• 1四川大學(xué)華西醫(yī)院衛(wèi)生部移植科學(xué)與移植免疫重點(diǎn)實(shí)驗(yàn)室（成都，610041）;;
  • 2四川大學(xué)生命科學(xué)學(xué)院;

目的 建立恒河猴外周血CD4+CD25+調(diào)節(jié)性T淋巴細(xì)胞（regulatory T cells，Tregs）快速有效的分離純化方法。 方法4～5歲健康恒河猴10只，雌性4只，雄性6只，體重5～8 kg；于大隱靜脈抽取外周血，每只抽取8 mL。密度梯度離心法分離外周血單個核細(xì)胞（peripheral blood mononuclear cell，PBMC），分別采用非人靈長類Tregs分離試劑盒的生物素標(biāo)記的混合抗體和磁珠標(biāo)記的抗生物素抗體陰性分選，以及大鼠抗人CD4-活化蛋白C（activated protein C，APC）和抗APC多選試劑盒中的磁珠標(biāo)記的抗APC抗體陽性分選出 CD4+ T淋巴細(xì)胞，比較兩種方法獲得細(xì)胞的得率、活性及純度；選擇得率、活性和純度較高的分選方法獲得的CD4+ T淋巴細(xì)胞，用磁珠標(biāo)記的抗人CD25抗體進(jìn)行陽性分選，獲得 CD4+CD25+ Tregs，流式細(xì)胞儀檢測純度、活性及FoxP3表達(dá)水平，以及Tregs對刀豆蛋白（concanavalin A，ConA）刺激的自體CD4+CD25-效應(yīng)性T淋巴細(xì)胞（effective T cells，Teffs）增殖的抑制功能。 結(jié)果CD4+ T淋巴細(xì)胞陽性分選和陰性分選后，細(xì)胞活性均達(dá)95%左右，差異無統(tǒng)計(jì)學(xué)意義（P  gt; 0.05），但陽性分選后CD4+ T淋巴細(xì)胞得率和純度均顯著高于陰性分選（P  lt; 0.05）。后續(xù)CD4+CD25+ Tregs分選采用陽性分選法獲得的CD4+ T淋巴細(xì)胞。經(jīng)雙陽性分選收集的目的細(xì)胞中CD4+CD25+ Tregs占76.2% ± 8.6%，活細(xì)胞比例為93.3% ± 4.7%，F(xiàn)oxP3陽性細(xì)胞比例為74.2% ± 6.9%?；旌吓囵B(yǎng)后Tregs均對ConA刺激的Teffs的增殖有抑制作用。 結(jié)論免疫磁珠雙陽性分選法能有效分選出恒河猴外周血中有功能的CD4+CD25+ Tregs。

引用本文： 張爽,金熙,程漢佳,何斯榮,陳又南,龍丹,單娟,曾力,王成世,程驚秋,陸燕蓉. 恒河猴外周血調(diào)節(jié)性T淋巴細(xì)胞的分離純化及鑒定. 中國修復(fù)重建外科雜志, 2012, 26(10): 1237-1241. doi: 復(fù)制

1. Fan H, Cao P, Game DS, et al. Regulatory T cell therapy for induction of clinical organ transplantation tolerance. Semin Immunol, 2011, 23(6): 453-461.
2. Sagoo P, Lombardi G, Lechler RI. Relevance of regulatoty T cell promotion of donor-specific tolerance in solid organ transplantation. Front Immunol, 2012, 3: 184.
3. Dummer CD, Carpio VN, Gonçalves LF, et al. FOXP3+ regulatory T cells: from suppression of rejection to induction of renal allograft tolerance. Transpl Immunol, 2012, 26(1): 1-10.
4. Sakaguchi S, Yamaguchi T, Numura T, et al. Regulatory T cells and immune tolerance. Cell, 2008, 133(5): 775-787.
5. Riley JL, June CH, Blazar BR, et al. Human T regulatory cells therapy: take a billion or so and call me in the morning. Immunity, 2009, 30(5): 656-665.
