二甲双胍增强放射对CT26WT细胞及小鼠移植瘤效应机制研究  

Study of the mechanism of anti-tumor effect of Metformin-enhanced radiotherapy in CT26WT cell lines or mouse models with transplanted tumors

作　　者：戴夕超 陶累累 方婷婷 陈平 孙海军 吴志峰 戴夕春[3] Dai Xichao;Tao Leilei;Fang Tingting;Chen Ping;Sun Haijun;Wu Zhifeng;Dai Xichun(Department of Oncology,Yancheng First People′s Hospital,Yancheng 224000,China;Intensive Care Unit,Suqian First Hospital,Suqian 223800,China;Department of Oncology,Hongze County people′s Hospital,Huai′an 223001,China)

机构地区：[1]盐城市第一人民医院肿瘤科,224000 [2]宿迁市第一人民医院重症医学科,223800 [3]洪泽县人民医院肿瘤科,淮安223001

出　　处：《中华放射肿瘤学杂志》2020年第3期203-206,共4页Chinese Journal of Radiation Oncology

基　　金：国家自然科学青年基金(81802999)。

摘　　要：目的探讨二甲双胍(Met)联合放射对结肠癌CT26WT细胞及移植瘤的抑制作用及其机制研究。方法利用CellTiter-Glo化学发光细胞活性试验检测0.5、1.0、5.0、10.0μmol/L的Met对CT26WT细胞活力影响,克隆形成试验检测对照组、10.0μmol/L的Met、15Gy照射、15Gy+10.0μmol/L的Met组对CT26WT细胞的增殖抑制作用。构建Bablc小鼠皮下移植瘤模型,肿瘤体积>150mm3随机分对照组、单纯15Gy照射、Met组、15Gy+Met组,照射前24h给予小鼠750 mg/kg的Met,定期测量肿瘤体积及小鼠体重绘制肿瘤生长曲线及生存时间曲线。蛋白质印迹法检测上述处理条件下CT26WT细胞及移植瘤组织中P-H2AX、Sting蛋白表达;并利用免疫组化方法检测移植瘤组织中CD8a(+)T细胞的浸润情况。结果0、0.5、1.0、5.0、10.0μmol/L的Met的相对细胞存活率分别为100%、87.9%、87.8%、87.3%、76.5%(P<0.05),其中10.0μmol/L较5.0μmol/L抑制作用更强(P<0.001)。克隆形成实验结果显示对照组、Met组、15Gy组、15Gy+Met组细胞克隆形成率分别为34.0%、24.0%、22.3%、14.0%(P<0.001)。与对照组比较,Met组、15Gy组、15Gy+Met组细胞内Sting蛋白表达分别增加2.99、1.37、4.41倍(P<0.001、<0.01、<0.001)。15Gy+Met组P-H2AX蛋白表达较15Gy组增加1.43倍(P<0.001)。移植瘤体积15Gy+Met组较对照组生长缓慢,最终结果为(1007.0±388.5)、(2639.0±242.9)mm3,(P<0.05),15Gy+Met组小鼠总生存期较对照组增加(48d︰32d,P<0.001)。移植瘤组织中P-H2AX、Sting蛋白表达量在15Gy+Met组较对照组分别增加8.8、1.6倍(P均<0.001)。15Gy+Met组CD8a(+)T细胞浸润在较对照组明显增高(P<0.01)。结论Met与放射联合能协同抑制结肠癌细胞增殖、克隆形成,可能作用机制是通过加重DNA损伤、激活Sting信号通路导致肿瘤组织中CD8a(+)T细胞增加加强对肿瘤细胞的杀伤作用。Objective To investigate the inhibitory effect and mechanism of Metformin(Met)combined with irradiation in CT26WT cell lines or mouse models with transplanted tumors.Methods CT26WT cell line was treated with 0.5μmol/L,1.0μmol/L,5.0μmol/L and 10.0μmol/L Met,and CellTiter Glo kit was used to detect the inhibitory effect of Met at different concentrations on the viability of CT26WT cells.CT26WT cell line was treated with the control,Met(10μmol/L),15Gy irradiation and 15Gy irradiation+Met(10μmol/L).Clone formation assay was employed to detect the cell proliferation activity.Bablc mouse models of transplanted tumors(tumor size>150 mm3)were established and randomly divided into the control,15Gy irradiation,Met and 15Gy irradiation+Met groups.Mice were given with 750 mg/kg Met at 24 h before irradiation.Transplanted tumor volume was measured regularly to delineate the growth curve of transplanted tumors and survival curve.The expression levels of P-H2AX and Sting proteins in CT26WT cells and transplanted tumors were detected by Western blot.The infiltration of CD8a(+)T cells in transplanted tumor tissues was detected by immunohistochemistry.Results The relative cell survival rate was 100%,87.9%,87.8%,87.3%and 76.5%in the 0,0.5,1.0,5.0 and 10.0μmol/L Met groups,respectively(all P<0.05).The inhibitory effect of 10.0μmol/L was significantly stronger than that of 5.0μmol/L(P<0.001).The colone formation rate 34.0%,24.0%,22.3%and 14.0%in the control,Met,15Gy irradiation,Met+15Gy irradiation groups,respectively(all P<0.001).Western blot showed that compared with the control group,the expression of Sting protein was increased by 2.99-fold after Met treatment(P<0.001),and increased by 1.37-fold and 4.41-fold in the 15Gy irradiation and 15Gy irradiation+Met groups(both P<0.01).Compared with the 15Gy irradiation group,the expression of P-H2AX protein was significantly increased by 1.43 times after treatment with 15Gy+Met(P<0.001).The transplanted tumor growth curve showed that the transplanted tumor growth in the 15Gy+Met

关 键 词：二甲双胍 Sting基因 H2AX基因 CT26WT细胞系 移植瘤/Bablc小鼠 

分 类 号：R735[医药卫生—肿瘤]