攜帶IL-13基因大鼠肝干細胞系的建立_《中國普外基礎與臨床雜志》

作者：

陳剛 ¹ , 韓江 ² , 劉永康 ¹ , 艾軍華 ¹ , 董家鴻 ¹

1.第三軍醫大學西南醫院全軍肝膽外科研究所西南醫院肝膽外科(重慶400038);;
2.成都軍區昆明總醫院肝膽外科(昆明650032);

關鍵詞：

慢病毒白介素-13 肝卵圓細胞肝干細胞載體構建

視頻：

導出 下載 收藏 掃碼 引用

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

目的　構建包含IL-13基因片段的慢病毒載體，培養IL-13基因工程大鼠肝干細胞(肝卵圓細胞,HOC,WB-F344細胞)，將IL-13基因重組至WB-F344細胞中，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)載體構建包含大鼠IL-13基因片段的質粒；制備慢病毒穿梭質粒及其輔助包裝元件載體質粒，四質粒載體(組成為pRsv-REV、pMDlg-pRRE、pMD2G及目的穿梭質粒)分別進行高純度無內毒素抽提，共轉染293T細胞，培養48 h后，收集富含慢病毒顆粒的細胞上清液，對其濃縮后得到高滴度的慢病毒濃縮液，定量PCR檢測病毒滴度。將構建好的慢病毒感染WB-F344細胞，5 d后采用real-time PCR檢測WB-F344細胞中IL-13的表達水平，Western blot檢測重組蛋白表達水平。
結果　成功構建了IL-13過表達慢病毒載體，并獲得IL-13基因工程大鼠肝干細胞；將IL-13基因重組至WB-F344細胞基因組中，并可在細胞中進行轉錄，慢病毒感染WB-F344細胞存在IL-13 mRNA表達，可分泌大鼠IL-13。
結論　構建的基因工程WB-F344細胞能顯著分泌大鼠IL-13，感染后的WB-F344細胞的IL-13表達水平顯著高于感染前，滿足動物實驗要求。

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

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對牛主動脈內皮細胞表達黏附分子的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2005；　12(5): 469-472.
15.	陳剛, 晉云, 李雪松, 等. 保護性基因A20在血管內皮細胞中的誘導表達 [Ｊ]. 細胞與分子免疫學雜志, 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. 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對牛主動脈內皮細胞表達黏附分子的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2005；　12(5): 469-472.
15. 陳剛, 晉云, 李雪松, 等. 保護性基因A20在血管內皮細胞中的誘導表達 [Ｊ]. 細胞與分子免疫學雜志, 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.

《中國普外基礎與臨床雜志》

攜帶IL-13基因大鼠肝干細胞系的建立

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

《中國普外基礎與臨床雜志》

攜帶IL-13基因大鼠肝干細胞系的建立

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

摘要全文圖表視頻參考文獻施引文獻補充材料