6. 熊競, 劉克劍, 吳芳新, 等. 健康中國恒河猴各淋巴組織中Ｔ淋巴細(xì)胞表型及分布. 中國畜牧獸醫(yī), 2012, 39(2): 86-91.
7. 陳磊, 李楠, 王學(xué)廉, 等. 微電極引導(dǎo)偏側(cè)帕金森病猴腦深部電極植入方法的研究. 中國微侵襲神經(jīng)外科雜志, 2012, 17(1): 32-35.
8. Anderson A, Martens CL, Hendrix R, et al. Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses. Am J Transplant, 2008, 8(11): 2252-2263.
9. Yan H, Ding CG, Tian PX, et al. Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+CD25+ Treg cells. J Zhejiang Univ Sci B, 2009, 10(12): 928-932.
10. Bresatz S, Sadlon T, Millard D, et al. Isolation, propagation and characterization of cord blood derived CD4+CD25+ regulatory T cells. J Immunol Methods, 2007, 327(1-2): 53-62.
11. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor FoxP3. Science, 2003, 299(5609): 1057-1061.
12. 陳祚珈, 高雅懿, 李志遠(yuǎn), 等. FOXP3+調(diào)節(jié)性T細(xì)胞. 生命科學(xué), 2010, 22(6): 515-528.
13. 胡義平, 李曉娟, 劉叔文. ConA對CD4+CD25+調(diào)節(jié)性T細(xì)胞早期活化和功能的影響. 細(xì)胞與分子免疫學(xué)雜志, 2010, 26(2): 118-120, 124.
14. 呂夢捷, 曾耀英, 宋兵. 人參皂苷Rb1 對小鼠T淋巴細(xì)胞體外活化、增殖及凋亡的影響. 中草藥, 2011, 42(4): 743-748.
15. Shevach EM. Regulatory T cells in autoimmunity. Ann Rev Immunol, 2000, 18: 423-449.
16. Campbell DJ, Ziegler SF. FOXP3 modifies the phenotypic and functional properties of regulatory T cells. Nat Rev Immunol, 2007, 7(4): 305-310.
17. Carrigan SO, Yang YJ, Issekutz T, et al. Depletion of natural CD4+CD25+ T regulatory cells with anti-CD25 antibody does not change the course of Pseudomonas aeruginosa-induced acute lung infection in mice. Immunobiology, 2009, 214(3): 211-222.
18. Dons EM, Raimondi G, Cooper DK, et al. Non-human primate regulatory T cells: current biology and implications for transplantation. Transplantation, 2010, 90(8): 811-816.
19. Porter CM, Horvath-Arcidiacono JA, Singh AK, et al. Characterization and expansion of baboon CD4+CD25+ Treg cells for potential use in a non-human primate xenotransplantation model. Xenotransplantation, 2007, 14(4): 298-308.
20. Wang J, Huizinga TW, Toes RE. De novo generation and enhanced suppression of human CD4+CD25+ regulatory T cells by retinoic acid. J Immunol, 2009, 183(6): 4119-4126.
21. Fantini MC, Becker C, Monteleone G, et al. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol, 2004, 172(9): 5149-5153.
22. Mucida D, Park YJ, Kim G, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science, 2007, 317(5835): 256-260.
23. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other. Trends Immunol, 2008, 29(9): 429-435.
24. Zhou X, Kong N, Zou H, et al. Therapeutic potential of TGF-β-induced CD4(+) Foxp3(+) regulatory T cells in autoimmune diseases. Autoimmunity, 2011, 44(1): 43-50.
  1. 1. Fan H, Cao P, Game DS, et al. Regulatory T cell therapy for induction of clinical organ transplantation tolerance. Semin Immunol, 2011, 23(6): 453-461.
  2. 2. Sagoo P, Lombardi G, Lechler RI. Relevance of regulatoty T cell promotion of donor-specific tolerance in solid organ transplantation. Front Immunol, 2012, 3: 184.
  3. 3. Dummer CD, Carpio VN, Gonçalves LF, et al. FOXP3+ regulatory T cells: from suppression of rejection to induction of renal allograft tolerance. Transpl Immunol, 2012, 26(1): 1-10.
