结核杆菌抗原诱导活化的人γδT细胞凋亡机制探讨
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摘要
目的 (1)建立结核杆菌抗原(Mtb-Ag)再刺激活化的人γδT细胞凋亡模型；(2)观察不同浓度乙醇和二甲基亚砜(DMSO)致γδT细胞和αβT细胞凋亡及对细胞活性的影响：(3)探讨神经酰胺(C2-Cer)诱导γδT细胞和αβT细胞凋亡作用的不同之处；(4)应用信号分子阻断剂观察PI3K，MAPK和PKC等信号传导途径在Mtb-Ag再刺激诱导γδT细胞凋亡中的作用。
方法 (1)正常人外周血单个核细胞分别用Mtb-Ag和CD3单抗(CD3mAb)刺激并加IL-2培养扩增，分别获得Mtb-Ag激活的T细胞(MtbAT)(γδT细胞为主)和CD3mAb激活的T细胞(CD3AT)(αβT细胞为主)。(2)培养15d～25d的MtbAT用3种浓度Mtb-Ag分别再刺激24h后，应用Annexin-V-FITC／PI染色流式细胞术(FCM)检测γδT细胞凋亡。(3)MtbAT用Mtb-Ag(10.0μg／ml)分别再刺激3h、6h、12h、24h后，检测γδT细胞凋亡情况。(4)用4种浓度乙醇和DMSO分别作用于MtbAT和CD3AT 4h、24h后，应用Annexin-V-FITC／PI染色FCM检测γδT细胞和αβT细胞凋亡，应用MTT比色法分析两类T细胞活性。(5)用4种浓度C2-Cer分别作用于MtbAT和CD3AT 3h后，检测γδT细胞和αβT细胞凋亡及细胞活性。(6)MtbAT预先用LY294002(P13K途径抑制剂)、SB203580(p38途径抑制剂)、Rottlerin(PKC抑制剂)分别处理60min，然后用Mtb-Ag(10.0μg／ml)再刺激3h后，检测γδT细胞凋亡。
结果 (1)MtbAT中γδT细胞比例可达70％～90％，CD3AT中αβT细胞比例＞90％。(2)与对照组相比，5.0μg／ml MtbAg诱导γδT细胞凋亡不显著(P＞0.05)，凋亡
安徽医科大学硕士学位论文
细胞比例仅增加8.05%;10.0协留ml和20.0协岁ml MtbAg均显著诱一导协T细胞凋亡
(P 导协T细胞凋亡方面无显著性差异(P>0.05)。‘3)与对照组相比，’10.0林留ml
Mth一Ag再刺激MthAT3h、6h、12h、24h均可显著诱导协T细胞凋亡(P<0 .01)，
凋亡细胞比例在44.21%一51 .76%之间;10.0卜留ml Mtb一Ag再刺激MthAT3h实验组
与再刺激MtbAT 24h实验组相比，在诱导协T细胞凋亡方面无显著性差异
(P>0.05)，后者的凋亡细胞比例仅比前者增加7.55%。(4)与对照组相比，除
浓度为1.0%及2.0%DMSO作用于apT细胞24h诱导其凋亡显著(P<0.05)及浓
度为2.0%DMSO作用于apT细胞4h对细胞活性抑制显著(P<0 .05)外，低浓度
乙醇及DMsO(三2.0%)诱导协T细胞和。pT细胞凋亡及抑制其细胞活性作用均
不显著(P>0.05);与对照组相比，除5.0%乙醇作用于apT细胞4h及5.0%DMSO
作用于协T细胞4h诱导细胞凋亡不显著(P>0.05)外，高浓度乙醇和DMSO(5 .0%)
诱导协T细胞和apT细胞凋亡及抑制二者细胞活性作用均显著增加(P<0 .05)。
(5)与对照组比较，低浓度CZ一cer(毛10 0 mol/L)诱导协T细胞和apT细胞凋
亡及抑制其细胞活性作用均不显著(P>0.05);高浓度cZ一Cer诱导泌T细胞和apT
细胞凋亡及抑制其细胞活性作用均存在显著差异(P<0.05)，100协mol/L的CZ一Cer
诱导协T细胞、仪pT细胞凋亡比例分别增加104%、P>。.必;及73.5%(P<0.01)，
活细胞区(Rl区)的细胞比例分别为对照组的88.1%(P>.05)和7.8%(P<0.01);
而500 p mol几时诱导协T细胞和a日T细胞凋亡比例分别增加71.9%和73.8%
(P<0 .01)，Rl区的细胞比例分别为对照组的36.5%和2.7%(P<0 .01)。
(6)Mtb一Ag再刺激Mtb灯诱导丫6T细胞凋亡可分别被sBZo35so(50.0林mol/L)
和Rortlerin(40.0林mof几)几乎完全抑制，凋亡抑制率分别为916%及98.6%;而
LY294002(10.0林mol/L)可部分抑制Mth一Ag再刺激诱导的协T细胞凋亡，凋亡抑
制率为43.1%。
结论(l)通过用较高浓度Mth一Ag(10.0林留ml)再刺激活化的Y6T细胞3h后，
可建立Mth一Ag再刺激活化的人Y6T细胞凋亡模型。(2)高浓度乙醇和DMsO
(5 .0%)对协T细胞和apT细胞均有诱导凋亡作用，且对二者活性均有抑制作用。
3
安徽医科大学硕士学位论文
(3)C2一Cer能诱导声i细胞和。pT细胞发生凋亡，诱导协T细胞发生凋亡所需
CZ一Cer浓度明显高于诱导apT细胞所需浓度。(4)信号分子阻断剂的实验证
