高表达SMS1效应性T细胞对卵巢癌细胞凋亡影响机制研究
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摘要
目的神经鞘磷脂酶1(sphingomyelin synthesis1,SMS1)是催化鞘磷脂生物合成的关键酶之一,与细胞凋亡密切相关,在免疫抗肿瘤研究中较为鲜见。本研究探讨高表达SMS1效应T细胞对卵巢癌细胞增殖和凋亡活性的影响。方法采用密度梯度离心法收集人外周血单核细胞(peripheral blood mononuclear cells,PBMC),体外采用协同抗CD3抗体、CD28抗体、重组白介素2(interleukin 2,IL-2)和重组IL-15刺激活化T细胞。构建pCMV.sport 6-SMS1,并将SMS1基因转染或空质粒至T细胞,分为SMS1转染组和对照组。经甲胎蛋白(alpha-fetal protein,AFP)刺激活化,分别与SKOV3、OVCAR3、A2780细胞共培养。采用四唑盐(methyl thiazolyl tetrazolim,MTT)比色法和流式细胞术检测细胞增殖和凋亡活性。采用ELISA试剂盒检测共培养上清中γ干扰素(interferonγ,IFNγ)和IL-2含量。结果 SMS1基因转染T细胞经肿瘤抗原刺激后充分活化,T细胞受体(T cell receptor,TCR)+细胞比例为(32.83±2.17)%,高于对照组,n=8,t=29.941,P<0.001;SMS1转染组CD45RO+细胞比例为(31.23±3.41)%,同样高于对照组,n=8,t=18.715,P<0.001。qRT-PCR方法检测,AFP表达量SKOV3细胞为(0.72±0.13),OVCAR3细胞为(0.75±0.14),A2780细胞为(0.38±0.10),SKOV3细胞和OVCAR细胞AFP表达量高于A2780细胞,n=8,F=21.798,P<0.001。SMS1转染效应性T细胞对SKOV3、OVCAR3、A2780细胞的杀伤率以及促凋亡率高于对照组;且呈效靶比依赖性。但SMS1转染效应性T细胞对A2780细胞抑制生长的作用和促凋亡作用明显低于SKOV3和OVCAR3细胞,F=189.390,P<0.001。SMS1转染组靶细胞与卵巢癌细胞40∶1共培养24h后,培养上清中IFN-γ的含量SKOV3细胞为(32.74±10.18)ng/mL,OVCAR3细胞为(34.49±12.72)ng/mL,A2780细胞为(18.65±6.37)ng/mL,n=8,F=5.91,P<0.05;另外,培养上清中IL-2的含量SKOV3细胞为(2 378.64±579.23)pg/mL,OVCAR3细胞为(2 057.85±492.73)pg/mL,A2780细胞为(1 274.68±242.14)pg/mL,n=8,F=7.91,P<0.05。结论上调SMS1基因表达后可诱导T细胞分化并识别肿瘤抗原,从而促进卵巢癌细胞的凋亡。
OBJECTIVE Sphingomyelin synthesis1(SMS1),the last key enzyme in the sphingomyelin biosynthetic pathway,is closely related with apoptosis of cells.There is almost none research about effect of SMS1 on anti-tumor immunity.The present study aimed to investigate the effect and mechanism of T cell transferred by SMS1 on apoptosis of ovarian cancer cells SKOV3,OVCAR3 and A2780.METHODS Human peripheral monocytes(PBMC)were isolated by Ficoll-plaque plus density gradient centrifugation of buffy coats from healthy donors,and activated by anti-CD3,anti-CD28,rIL-2 and rIL-15.T cells transferred by SMS1 were co-cultured with SKOV3,OVCAR3 or A2780,respectively.The proliferation and apoptosis of target cells was detected by MTT or FCM.The supernatant levels of interferonγ(IFN-γ)and IL-2 were detected by ELISA.RESULTS The frecuency of TCRγδ+ T cells in SMS1 transferred T cells after activated by stitumulation of tumor antigens AFP was(32.83±2.17)%,and higher than that in control group(n=8,t=29.941,P<0.001).The frecuency of CD45 RO+cells in SMS1 transferred T cells after activated by stitumulation of tumor antigens AFP was(31.23±3.41)%,and higher than that in control group(n=8,t=18.715,P<0.001).The expression of AFP in A2780 cells was(0.38±0.10),which was lower than that in SKOV3(0.72±0.13)or OVCAR3 cells(0.75±0.14),n=8,F=21.798,P<0.001.The effect of SMS1 transferred T cells on proliferation and apoptosis of SKOV3,OVCAR3 and A2780 cells was higher than that of the control group(F=189.390,P<0.001),which was effect-target ratio-independent.After co-culturing with SMS1 transferred T cells for 24 h,the supernatant level of IFN-γin SKOV3 cells was(32.74±10.18)ng/ml,the supernatant level of IFN-γin OVCAR3 cells was(34.49±12.72)ng/ml,the supernatant level of IFN-γin A2780 cells was(18.65±6.37)ng/ml,the difference was statistically significant,n=8,F=5.91,P<0.05.The supernatant level of IL-2 in SKOV3 cells was(2 378.64±579.23)pg/ml,the supernatant level of IL-2 in OVCAR3 cells was(2 057.85±492.73)pg/ml,the supernatant level of IL-2 in A2780 cells was(1 274.68±242.14)pg/ml,the difference was statistically significant,n=8,F=7.91,P<0.05.CONCLUSION It's suggested that the tumor antigen identification of SMS1 transferred T cells can be stronger in order to enhance anti-tumor immunity.
