高表达SMS1效应性T细胞对卵巢癌细胞凋亡影响机制研究  

Effect of T cell transferred by SMS1 on apoptosis of ovarian cancer cells

作　　者：韩蕾 林昌岫 孙雨鸿 杭月 付云柯 萨帝什 黄成日 HAN Lei;LIN Chang-jriu;SUN Yu-hong;HANG Yue;FU Yun-ke;SA Dis hen;HUANG Cheng-ri(Affiliated Hospital of Yanbian University .Yanbian 133000,P. R. China)

机构地区：[1]延边大学附属医院妇产科,吉林延边133000 [2]延边大学附属医院中心实验室,吉林延边133000

出　　处：《中华肿瘤防治杂志》2019年第10期682-689,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81360382).

摘　　要：目的神经鞘磷脂酶1 (sphingomyelin synthesisl,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SK()V3、()VCAR3、A2780细胞共培养。采用四哇盐(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了干扰素仃nterferon y,IFNy)和II「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,72=8,/= 29. 941,P<0. 001;SMSl 转染组 CD45RO’细胞比例为(31. 23 + 3. 41)%,同样高于对照组皿=8,/=1& 715,P<0. 001o 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),SK()V3 细胞和 OVCAR 细胞 AFP 表达量高于 A2780 细胞皿= 8,F=21. 798,PV0. 001oSMS1转染效应性T细胞对SK()V3、()VCAR3、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. 001o SMS1转染组靶细胞与卵巢癌细胞40 : 1共培养24 h后,培养上清中IFN-y的含量SKOV3细胞为(32. 74±10. 18) ng/mL,()VCAR3 细胞为(34. 49±12. 72) ng/mL, A2780 细胞为(18. 65 土 6. 37) ng/mL,n = 8, F =5.91,PV0.05;另夕卜,培养上清中 IL-2 的含量 SKOV3 细胞为(2 37& 64 + 579. 23) pg/mL,()VCAR3 细胞为(2 057. 85 土492.73) pg/mL,A2780 细胞为(1 OBJECTIVE Sphingomyelin synthesisl (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 interferon y(IFN-y) and IL-2 were detected by ELISA. RESULTS The frecuency of TCRyS+ 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,/= 29. 941, PVO. 001). The frecuency of CD45RO+ 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= 1& 715,PVO. 001). The expression of AFP in A2780 cells was (0. 38dz0. 10), which was lower than that in SKOV3 (0. 72±0. 13) or OVCAR3 cells (0. 75± 0. 14),n = 8, F= 21. 798, PVO. 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-y in SKOV3 cells was (32. 74±10. 18) ng/ml,the supernatant level of IFN-y in OVCAR3 cells was (34. 49士 12. 72) ng/ml,the supernatant level of IFN-y in A2780 cells was (1& 65 ±6. 37) ng/ml, the difference was statistically significant, n = 8, F= 5. 91, PV 0. 05. The supernatant level of IL-2 in SKOV3 cells was (2 37& 64 zb 579. 23) pg/ml,

关 键 词：神经鞘磷脂酶1 卵巢癌 过继性T细胞 免疫逃逸 转染 

分 类 号：R737.31[医药卫生—肿瘤]