明，MAPK途径的P38通路，PI3K途径和PKC均参与了Mtb一Ag再刺激己活化协T
细胞凋亡过程。
Objective (1) To establish the model of apoptosis of activated human y8T cells induced by reatimulating with Mycobacterium tuberculosis antigen (Mtb-Ag). (2) To investigate effects of different concentrations of ethanol and dimethyl sulfoxide (DMSO) on the apoptosis and the viability of γδT cells and αβT cells. (3) To investigate the difference of ceramide (C2-Cer) in induction of apoptosis of γδT cells and αβT cells. (4) To investigate the roles of PKC, PI3K and MAPK pathways in the apoptosis of activated human γδT cells induced by restimualting wih Mtb-Ag.
Methods (1) The peripheral blood mononuclear lymphocytes (PBMC) of healthy donors were stimulated with Mtb-Ag and CD3mAb, cultured in IL-2 containing media, to generate Mtb-Ag activated T cells (MtbAT) (γδT cells were predominant) and CD3mAb activated T cells (CD3AT) (αβT cells were predominant) respectively. (2) MtbAT which were cultured about 15d to 25d were restimulated with three concentrations of Mtb-Ag for 24 hours, and the apoptosis of roT cells were measured by flowcytometry (FCM) using Annexin-V-FITC/PI staining. (3) Mtb-AT were restimulating with Mtb-Ag (10ug/ml) for 3 hours, 6 hours, 12 hours, and 24 hours, the apoptosis of γδT cells were detected. (4) MtbAT and CD3AT were treated with four concentrations of ethanol and DMSO for 4 hours or 24 hours, the apoptosis of γδT cells and αβT cells were measured by FCM using Annexin-V-FITC/PI staining, and the
viability of T cells were detected by MTT assay. (5) MtbAT and CD3AT were treated with four concentrations of C2-Cer for 3 hours, the apoptosis of γδT cells and αβT cells were measured, and the viability of T cells was detected. (6) Mtb-AT cells, were pretreated with SB203580 (an inhibitor for P38 pathway), LY294002 (an inhibitor for PI3K pathway) and Rottlerin (an inhibitor for PKC) respectively for 60 minutes, and restimulating with Mtb-Ag for 3 hours, the apoptosis of γδT cells were detected. Results (1) The percentages of γδT cells in MtbAT were from 70% to 90%, and aBT cells in CD3AT were above 90%. (2) The percentages of apoptosis of γδT cells