摘要
目的探讨不同剂量电离辐射对人γδT细胞体外增殖与细胞毒活性的影响及其相关分子机制。方法体外培养扩增人γδT细胞,第10天观察细胞形态特征,流式细胞术检测人γδT细胞百分含量。分别采用0.08、0.50、2.00、4.00、6.00 Gy X射线辐射人γδT细胞作为各辐照剂量组,以0.00 Gy组作为未辐射对照组。继续孵育24、48 h,收集细胞,在倒置相差显微镜下进行活细胞计数,乳酸脱氢酶释放法检测各组人γδT细胞对HepG2细胞的杀伤活性;流式细胞术检测各组穿孔素、颗粒酶B及CD107a的表达。结果经10 d扩增后人γδT细胞百分含量为81.22%±3.51%,经磁珠阳性分选纯化后γδT细胞百分含量为96.76%±1.37%。0.08 Gy剂量组人γδT细胞增殖最为显著,24 h的扩增倍数为1.32±0.07(P<0.05),48 h的扩增倍数为2.22±0.11(P<0.01);辐射后培养24 h的2.00 Gy剂量组、4.00 Gy剂量组人γδT细胞的杀伤活性分别为45.33%±3.22%、42.42%±3.54%,辐射后培养48 h的2.00 Gy剂量组、4.00 Gy剂量组人γδT细胞的杀伤活性分别为47.33%±3.81%、43.85%±3.39%,均显著高于未辐射对照组(分别P<0.01、P<0.05)。2.00 Gy剂量组或4.00 Gy剂量组人γδT细胞在辐照后培养48 h,细胞胞内抗原穿孔素分别为85.88%±3.37%、82.96%±3.55%,均显著高于未辐射对照组(P<0.01、P<0.05);颗粒酶B表达分别为88.30%±4.26%、88.57%±3.92%,均显著高于未辐射对照组(P<0.01);2.00 Gy剂量组人γδT细胞在辐照后培养48 h,细胞表面抗原CD107a表达为90.45%±2.56%,高于未辐射对照组(P<0.01)。结论单次0.08 Gy电离辐射能够显著促进体外人γδT细胞增殖能力,单次2.00 Gy或4.00 Gy电离辐射可显著增强人γδT细胞对肝癌HepG2细胞的肿瘤杀伤活性;其机制可能与电离辐射促进人γδT细胞穿孔素、颗粒酶B及CD107a的表达相关。
Objective To investigate the effect of different doses of ionizing radiation on the proliferation,cytotoxic activity and related molecular mechanisms of humanγδT cells in vitro.Methods HumanγδT cells were expanded in vitro.On day10,the cell morphological characteristics were observed and percentage ofγδT cells were measured by flow cytometry.With0.00 Gy dosage as the irradiation control group,humanγδT cells were exposed to radiation by X-ray doses of 0.08 Gy,0.50 Gy,2.00 Gy,4.00 Gy and 6.00 Gy,respectively.After incubation for 24 hours or 48 hours,the cells were collected and the number of living cells was counted under microscope.Lactate dehydrogenase release method was used to detect the killing activity of humanγδT cells against hepatoma cells(HepG2).The expressions of perforin,granzyme B and CD107a in each group cells were detected by flow cytometry.Results The percentage of humanγδT cells was 81.22%±3.51%after 10 days culture expansion.After positive sorting and purification by magnetic beads,the percentage was 96.76%±1.37%.The proliferation of humanγδT cells in 0.08 Gy radiation dosage group was the most significant.The expansion multiple after 24 hours was1.32±0.07(P<0.05),and the expansion multiple after 48 hours was 2.22±0.11(P<0.01).The killing activities of humanγδT cells after 24 hours radiation culture in 2.00 Gy and 4.00 Gy radiation dose groups against HepG2 were 45.33%±3.22%and42.42%±3.54%,respectively.Meanwhile,the killing activities of humanγδT cells after 48 hours radiation culture in 2.00 Gy and 4.00 Gy radiation dose groups against HepG2 were 47.33%±3.81%and 43.85%±3.39%,respectively.They were both significantly higher than those of the corresponding control group(P<0.01 and P<0.05,respectively).HumanγδT cells in 2.00 Gy dose group or 4.00 Gy dose group were cultured for 48 hours after irradiation,the expressions of intracellular antigen perforin were85.88%±3.37%and 82.96%±3.55%,which were significantly higher than those in control group(P<0.01 and P<0.05,respectively);the expressions of intracellular antigen granzyme B were 88.30%±4.26%and 88.57%±3.92%,which were significantly higher than those in control group(both P<0.01).The 2.00 Gy dose group humanγδT cells cultured for 48 hours after irradiation,the expression of cell surface antigen CD107a was 90.45%±2.56%,which was significantly higher than that in control group(P<0.01).Conclusion A single dose of 0.08 Gy ionizing radiation can significantly promote the proliferation of humanγδT cells in vitro.A single dose of 2.00 Gy or 4.00 Gy ionizing radiation can significantly enhance the tumor-killing activity of humanγδT cells against HepG2 cells in vitro,with the possible mechanism related to ionizing radiation promoting the expression of perforin,granzyme B and CD107a in humanγδT cells.
作者
王路路
周忠海
陈玲
杨阳
陈复兴
孙蕾清
吕小婷
冯守信
WANG Lu-lu;ZHOU Zhong-hai;CHEN Ling;YANG Yang;CHEN Fu-xing;SUN Lei-qing;LÜXiao-ting;FENG Shou-xin(School of Graduate,Xuzhou Medical University,Xuzhou 221004,Jiangsu,China;Department of Medi-cal Imaging,the 71th Group Army Hospital of CPLA Army/the Affiliated Huaihai Hospital of Xuzhou Medical University,Xuzhou 221004,Jiangsu,China;Department of Clinical Laboratory and Pathology,the 71th Group Army Hospital of CPLA Army/the Affiliated Huaihai Hospital of Xuzhou Medical University,Xuzhou 221004,Jiangsu,China;Department of Pharmacy,the 71th Group Army Hospital of CPLA Army/the Affiliated Huaihai Hospital of Xuzhou Medical University,Xuzhou 221004,Jiangsu,China;Gamma Knife Treatment Center,the Affiliated Hospital of Xuzhou Medical University,Xuzhou 221002,Jiangsu,China)
出处
《生物医学工程与临床》
CAS
2022年第6期682-688,共7页
Biomedical Engineering and Clinical Medicine
基金
中国人民解放军原南京军区医学科技创新经费资助项目(15MS045)。
关键词
电离辐射
不同剂量
ΓΔT细胞
贴壁细胞
增殖活性
细胞毒活性
ionizing radiation
different doses
γδT cell
adherent cell
proliferative activity
cytotoxic activity
作者简介
王路路(1986-),女,江苏徐州市人,硕士研究生,主管技师,主要从事肿瘤放射治疗工作。电话:15950662149。E-mail:anglia19861010@163.com;通信作者:冯守信(1962-),男,江苏沛县人,研究生,主任医师,硕导,主要从事体部恶性肿瘤伽马刀治疗及影像诊断研究。电话:18905207889。E-mail:fengshouxin@sina.com。
