唑来膦酸刺激人PBMCs体外扩增γδT细胞及其在肝癌非特异性免疫中的作用
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摘要
第一部分:唑来膦酸刺激人外周血γδT细胞增殖
目的:探讨唑来膦酸刺激人外周血γδT细胞的增殖作用
方法:抽取10例健康捐献者外周血10ml,以密度梯度离心法分离外周血单个核细胞。选用第三代双膦酸盐类药物唑来膦酸作为刺激因子,以不同浓度的唑来膦酸联合IL-2刺激外周血γδT细胞增殖。细胞培养12-14天后,收集细胞,以流式细胞仪检测相关表型, 3H-TdR掺入法测定细胞增殖程度。
结果:培养12-14天后,外周血γδT细胞增殖明显,与对照组相比较,实验组γδT表型比例升高显著(对照组9.7%、实验组80.8%、P<0.01)。CD3升高显著(对照组71.2%、实验组96.2%、P<0.01)。3H-TdR掺入法测定显示实验组细胞增殖显著:(空白对照组CMP值:111.1、实验组CMP值:17488.6、P<0.01)。在所有唑来膦酸浓度中,以1μΜ浓度时,γδT细胞增殖最为明显。
结论:用唑来膦酸能促进人外周血γδT细胞的增殖,得到大量高纯度的γδT细胞。
第二部分:扩增的γδT细胞对人肝癌细胞的杀伤效应
目的:探讨以唑来膦酸刺激增殖的γδT细胞对人肝癌细胞的杀伤作用
方法:同实验一法获取健康人外周血γδT细胞,以γδT细胞作为效应细胞,分别以人肝癌细胞株SMCC-7721、MHCC-97、HepG2作为靶细胞。按照效靶比20:1、10:1共同培养48小时后终止实验,以SRB法测定效应细胞对靶细胞的杀伤效能。
结果:γδT细胞对三种肿瘤细胞均表现出强大的杀伤作用,效靶比20:1时,γδT细胞对肝癌细胞杀伤率最高达98.7%,效靶比10:1时,γδT细胞对肝癌细胞杀伤率最高达92.6%。同一效靶比时,γδT细胞对不同细胞株杀伤作用无显著差异(P>0.05),不同效靶比时,γδT细胞对同一细胞株杀伤作用有显著差异(P<0.05)。
结论:以唑来膦酸刺激扩增的γδT细胞在体外对肝癌细胞有显著的杀伤作用
PartⅠγδT lymphocyte multiplication stimulated by zoledronic acid
Objective To study the effect ofγδT lymphocyte multiplication stimulated by zoledronic acid .
Methods: Take 10ml peripheral blood of healthy adults, obtain peripheral blood mononuclear cell by centrifugation on a Ficoll-Hypaque density gradient. Take zoledronic acid the Third-generation Biphosphonate as the stimulating factor.Exert different concentration zoledronic acid combined with IL-2 to stimulate peripheral bloodγδT cells proliferation. Collecting cells after 12-14 days, analyze correlated phaenotypes by flow cytometry. The quantity of cells was determined by method of 3H-TdR incorporation.
Results: After cultured 12-14 days ,peripheral bloodγδT cells obviously multiplied. Confer with the control group, proportion ofγδT phaenotype in experimental gtoup obviously heightened(control group 9.7%、experimental gtoup 80.8%、P<0.01). proportion of CD3 phaenotype in experimental gtoup also obviously heightened(control group 71.2%、experimental gtoup 96.2%、P<0.01)。Cells in experimental gtoup obviously multiplied determined by method of 3H-TdR incorporation(CMP of control group 9961.5、CMP of experimental gtoup 17488.6、P<0.01).In all of the zoledronic acid concentration,γδT cells multiplied most obviously in 1μΜzoledronic acid.γδT cells.
Conclusions: Peripheral bloodγδT lymphocyte can be stimulated and multiplied by zoledronic acid ,and can obtain quantity of highly purifiedγδTcells.
PartⅡThe cytotoxic activity of the multipliedγδT lymphocyte to hepatoma carcinoma cell.
Objective To investigate the cytotoxic activity ofγδT lymphocyte stimulated and multiplied by zoledronic acid to hepatoma carcinoma cell.