restimulated with Mtb-Ag (5.0ug/ml) only increased 8.05%, appeared to be weak (P>0.05) in contrast to control. But both 10.0ug/ml and 20.0ug/ml Mtb-Ag significantly induced the apoptosis of γδT cells (P<0.01), the percentages of apoptosis of γδT cells increased respectively 35.63% and 38.83%, and there were not apparent difference between them (P>0.05) as compared with control. (3) Compared with control, the apoptosis of γδT cells could be significantly induced by restimulating MtbAT with Mtb-Ag (10.0ug/ml) for 3 hours, 6 hours, 12 hours or 24 hours (P<0.01), the percentages of apoptosis of γδT cells were among 44.21% to 51.76%. There weren't profound difference (P>0.05) in the percentages of apoptosis of γδT cells restimulated by Mtb-Ag (10.0ug/ml) between for 3 hours and for 24 hours, the percentages of apoptosis of the latter is higher than the former about 7.55%. (4) Besides 1.0% and 2.0% DMSO in media for 24 hours induced profoundly apoptosis (P<0.05) on αβT cells, and 2.0% DMSO in media for 4 hours inhibited significantly the viability of αβT cells (P<0.05), the apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by lower concentration of ethanol and DMSO (< 2.0%) appeared to be weak (P>.05) in contrast to control. With the exception of inapparent apoptosis (P>0.05) of αβT cells induced by 5.0% ethanol in media for 4 hours and γδT cells induced by 5.0% DMSO in media for 4 hours, the apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by higher concentration of ethanol and DMSO (5.0%) appeared to be significant increase (P<0.05) as compared with control. (5) The apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by lower concentration
of C2-Cer (< 10umol/L) appeared to be weak (P>. 05) in contrast to control. There were significant differences in the apoptosis of yST cells and αβT cells induced by highe
方法 (1)正常人外周血单个核细胞分别用Mtb-Ag和CD3单抗(CD3mAb)刺激并加IL-2培养扩增，分别获得Mtb-Ag激活的T细胞(MtbAT)(γδT细胞为主)和CD3mAb激活的T细胞(CD3AT)(αβT细胞为主)。(2)培养15d～25d的MtbAT用3种浓度Mtb-Ag分别再刺激24h后，应用Annexin-V-FITC／PI染色流式细胞术(FCM)检测γδT细胞凋亡。(3)MtbAT用Mtb-Ag(10.0μg／ml)分别再刺激3h、6h、12h、24h后，检测γδT细胞凋亡情况。(4)用4种浓度乙醇和DMSO分别作用于MtbAT和CD3AT 4h、24h后，应用Annexin-V-FITC／PI染色FCM检测γδT细胞和αβT细胞凋亡，应用MTT比色法分析两类T细胞活性。(5)用4种浓度C2-Cer分别作用于MtbAT和CD3AT 3h后，检测γδT细胞和αβT细胞凋亡及细胞活性。(6)MtbAT预先用LY294002(P13K途径抑制剂)、SB203580(p38途径抑制剂)、Rottlerin(PKC抑制剂)分别处理60min，然后用Mtb-Ag(10.0μg／ml)再刺激3h后，检测γδT细胞凋亡。
