恒河猴外周血調節性T淋巴細胞的分離純化及鑒定_《中國修復重建外科雜志》

作者：

張爽 ¹ , 金熙 ¹ , 程漢佳 ² , 何斯榮 ¹ , 陳又南 ¹ , 龍丹 ¹ , 單娟 ¹ , 曾力 ¹ , 王成世 ¹ , 程驚秋 ¹ , 陸燕蓉 ¹

1四川大學華西醫院衛生部移植科學與移植免疫重點實驗室（成都，610041）;;
2四川大學生命科學學院;

關鍵詞：

調節性T淋巴細胞效應性T淋巴細胞免疫磁珠分選淋巴細胞分離恒河猴

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目的建立恒河猴外周血CD4+CD25+調節性T淋巴細胞（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記的混合抗體和磁珠標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記的抗APC抗體陽性分選出 CD4+ T淋巴細胞，比較兩種方法獲得細胞的得率、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淋巴細胞，用磁珠標記的抗人CD25抗體進行陽性分選，獲得 CD4+CD25+ Tregs，流式細胞儀檢測純度、活性及FoxP3表達水平，以及Tregs對刀豆蛋白（concanavalin A，ConA）刺激的自體CD4+CD25-效應性T淋巴細胞（effective T cells，Teffs）增殖的抑制功能。結果CD4+ T淋巴細胞陽性分選和陰性分選后，細胞活性均達95%左右，差異無統計學意義（P gt; 0.05），但陽性分選后CD4+ T淋巴細胞得率和純度均顯著高于陰性分選（P lt; 0.05）。后續CD4+CD25+ Tregs分選采用陽性分選法獲得的CD4+ T淋巴細胞。經雙陽性分選收集的目的細胞中CD4+CD25+ Tregs占76.2% ± 8.6%，活細胞比例為93.3% ± 4.7%，FoxP3陽性細胞比例為74.2% ± 6.9%。混合培養后Tregs均對ConA刺激的Teffs的增殖有抑制作用。結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. 恒河猴外周血調節性T淋巴細胞的分離純化及鑒定. 中國修復重建外科雜志, 2012, 26(10): 1237-1241. doi: 復制

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淋巴細胞表型及分布. 中國畜牧獸醫, 2012, 39(2): 86-91.
7.	陳磊, 李楠, 王學廉, 等. 微電極引導偏側帕金森病猴腦深部電極植入方法的研究. 中國微侵襲神經外科雜志, 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, 等. FOXP3+調節性T細胞. 生命科學, 2010, 22(6): 515-528.
13.	胡義平, 李曉娟, 劉叔文. ConA對CD4+CD25+調節性T細胞早期活化和功能的影響. 細胞與分子免疫學雜志, 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. 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淋巴細胞表型及分布. 中國畜牧獸醫, 2012, 39(2): 86-91.
7. 陳磊, 李楠, 王學廉, 等. 微電極引導偏側帕金森病猴腦深部電極植入方法的研究. 中國微侵襲神經外科雜志, 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, 等. FOXP3+調節性T細胞. 生命科學, 2010, 22(6): 515-528.
13. 胡義平, 李曉娟, 劉叔文. ConA對CD4+CD25+調節性T細胞早期活化和功能的影響. 細胞與分子免疫學雜志, 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.

《中國修復重建外科雜志》

恒河猴外周血調節性T淋巴細胞的分離純化及鑒定

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《中國修復重建外科雜志》

恒河猴外周血調節性T淋巴細胞的分離純化及鑒定

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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