摘要
目的探讨脂肪量和肥胖相关蛋白(FTO)及其抑制剂对弥漫大B细胞淋巴瘤(DLBCL)利妥昔单抗耐药的影响。方法选取2种“双重打击”淋巴瘤衍生的细胞系,活化B细胞样(ABC型)OCI-LY8(LY8)和生发中心B细胞样(GCB型)OCI-LY18(LY18),采用梯度加药法构建利妥昔单抗耐药细胞系(LY8R、LY18R);运用CCK-8法检测不同浓度利妥昔单抗分别干扰亲本及耐药细胞后的细胞存活率并计算半数抑制浓度(IC_(50))值;AnnexinⅤ/PI双染法检测亲本及耐药细胞的凋亡率;RT-qRCR、免疫荧光和Western blot检测在耐药细胞株中去甲基化酶FTO的表达情况;在耐药细胞株中加入甲氯芬酸钠(MA)抑制FTO表达,用RT-qRCR和Western blot验证抑制情况,并通过Western blot分析抑制耐药株中FTO后,c-Myc和CEBPA的表达情况。结果与亲本细胞比较,在利妥昔单抗耐药的细胞株中,去甲基化酶FTO、c-Myc表达均增加,CEBPA表达降低,差异均有统计学意义(P<0.01)。耐药细胞株经MA处理后,FTO、c-Myc表达降低,CEBPA表达增高,差异均有统计学意义(P<0.01)。结论FTO在耐药株中表达明显增高,MA可抑制DLBCL耐药细胞中FTO表达,可能对利妥昔单抗耐药的DLBCL患者有治疗的潜能。
Objective To explore the effect of FTO(fat mass and obesity-associated protein)and inhibitor on rituximab resistance in DLBCL(diffuse large B cell lymphoma).Methods Two cell lines derived from“double strike”lymphoma were selected,activated B-cell-like(ABC)OCI-LY8(LY8)and germinal center B-cell-like(GCB)OCI-LY18(LY18),and rituximab-resistant DLBCL cell lines(LY8R and LY18R)were constructed by“concentration gradient dosing method”.The CCK-8 method was used to detect the cell survival rate of different concentrations of rituximab after interfering with the parental and drug-resistant cells,and the half maximal inhibitory concentration(IC_(50))was calculated;AnnexinⅤ/PI double staining method was used to detect the apoptotic rate of parent cells and drug-resistant cells;qRT-RCR,immunofluorescence and Western blot were used to detect the expression of demethylase FTO in rituximab-resistant cells.The expression of FTO was inhibited by adding meclofenamic acid(MA)to drug-resistant cell lines,and the inhibition was verified by RT-qRCR and Western blot.The expression of c-Myc and CEBPA in drug-resistant strains after inhibiting FTO was detected by using Western blot.Results Compared with the parent cells,the expression of demethylase FTO and c-Myc increased,and the expression of CEBPA decreased in the rituximab-resistant cell lines,with statistical significance(P<0.01).After the drug-resistant cell lines treated with MA,the expression of FTO and c-Myc decreased and CEBPA increased,all with statistically significant differences(P<0.01).Conclusion The expression of FTO in drug-resistant DLBCL significantly increases,and MA inhibits the expression of FTO in drug-resistant cells,which may have therapeutic potential for rituximab-resistant DLBCL.
作者
顾季炜
施梅
宋国齐
Gu Jiwei;Shi Mei;Song Guoqi(Medical School of Nantong University,Nantong 226001;Dept of Haematology,Affiliated Hospital of Nantong University,Nantong 226001)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第5期760-765,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81770214)。