结果 (1)MtbAT中γδT细胞比例可达70％～90％，CD3AT中αβT细胞比例＞90％。(2)与对照组相比，5.0μg／ml MtbAg诱导γδT细胞凋亡不显著(P＞0.05)，凋亡
安徽医科大学硕士学位论文
细胞比例仅增加8.05%;10.0协留ml和20.0协岁ml MtbAg均显著诱一导协T细胞凋亡
(P
Mth一Ag再刺激MthAT3h、6h、12h、24h均可显著诱导协T细胞凋亡(P<0 .01)，
凋亡细胞比例在44.21%一51 .76%之间;10.0卜留ml Mtb一Ag再刺激MthAT3h实验组
与再刺激MtbAT 24h实验组相比，在诱导协T细胞凋亡方面无显著性差异
(P>0.05)，后者的凋亡细胞比例仅比前者增加7.55%。(4)与对照组相比，除
浓度为1.0%及2.0%DMSO作用于apT细胞24h诱导其凋亡显著(P<0.05)及浓
度为2.0%DMSO作用于apT细胞4h对细胞活性抑制显著(P<0 .05)外，低浓度
乙醇及DMsO(三2.0%)诱导协T细胞和。pT细胞凋亡及抑制其细胞活性作用均
不显著(P>0.05);与对照组相比，除5.0%乙醇作用于apT细胞4h及5.0%DMSO
作用于协T细胞4h诱导细胞凋亡不显著(P>0.05)外，高浓度乙醇和DMSO(5 .0%)
诱导协T细胞和apT细胞凋亡及抑制二者细胞活性作用均显著增加(P<0 .05)。
(5)与对照组比较，低浓度CZ一cer(毛10 0 mol/L)诱导协T细胞和apT细胞凋
亡及抑制其细胞活性作用均不显著(P>0.05);高浓度cZ一Cer诱导泌T细胞和apT
细胞凋亡及抑制其细胞活性作用均存在显著差异(P<0.05)，100协mol/L的CZ一Cer
诱导协T细胞、仪pT细胞凋亡比例分别增加104%、P>。.必;及73.5%(P<0.01)，
活细胞区(Rl区)的细胞比例分别为对照组的88.1%(P>.05)和7.8%(P<0.01);
而500 p mol几时诱导协T细胞和a日T细胞凋亡比例分别增加71.9%和73.8%
(P<0 .01)，Rl区的细胞比例分别为对照组的36.5%和2.7%(P<0 .01)。
(6)Mtb一Ag再刺激Mtb灯诱导丫6T细胞凋亡可分别被sBZo35so(50.0林mol/L)
和Rortlerin(40.0林mof几)几乎完全抑制，凋亡抑制率分别为916%及98.6%;而
LY294002(10.0林mol/L)可部分抑制Mth一Ag再刺激诱导的协T细胞凋亡，凋亡抑
制率为43.1%。
结论(l)通过用较高浓度Mth一Ag(10.0林留ml)再刺激活化的Y6T细胞3h后，
可建立Mth一Ag再刺激活化的人Y6T细胞凋亡模型。(2)高浓度乙醇和DMsO
(5 .0%)对协T细胞和apT细胞均有诱导凋亡作用，且对二者活性均有抑制作用。
3
安徽医科大学硕士学位论文
(3)C2一Cer能诱导声i细胞和。pT细胞发生凋亡，诱导协T细胞发生凋亡所需
CZ一Cer浓度明显高于诱导apT细胞所需浓度。(4)信号分子阻断剂的实验证
明，MAPK途径的P38通路，PI3K途径和PKC均参与了Mtb一Ag再刺激己活化协T
细胞凋亡过程。
Objective (1) To establish the model of apoptosis of activated human y8T cells induced by reatimulating with Mycobacterium tuberculosis antigen (Mtb-Ag). (2) To investigate effects of different concentrations of ethanol and dimethyl sulfoxide (DMSO) on the apoptosis and the viability of γδT cells and αβT cells. (3) To investigate the difference of ceramide (C2-Cer) in induction of apoptosis of γδT cells and αβT cells. (4) To investigate the roles of PKC, PI3K and MAPK pathways in the apoptosis of activated human γδT cells induced by restimualting wih Mtb-Ag.