Methods: Obtain peripheral bloodγδT cells of healthy adults by the method of experiment one .TakeγδT cell as effector cell,and hepatoma carcinoma cell(SMCC-7721、MHCC-97、Hep - G2) as target cell. Mixed culture such cells according to the efffector/target cell ratio(E/T 10:1,20:1).Stop experiment after 48 hours. Determine the cytotoxic activity of effector cells to target cells by method of SRB.
Results:γδT cell display powerful cytotoxic activity to the three tumour cells. The percentage of cytotoxin reached 98.7% when E/T was 20:1 and reached 92.6% when E/T was 10:1. The cytotoxic activity ofγδT cell to different tumour cells has no significant deviation in same E/T(P>0.05). The cytotoxic activity ofγδT cell to same tumour cells has significant deviation in different E/T(P<0.05)。
Conclusions: TheγδT lymphocyte stimulated and multiplied by zoledronic acid has significant cytotoxic activity to hepatoma carcinoma cell in vitro.
目的:探讨唑来膦酸刺激人外周血γδT细胞的增殖作用
方法:抽取10例健康捐献者外周血10ml,以密度梯度离心法分离外周血单个核细胞。选用第三代双膦酸盐类药物唑来膦酸作为刺激因子,以不同浓度的唑来膦酸联合IL-2刺激外周血γδT细胞增殖。细胞培养12-14天后,收集细胞,以流式细胞仪检测相关表型, 3H-TdR掺入法测定细胞增殖程度。
结果:培养12-14天后,外周血γδT细胞增殖明显,与对照组相比较,实验组γδT表型比例升高显著(对照组9.7%、实验组80.8%、P<0.01)。CD3升高显著(对照组71.2%、实验组96.2%、P<0.01)。3H-TdR掺入法测定显示实验组细胞增殖显著:(空白对照组CMP值:111.1、实验组CMP值:17488.6、P<0.01)。在所有唑来膦酸浓度中,以1μΜ浓度时,γδT细胞增殖最为明显。
结论:用唑来膦酸能促进人外周血γδT细胞的增殖,得到大量高纯度的γδT细胞。
第二部分:扩增的γδT细胞对人肝癌细胞的杀伤效应
目的:探讨以唑来膦酸刺激增殖的γδT细胞对人肝癌细胞的杀伤作用
方法:同实验一法获取健康人外周血γδT细胞,以γδT细胞作为效应细胞,分别以人肝癌细胞株SMCC-7721、MHCC-97、HepG2作为靶细胞。按照效靶比20:1、10:1共同培养48小时后终止实验,以SRB法测定效应细胞对靶细胞的杀伤效能。
结果:γδT细胞对三种肿瘤细胞均表现出强大的杀伤作用,效靶比20:1时,γδT细胞对肝癌细胞杀伤率最高达98.7%,效靶比10:1时,γδT细胞对肝癌细胞杀伤率最高达92.6%。同一效靶比时,γδT细胞对不同细胞株杀伤作用无显著差异(P>0.05),不同效靶比时,γδT细胞对同一细胞株杀伤作用有显著差异(P<0.05)。
结论:以唑来膦酸刺激扩增的γδT细胞在体外对肝癌细胞有显著的杀伤作用
PartⅠγδT lymphocyte multiplication stimulated by zoledronic acid
Objective To study the effect ofγδT lymphocyte multiplication stimulated by zoledronic acid .
Methods: Take 10ml peripheral blood of healthy adults, obtain peripheral blood mononuclear cell by centrifugation on a Ficoll-Hypaque density gradient. Take zoledronic acid the Third-generation Biphosphonate as the stimulating factor.Exert different concentration zoledronic acid combined with IL-2 to stimulate peripheral bloodγδT cells proliferation. Collecting cells after 12-14 days, analyze correlated phaenotypes by flow cytometry. The quantity of cells was determined by method of 3H-TdR incorporation.