OBJECTIVE Sphingomyelin synthesis1(SMS1),the last key enzyme in the sphingomyelin biosynthetic pathway,is closely related with apoptosis of cells.There is almost none research about effect of SMS1 on anti-tumor immunity.The present study aimed to investigate the effect and mechanism of T cell transferred by SMS1 on apoptosis of ovarian cancer cells SKOV3,OVCAR3 and A2780.METHODS Human peripheral monocytes(PBMC)were isolated by Ficoll-plaque plus density gradient centrifugation of buffy coats from healthy donors,and activated by anti-CD3,anti-CD28,rIL-2 and rIL-15.T cells transferred by SMS1 were co-cultured with SKOV3,OVCAR3 or A2780,respectively.The proliferation and apoptosis of target cells was detected by MTT or FCM.The supernatant levels of interferonγ(IFN-γ)and IL-2 were detected by ELISA.RESULTS The frecuency of TCRγδ+ T cells in SMS1 transferred T cells after activated by stitumulation of tumor antigens AFP was(32.83±2.17)%,and higher than that in control group(n=8,t=29.941,P<0.001).The frecuency of CD45 RO+cells in SMS1 transferred T cells after activated by stitumulation of tumor antigens AFP was(31.23±3.41)%,and higher than that in control group(n=8,t=18.715,P<0.001).The expression of AFP in A2780 cells was(0.38±0.10),which was lower than that in SKOV3(0.72±0.13)or OVCAR3 cells(0.75±0.14),n=8,F=21.798,P<0.001.The effect of SMS1 transferred T cells on proliferation and apoptosis of SKOV3,OVCAR3 and A2780 cells was higher than that of the control group(F=189.390,P<0.001),which was effect-target ratio-independent.After co-culturing with SMS1 transferred T cells for 24 h,the supernatant level of IFN-γin SKOV3 cells was(32.74±10.18)ng/ml,the supernatant level of IFN-γin OVCAR3 cells was(34.49±12.72)ng/ml,the supernatant level of IFN-γin A2780 cells was(18.65±6.37)ng/ml,the difference was statistically significant,n=8,F=5.91,P<0.05.The supernatant level of IL-2 in SKOV3 cells was(2 378.64±579.23)pg/ml,the supernatant level of IL-2 in OVCAR3 cells was(2 057.85±492.73)pg/ml,the supernatant level of IL-2 in A2780 cells was(1 274.68±242.14)pg/ml,the difference was statistically significant,n=8,F=7.91,P<0.05.CONCLUSION It's suggested that the tumor antigen identification of SMS1 transferred T cells can be stronger in order to enhance anti-tumor immunity.
引文
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[14] Simons K,Ikonen E.Functional rafts in cell membranes[J].Nature,1997,387(6633):569-572.
[15]黄成日.脂质筏神经鞘磷脂在T细胞活化中的作用[J].中国免疫学杂志,2014,30(11):1574-1577.
[16] Balagopalan L,Ashwell BA,Bernot KM,et al.Enhanced T-cell signaling in cells bearing linker for activation of T-cell(LAT)molecules resistant to ubiquitylation[J].Proc Natl Acad Sci,2011,108(7):2885-2890.
[17]熊艳,任玉萍,刘小彭,等.酸性鞘磷脂酶对免疫细胞的调节及与疾病的关系[J].细胞与分子免疫学杂志,2016,32(7):1003-1006.
[18] Wu S,Chen X,Tang Y,et al.Delivery of Tapasin-modified CTL epitope peptide via cytoplasmic transduction peptide induces CTLs by JAK/STAT signaling pathway in vivo[J].Acta Biochim Biophys Sin(Shanghai),2018,50(2):181-190.
[19] Kapnick SM,Stinchcombe JC,Griffiths GM,et al.Inducible T Cell kinase regulates the acquisition of cytolytic capacity and degranulation in CD8+CTLs[J].J Immunol,2017,198(7):2699-2711.