Methods (1) The peripheral blood mononuclear lymphocytes (PBMC) of healthy donors were stimulated with Mtb-Ag and CD3mAb, cultured in IL-2 containing media, to generate Mtb-Ag activated T cells (MtbAT) (γδT cells were predominant) and CD3mAb activated T cells (CD3AT) (αβT cells were predominant) respectively. (2) MtbAT which were cultured about 15d to 25d were restimulated with three concentrations of Mtb-Ag for 24 hours, and the apoptosis of roT cells were measured by flowcytometry (FCM) using Annexin-V-FITC/PI staining. (3) Mtb-AT were restimulating with Mtb-Ag (10ug/ml) for 3 hours, 6 hours, 12 hours, and 24 hours, the apoptosis of γδT cells were detected. (4) MtbAT and CD3AT were treated with four concentrations of ethanol and DMSO for 4 hours or 24 hours, the apoptosis of γδT cells and αβT cells were measured by FCM using Annexin-V-FITC/PI staining, and the
viability of T cells were detected by MTT assay. (5) MtbAT and CD3AT were treated with four concentrations of C2-Cer for 3 hours, the apoptosis of γδT cells and αβT cells were measured, and the viability of T cells was detected. (6) Mtb-AT cells, were pretreated with SB203580 (an inhibitor for P38 pathway), LY294002 (an inhibitor for PI3K pathway) and Rottlerin (an inhibitor for PKC) respectively for 60 minutes, and restimulating with Mtb-Ag for 3 hours, the apoptosis of γδT cells were detected. Results (1) The percentages of γδT cells in MtbAT were from 70% to 90%, and aBT cells in CD3AT were above 90%. (2) The percentages of apoptosis of γδT cells restimulated with Mtb-Ag (5.0ug/ml) only increased 8.05%, appeared to be weak (P>0.05) in contrast to control. But both 10.0ug/ml and 20.0ug/ml Mtb-Ag significantly induced the apoptosis of γδT cells (P<0.01), the percentages of apoptosis of γδT cells increased respectively 35.63% and 38.83%, and there were not apparent difference between them (P>0.05) as compared with control. (3) Compared with control, the apoptosis of γδT cells could be significantly induced by restimulating MtbAT with Mtb-Ag (10.0ug/ml) for 3 hours, 6 hours, 12 hours or 24 hours (P<0.01), the percentages of apoptosis of γδT cells were among 44.21% to 51.76%. There weren't profound difference (P>0.05) in the percentages of apoptosis of γδT cells restimulated by Mtb-Ag (10.0ug/ml) between for 3 hours and for 24 hours, the percentages of apoptosis of the latter is higher than the former about 7.55%. (4) Besides 1.0% and 2.0% DMSO in media for 24 hours induced profoundly apoptosis (P<0.05) on αβT cells, and 2.0% DMSO in media for 4 hours inhibited significantly the viability of αβT cells (P<0.05), the apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by lower concentration of ethanol and DMSO (< 2.0%) appeared to be weak (P>.05) in contrast to control. With the exception of inapparent apoptosis (P>0.05) of αβT cells induced by 5.0% ethanol in media for 4 hours and γδT cells induced by 5.0% DMSO in media for 4 hours, the apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by higher concentration of ethanol and DMSO (5.0%) appeared to be significant increase (P<0.05) as compared with control. (5) The apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by lower concentration
of C2-Cer (< 10umol/L) appeared to be weak (P>. 05) in contrast to control. There were significant differences in the apoptosis of yST cells and αβT cells induced by highe
引文
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32．杜春青，张捷，张尚立，等．二甲基亚砜对血管内皮细胞生长和凋亡的影响．中国现代普通外科进展，2003；6(4)：208～214