Results: After cultured 12-14 days ,peripheral bloodγδT cells obviously multiplied. Confer with the control group, proportion ofγδT phaenotype in experimental gtoup obviously heightened(control group 9.7%、experimental gtoup 80.8%、P<0.01). proportion of CD3 phaenotype in experimental gtoup also obviously heightened(control group 71.2%、experimental gtoup 96.2%、P<0.01)。Cells in experimental gtoup obviously multiplied determined by method of 3H-TdR incorporation(CMP of control group 9961.5、CMP of experimental gtoup 17488.6、P<0.01).In all of the zoledronic acid concentration,γδT cells multiplied most obviously in 1μΜzoledronic acid.γδT cells.
Conclusions: Peripheral bloodγδT lymphocyte can be stimulated and multiplied by zoledronic acid ,and can obtain quantity of highly purifiedγδTcells.
PartⅡThe cytotoxic activity of the multipliedγδT lymphocyte to hepatoma carcinoma cell.
Objective To investigate the cytotoxic activity ofγδT lymphocyte stimulated and multiplied by zoledronic acid to hepatoma carcinoma cell.
Methods: Obtain peripheral bloodγδT cells of healthy adults by the method of experiment one .TakeγδT cell as effector cell,and hepatoma carcinoma cell(SMCC-7721、MHCC-97、Hep - G2) as target cell. Mixed culture such cells according to the efffector/target cell ratio(E/T 10:1,20:1).Stop experiment after 48 hours. Determine the cytotoxic activity of effector cells to target cells by method of SRB.
Results:γδT cell display powerful cytotoxic activity to the three tumour cells. The percentage of cytotoxin reached 98.7% when E/T was 20:1 and reached 92.6% when E/T was 10:1. The cytotoxic activity ofγδT cell to different tumour cells has no significant deviation in same E/T(P>0.05). The cytotoxic activity ofγδT cell to same tumour cells has significant deviation in different E/T(P<0.05)。
Conclusions: TheγδT lymphocyte stimulated and multiplied by zoledronic acid has significant cytotoxic activity to hepatoma carcinoma cell in vitro.
引文
1. 杨萍萍 γδT 细胞概述 中国医学科学院学报 2003,1:109-111
2. Fisch P,Moris A,Rammensee H-G,et al.Inhibitory MHC class Ⅰ receptors an gammadelta T cells in tumor immunity and autoimmunity.Immunol Today,2000,21: 187-192
3. ZOCCHI MR,FERRARINI M,CASORATI G,et al. T-cell receptor Vδ gene usage by tumor reactive γδ T lymphocytes infiltrating human lung cancer[J].Immunology,1994,81:234-239.
4. Zocchi E. Polvani C. Guida L. Glyoxylic acid prevents NAD+ and NADH depletion in K562 cells cultured at limiting dilution. [J] Biochemical & Biophysical Research Communications. 1990,173:179-185.
5. Eisenreich W, Schwarz M, Cartayrade A, Arigoni D, Zenk MH, Bacher A. The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms. Chem Biol 1998,5:221–233.
6. Ferrick DA,etal.Differential Production Of Interferongamma and interleukin-4 in response to Th1 and Th2 stimulating pathogens by gammadelts T cells in vivo.Nture1995,373:255
7. HsiehB.etal. Evidence for immunoregulation by gammdelta+Tcells.J Immunol 1996,156:232
8. Allison TJ , Garboczi DN. Structure of T cell receptors and recognition of non-peptide antigens. Mol Immunol , 2001 , 38 : 1051-1061.
9. Hubert P,Lang V,Debre P,etal.Tyrosine phosphorylation and recruitment of ZAP 70 to the CD3 TCR complex are defective after CD2 stimulation.[J] Immunol,1996, 157:4322-4332.
10. Schade AE,Levine AD.Lipid raf the terogeneity in human periphera Blood T lymphoblasts:a mechanism for regulating the initiation of TCR signal transduction. JImmunol,2002,168:2233-2239.
11. Mary Poupot,Jean-Jacques Founie. Non-peptide antigens activating human vγ9/vδ2 T Lymphocytes .Jmmunology Letters 2004, 95:129-138
12. Kunzmann, V. Bauer, E. Feurle, J. Weissinger, F. Tony, H P. Wilhelm, M Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 2000 , 96:384
1. 赵温利,李柏青,田志刚.γδT 细胞与肿瘤免疫[J].国外医学 肿瘤学分册,1997,24:149-152.
