xué)雜志, 2007; 23(12): 1200-1202.
  16. 16. Qin AL, Zhou XQ, Zhang W, et al. Characterization and enrichment of hepatic progenitor cells in adult rat liver [Ｊ]. World J Gastroenterol, 2004; 10(10): 1480-1486.
  17. 17. Strain AJ, Crosby HA, Nijjar S, et al. Human liver-derived stem cells [Ｊ]. Semin Liver Dis, 2003; 23(4): 373-384.
  18. 18. Wang X, Foster M, Al-Dhalimy M, et al. The origin and liver repopulating capacity of murine oval cells [Ｊ]. Proc Natl Acad Sci USA, 2003; 100 Suppl 1: 11881-11888.
  19. 19. Sell S. Heterogeneity and plasticity of hepatocyte lineage cells [Ｊ]. Hepatology, 2001; 33(3): 738-750.
  20. 20. Oh SH, Hatch HM, Petersen BE. Hepatic oval ‘stem’ cell in liver regeneration [Ｊ]. Semin Cell Dev Biol, 2002; 13(6): 405-409.
  21. 21. Muller-Borer BJ, Cascio WE, Anderson PA, et al. Adult-derived liver stem cells acquire a cardiomyocyte structural and functional phenotype ex vivo [Ｊ]. Am J Pathol, 2004; 165(1): 135-145.
  22. 22. Chramostová K, Vondrácek J, Sindlerová L, et al. Polycyclic aromatic hydrocarbons modulate cell proliferation in rat hepatic epithelial stem-like WB-F344 cells [Ｊ]. Toxicol Appl Pharmacol, 2004; 196(1): 136-148.
  23. 23. Suzuki A, Nakauchi H, Taniguchi H. In vitro production of functionally mature hepatocytes from prospectively isolated hepatic stem cells [Ｊ]. Cell Transplant, 2003; 12(5): 469-473.
  24. 24. Poulsom R, Alison MR, Forbes SJ, et al. Adult stem cell plasticity [Ｊ]. J Pathol, 2002; 197(4): 441-456.
  25. 25. Yin L, Sun M, Ilic Z, et al. Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats [Ｊ]. Hepatology, 2002; 35(2): 315-324.
  26. 26. 〖ＪＰ３〗Zheng YW, Taniguchi H. Diversity of hepatic stem cells in the fetal and adult liver [Ｊ]. Semin Liver Dis, 2003; 23(4): 337-348.
  27. 27. Weber A. Immortalization of hepatic progenitor cells [Ｊ]. Pathol Biol (Paris), 2004; 52(2): 93-96.