[20] Thwin MM,Satyanarayanajois DS,Nagarajarao LM,et al.Novel peptide inhibitors of human secretory phospholipase A2with antiinflammatory activity:solution structure and molecular modeling[J].J Med.Chem,2007,50(25):5938-5950.
[21]石渊渊,李志强,谷敬丽,等.神经鞘磷脂合成酶基因沉默对细胞凋亡的影响[J].高等学校化学学报,2009,30(9):1759-1762.
[22] van der Luit AH,Budde M,Ruurs P,et al.Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[J].J Biol Chem,2002,277(42):39541-39547.
[2]卢淮武,谢玲玲,林仲秋.《2016NCCN卵巢癌临床实践指南(第1版)》解读[J].中国实用妇科与产科杂志,2015,31(8):761-768.
[3]王欣彦,杜振华,刘宁,等.“肿瘤生物治疗新进展——暨2016卵巢癌规范化、个体化、精准治疗高峰论坛”会议纪要[J].中国实用妇科与产科杂志,2016,32(11):1134-1139.
[4] Levental I,Grzybek M,Simons K.Raft domains of variable properties and compositions in plasma membrane vesicles[J].Proc Natl Acad Sci U S A,2001,108(28):11411-11416.
[5]赵瑾超,宋健,常旦琪,等.低密度脂蛋白诱导巨噬细胞Raw264.7脂质筏的聚集[J].武汉大学学报:医学版,2017,38(2):178-181,206.
[6] Wertel I,Okla K,Surówka J,et al.Why ovarian cancer cells escape from immune surveillance[J].Wiad Lek,2017,70(1):74-80.
[7] Odunsi K.Immunotherapy in ovarian cancer[J].Ann Oncol,2017,28(suppl_8):viii1-viii7.
[8] Bose CK.Immune checkpoints,their control by immunotherapy and ovarian cancer[J].Contemp Oncol/(Pozn),2017,21(3):189-196.
[9]王雄彪.TIL特异抗癌活性的研究[J].免疫学杂志,1993,5(2):133-136.
[10] de Wolf C,van de Bovenkamp M,Hoefnagel M.Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy[J].Cytotherapy,2018,20(5):601-622.
[11] Hughes E,Scurr M,Campbell E,et al.T-cell modulation by cyclophosphamide for tumour therapy[J].Immunology,2018,154(1):62-68.
[12] Singh N,Shi J,June CH,et al.Genome-editing technologies in adoptive T cell immunotherapy for cancer[J].Curr Hematol Malig Rep,2017,12(6):522-529.
[13] Nicolson GL,Schmidt GL.Structure of the Chromatium sulfur particle and its protein membrane[J].J Bacteriol,1971,105(3):1142-1148.
[14] Simons K,Ikonen E.Functional rafts in cell membranes[J].Nature,1997,387(6633):569-572.
[15]黄成日.脂质筏神经鞘磷脂在T细胞活化中的作用[J].中国免疫学杂志,2014,30(11):1574-1577.
[16] Balagopalan L,Ashwell BA,Bernot KM,et al.Enhanced T-cell signaling in cells bearing linker for activation of T-cell(LAT)molecules resistant to ubiquitylation[J].Proc Natl Acad Sci,2011,108(7):2885-2890.
[17]熊艳,任玉萍,刘小彭,等.酸性鞘磷脂酶对免疫细胞的调节及与疾病的关系[J].细胞与分子免疫学杂志,2016,32(7):1003-1006.
[18] Wu S,Chen X,Tang Y,et al.Delivery of Tapasin-modified CTL epitope peptide via cytoplasmic transduction peptide induces CTLs by JAK/STAT signaling pathway in vivo[J].Acta Biochim Biophys Sin(Shanghai),2018,50(2):181-190.
[19] Kapnick SM,Stinchcombe JC,Griffiths GM,et al.Inducible T Cell kinase regulates the acquisition of cytolytic capacity and degranulation in CD8+CTLs[J].J Immunol,2017,198(7):2699-2711.
[20] Thwin MM,Satyanarayanajois DS,Nagarajarao LM,et al.Novel peptide inhibitors of human secretory phospholipase A2with antiinflammatory activity:solution structure and molecular modeling[J].J Med.Chem,2007,50(25):5938-5950.
[21]石渊渊,李志强,谷敬丽,等.神经鞘磷脂合成酶基因沉默对细胞凋亡的影响[J].高等学校化学学报,2009,30(9):1759-1762.
[22] van der Luit AH,Budde M,Ruurs P,et al.Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis[J].J Biol Chem,2002,277(42):39541-39547.