2. LaadAD,ThomasML,FakihARetal.Human gammadelta T cell recognize heatshock protein 60 on oraltumorcells.IntJCancer,1999,8:709
3. Srivastava PK,Udono H,Blachere NE etal.Heatshock protein transfer Peptides during antigen processing and CTL priming.Immunogenetics 1994,39:934
4. BlinkEJ,TranpaniJA,JansDA. Perforin dependent nuclear targeting of granzymes:acentral role in the nuclear events of granule exocytosis mediate dapoptosis〔J〕.Immunol CellBiol,1999,77:206
5. Sayers TJ. Brooks AD. Ward JM. Hoshino T. Bere WE. Wiegand GW. Kelly JM. Smyth MJ. The restricted expression of granzyme M in human lymphocytes. [J] Journal of Immunology. 2001,166:765-771.
6. Kagi DF,Vignaux B,Ledermann K,etal.Fas and perforin Pathways as major mechanisms of T cell mediated cytotoxicity[J].Science,1994,265:528 -530.
7. Lin T. Brunner T. Tietz B. Madsen J. Bonfoco E. Reaves M. Huflejt M. Green DR. Fas ligand- mediated killing by intestinal intraepithelial lymphocytes. Participation in intestinal graft-versus-host disease. [J] Journal of Clinical Investigation. 1998,101:570-577.
8. 胡建国,侯彦强,陈 勇,等 颗粒外排与 Fas/Fas 配体途径在人 γδT细胞的肿瘤细胞毒活性中的作用 中国肿瘤生物治疗杂志 2001,8:260～263
9. Zamai L. Ahmad M. Bennett IM. Azzoni L. Alnemri ES. Perussia B. Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. [J] Journal of Experimental Medicine. 1998,188:2375-2380,.
10. Born W. Cady C. Jones-Carson J. Mukasa A. Immunoregulatory functions of gamma delta T cells. [J] Advances in Immunology. 1999, 71:77-144.
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14. Smyth MJ,Godfrey DI,Trapani JA.A fresh look at tumor immunosureillance and immunotherapy.Nature Immunol,2001,2:293-299
1. 杨萍萍 γδT 细胞概述 中国医学科学院学报 2003,1:109-111
2. Fisch P,Moris A,Rammensee H-G, etal. Inhibitory MHC class 1 receptors an gammadelta T cells in tumor immunity and autoimmunity.Immunol Today,2000,21: 187-192
3. ZOCCHI MR,FERRARINI M,CASORATI G,et al. T-cell receptor Vδ gene usage by tumor reactive γδ T lymphocytes infiltrating human lung cancer[J].Immunology,1994,81:234～239.
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23. Kunzmann, V. Bauer, E. Feurle, J. Weissinger, F. Tony, H P. Wilhelm, M Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 2000,96(2):384
24. Blink EJ,Tranpani JA,Jans DA.Perfor in dependent nuclear targeting of granzymes: a central role in the nuclear events of granule- exocytosis- mediated apoptosis. Immunol Cell Biol,1999,77:206
25. 胡建国,侯彦强,陈勇等.颗粒外排与 Fas/FasL 配体途径在人 γδT 细胞的肿瘤细胞毒活性中的作用[J].中国肿瘤生物治疗杂志,2000,8:260～262
26. Zamai L. Ahmad M. Bennett IM. Azzoni L. Alnemri ES. Perussia B. Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells[J]. Journal of Experimental Medicine. 1998,188:2375-2380,.
27. Born W. Cady C. Jones-Carson J. Mukasa A. Lahn M. O'Brien R. Immunoregulatory functions of gamma delta T cells. [Review] [J] Advances in Immunology. 1999,71:77-144.