  4. 4. Sakaguchi S, Yamaguchi T, Numura T, et al. Regulatory T cells and immune tolerance. Cell, 2008, 133(5): 775-787.
  5. 5. Riley JL, June CH, Blazar BR, et al. Human T regulatory cells therapy: take a billion or so and call me in the morning. Immunity, 2009, 30(5): 656-665.
  6. 6. 熊競, 劉克劍, 吳芳新, 等. 健康中國恒河猴各淋巴組織中Ｔ淋巴細(xì)胞表型及分布. 中國畜牧獸醫(yī), 2012, 39(2): 86-91.
  7. 7. 陳磊, 李楠, 王學(xué)廉, 等. 微電極引導(dǎo)偏側(cè)帕金森病猴腦深部電極植入方法的研究. 中國微侵襲神經(jīng)外科雜志, 2012, 17(1): 32-35.
  8. 8. Anderson A, Martens CL, Hendrix R, et al. Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses. Am J Transplant, 2008, 8(11): 2252-2263.
  9. 9. Yan H, Ding CG, Tian PX, et al. Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+CD25+ Treg cells. J Zhejiang Univ Sci B, 2009, 10(12): 928-932.
  10. 10. Bresatz S, Sadlon T, Millard D, et al. Isolation, propagation and characterization of cord blood derived CD4+CD25+ regulatory T cells. J Immunol Methods, 2007, 327(1-2): 53-62.
  11. 11. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor FoxP3. Science, 2003, 299(5609): 1057-1061.
  12. 12. 陳祚珈, 高雅懿, 李志遠(yuǎn), 等. FOXP3+調(diào)節(jié)性T細(xì)胞. 生命科學(xué), 2010, 22(6): 515-528.
  13. 13. 胡義平, 李曉娟, 劉叔文. ConA對CD4+CD25+調(diào)節(jié)性T細(xì)胞早期活化和功能的影響. 細(xì)胞與分子免疫學(xué)雜志, 2010, 26(2): 118-120, 124.
  14. 14. 呂夢捷, 曾耀英, 宋兵. 人參皂苷Rb1 對小鼠T淋巴細(xì)胞體外活化、增殖及凋亡的影響. 中草藥, 2011, 42(4): 743-748.
  15. 15. Shevach EM. Regulatory T cells in autoimmunity. Ann Rev Immunol, 2000, 18: 423-449.
  16. 16. Campbell DJ, Ziegler SF. FOXP3 modifies the phenotypic and functional properties of regulatory T cells. Nat Rev Immunol, 2007, 7(4): 305-310.
  17. 17. Carrigan SO, Yang YJ, Issekutz T, et al. Depletion of natural CD4+CD25+ T regulatory cells with anti-CD25 antibody does not change the course of Pseudomonas aeruginosa-induced acute lung infection in mice. Immunobiology, 2009, 214(3): 211-222.
  18. 18. Dons EM, Raimondi G, Cooper DK, et al. Non-human primate regulatory T cells: current biology and implications for transplantation. Transplantation, 2010, 90(8): 811-816.
  19. 19. Porter CM, Horvath-Arcidiacono JA, Singh AK, et al. Characterization and expansion of baboon CD4+CD25+ Treg cells for potential use in a non-human primate xenotransplantation model. Xenotransplantation, 2007, 14(4): 298-308.
  20. 20. Wang J, Huizinga TW, Toes RE. De novo generation and enhanced suppression of human CD4+CD25+ regulatory T cells by retinoic acid. J Immunol, 2009, 183(6): 4119-4126.
  21. 21. Fantini MC, Becker C, Monteleone G, et al. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol, 2004, 172(9): 5149-5153.
  22. 22. Mucida D, Park YJ, Kim G, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science, 2007, 317(5835): 256-260.
  23. 23. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other. Trends Immunol, 2008, 29(9): 429-435.
  24. 24. Zhou X, Kong N, Zou H, et al. Therapeutic potential of TGF-β-induced CD4(+) Foxp3(+) regulatory T cells in autoimmune diseases. Autoimmunity, 2011, 44(1): 43-50.