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47. Gschwendt M, Muller HJ, Kielbassa K, et al. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun, 1994; 199(1): 93～98
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21．李新燕，张学光，谢伟，等．人gdT细胞识别肿瘤抗原的分子机理研究．上海免疫学杂志，1997；17(4)：210～212
22．闵宏林，宋秀宇，陈勇，等．结核杆菌抗原活化的γδT细胞的抗肿瘤活性．蚌埠医学院学报，2000；25(5)：321～322
23．高学平，宋秀宇，陈勇，等．γδT细胞的细胞毒活性研究．肿瘤，2001；21(2)：94～97
24．李少华．抗原激活的T淋巴细胞凋亡的分子机制．国外医学免疫学分册，
2001; 24(1): 41～44
25. Baisch H, Bollmann H, Bornkessel S. Degradation of apoptotic cells and fragments in HL-60 suspension cultures after induction of apoptosis by camptothecin and ethanol. Cell Prolif, 1999;32(5): 303～319
26. Singh NP, Lai H, Khan A. Ethanol induced single strand DNA breaks in rat brain cells. Mutation Res (Genetic Toxicol ), 1995;345(3,4): 191～196
27. Ewald SJ, Shao H. Ethanol increases apoptotic cell death of thymocytes in vitro. Alc Clin Exp Res, 1993;17(3): 359～365
28. Slukvin Ⅱ, Jerrells TR. Different pathways of in vitro ethanol-induced apoptosis in thymocytes and splenic T and B lymphocytes. Immunopharmacology, 1995; 31(1):43～57
29. Singhal PC, Reddy K, Ding G, et al.Ethanol-induced macrophage apoptosis: the role of TGF-beta. J Immunol, 1999; 162(5): 3 031～3 036
30. Szabo G, Verma B, Catalano D. Selective inhibition of antigen-specific T lymphocyte proliferation by acute ethanol exposure: the role of impaired monocyte antigen presentation capacity and mediator production. Journal of Leukocyte Biology, 1993;54(6): 534～544
31. Slomiany BL, Piotrowski J, Piotrowski E, et al.Induction of buccalmucosal apoptosis with chronic alcohol ingestion. Biochem Mol Biol Int, 1998;44(2): 381～389
32．杜春青，张捷，张尚立，等．二甲基亚砜对血管内皮细胞生长和凋亡的影响．中国现代普通外科进展，2003；6(4)：208～214
33．张覃沐主编．抗肿瘤药物的药理与临床应用．郑州：河南医科大学出版社，1999：361～396
34．汪坤仁，薛绍白，柳惠图主编．细胞生物学．北京：北京师范大学出版社，1998：
35. Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science, 1996;274(13): 1855～1959
36. Pfeilschifler J, Huwiler A. Ceramides as key players in cellular stress response. News Physiol Sci, 2000; 15(2): 11～15
37. Gorak-stolinska P, Truman JP, Kemeny DM, et al. Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme β but is independent of TNF-alpha. J Leukoc Biol, 2001;70:756～766
38. Juo P, Kuo CJ, Yuan J, et al. Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Current Biology, 1998;8:1001～1008
39. Ward SG, Wilson A, Turner L, et al. Inhibition of CD28-mediated T cell costimulation by the phosphoinositide 3-kinase inhibitor wortmannin. Eur J Immunol,1995;25:526～532
40. Edmead CE, Patel YI, Wilson A, et al. Induction of activator protein (AP)-1 and muclear factor-κ B by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomylinase signals. J Immunol. 1996:157: 3290～3297
41．吴俊英，李柏青，侯彦强，等．PI3K在TCR和CD28协同激发的γδT细胞活化信号转导中的作用．中华微生物和免疫学杂志，2003；23(2)：123～126
42. Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem, 1998;67:481～507
43. Brunn, GJ, Williams J, Sabers C, et al. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J, 1996;15:5256～5267
44. Bommhardt U, Basson MA, Krummrei U, et al. Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus. J Immunol, 1999; 163(2): 715～722
45. Whitehurst CE, Geppert TD. MEK1 and the extracellular signal-regulated kinases are required for the stimulation of IL-2 gene transcription in T cells. J Immunol, 1996;156(3):1020～1029
46. Yu TK, Caudell EG, Smid C, et al. IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway. J Immunol, 2000;164(12): 6244～6251
47. Gschwendt M, Muller HJ, Kielbassa K, et al. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun, 1994; 199(1): 93～98