28. Carding SR,Egan PJ.The importance of gamma delta T cells in the resolutiong of pathogen-induced inflammatory immune responses. Immunol Rev.2002,173:98-108
29. Smyth MJ,Godfrey DI,Trapani JA.A fresh look at tumor immunosureillance and immunotherapy[J].Nature Immunol,2001,2:293-299
30. Gording SR,Egan Pj. Gammadelta T Cells:functional plasticity and heterogeneity [J]. Nature Rev Immunol,2002;2:336-345
2. Fisch P,Moris A,Rammensee H-G,et al.Inhibitory MHC class Ⅰ receptors an gammadelta T cells in tumor immunity and autoimmunity.Immunol Today,2000,21: 187-192
3. ZOCCHI MR,FERRARINI M,CASORATI G,et al. T-cell receptor Vδ gene usage by tumor reactive γδ T lymphocytes infiltrating human lung cancer[J].Immunology,1994,81:234-239.
4. Zocchi E. Polvani C. Guida L. Glyoxylic acid prevents NAD+ and NADH depletion in K562 cells cultured at limiting dilution. [J] Biochemical & Biophysical Research Communications. 1990,173:179-185.
5. Eisenreich W, Schwarz M, Cartayrade A, Arigoni D, Zenk MH, Bacher A. The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms. Chem Biol 1998,5:221–233.
6. Ferrick DA,etal.Differential Production Of Interferongamma and interleukin-4 in response to Th1 and Th2 stimulating pathogens by gammadelts T cells in vivo.Nture1995,373:255
7. HsiehB.etal. Evidence for immunoregulation by gammdelta+Tcells.J Immunol 1996,156:232
8. Allison TJ , Garboczi DN. Structure of T cell receptors and recognition of non-peptide antigens. Mol Immunol , 2001 , 38 : 1051-1061.
9. Hubert P,Lang V,Debre P,etal.Tyrosine phosphorylation and recruitment of ZAP 70 to the CD3 TCR complex are defective after CD2 stimulation.[J] Immunol,1996, 157:4322-4332.
10. Schade AE,Levine AD.Lipid raf the terogeneity in human periphera Blood T lymphoblasts:a mechanism for regulating the initiation of TCR signal transduction. JImmunol,2002,168:2233-2239.
11. Mary Poupot,Jean-Jacques Founie. Non-peptide antigens activating human vγ9/vδ2 T Lymphocytes .Jmmunology Letters 2004, 95:129-138
12. Kunzmann, V. Bauer, E. Feurle, J. Weissinger, F. Tony, H P. Wilhelm, M Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 2000 , 96:384
1. 赵温利,李柏青,田志刚.γδT 细胞与肿瘤免疫[J].国外医学 肿瘤学分册,1997,24:149-152.
2. LaadAD,ThomasML,FakihARetal.Human gammadelta T cell recognize heatshock protein 60 on oraltumorcells.IntJCancer,1999,8:709
3. Srivastava PK,Udono H,Blachere NE etal.Heatshock protein transfer Peptides during antigen processing and CTL priming.Immunogenetics 1994,39:934
4. BlinkEJ,TranpaniJA,JansDA. Perforin dependent nuclear targeting of granzymes:acentral role in the nuclear events of granule exocytosis mediate dapoptosis〔J〕.Immunol CellBiol,1999,77:206
5. Sayers TJ. Brooks AD. Ward JM. Hoshino T. Bere WE. Wiegand GW. Kelly JM. Smyth MJ. The restricted expression of granzyme M in human lymphocytes. [J] Journal of Immunology. 2001,166:765-771.
6. Kagi DF,Vignaux B,Ledermann K,etal.Fas and perforin Pathways as major mechanisms of T cell mediated cytotoxicity[J].Science,1994,265:528 -530.
7. Lin T. Brunner T. Tietz B. Madsen J. Bonfoco E. Reaves M. Huflejt M. Green DR. Fas ligand- mediated killing by intestinal intraepithelial lymphocytes. Participation in intestinal graft-versus-host disease. [J] Journal of Clinical Investigation. 1998,101:570-577.
8. 胡建国,侯彦强,陈 勇,等 颗粒外排与 Fas/Fas 配体途径在人 γδT细胞的肿瘤细胞毒活性中的作用 中国肿瘤生物治疗杂志 2001,8:260～263
9. Zamai L. Ahmad M. Bennett IM. Azzoni L. Alnemri ES. Perussia B. Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. [J] Journal of Experimental Medicine. 1998,188:2375-2380,.
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