地塞米松提高TRAIL基因修饰的脐带间充质干细胞对急性B淋巴细胞白血病细胞的杀伤作用及机制研究  

Killing effect of TRAIL gene-modified umbilical cord mesenchymal stem cells enhanced by dexamethasone and its mechanism

作　　者：王立华 李欣 徐立强 苗丽 米一 张晨亮 刘拥军 刘广洋 WANG Lihua;LI Xin;XU Liqiang;MIAO Li;MI Yi;ZHANG Chenliang;LIU Yongjun;LIU Guangyang(Department of Hematology,The Second Hospital of Hebei Medical University,Shijiazhuang 053600,China;Beijing Baylx Biotech Co.,Ltd.,Beijing 100176,China;Stem Cell Biology and Regenerative Medicine Institution,Yi-Chuang Institute of Bio-Industry,Beijing 100176,China)

机构地区：[1]河北医科大学第二医院血液科,河北石家庄053600 [2]北京贝来生物科技有限公司,北京100176 [3]北京市亦创生物技术产业研究院干细胞与再生医学研究所,北京100176

出　　处：《药物评价研究》2021年第8期1653-1659,共7页Drug Evaluation Research

基　　金：北京市科技计划课题(Z211100002521006);河北医科大学第二医院院基金(2h2201609)。

摘　　要：目的构建肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因修饰的脐带间充质干细胞(TRAIL-MSCs),并检测其联合地塞米松(DEX)对急性B淋巴细胞白血病(Nalm-6)细胞的影响。方法通过慢病毒载体将SPD-TRAIL基因转染UC-MSCs构建TRAIL-MSCs,使其能够表达十二聚体TRAIL蛋白(d TRAIL)。通过CCK-8法、流式细胞术、显微镜光镜下观察细胞形态及密度检测UC-MSCs、TRAIL-MSCs、DEX(25μmol/L)、DEX(25μmol/L)+UC-MSCs组和DEX(25μmol/L)+TRAIL-MSCs对Nalm-6细胞增殖、凋亡的影响;通过实时荧光定量PCR及Western blotting法检测各组Nalm-6细胞表面DR4、DR5 mRNA和蛋白表达。结果成功构建了能够稳定表达dTRAIL蛋白的TRAIL-MSCs工作库细胞。与对照组比较,UC-MSCs、TRAIL-MSCs显著抑制Nalm-6细胞增殖(P<0.01),但抑制率均在30%以下,DEX抑制率约为43%,而DEX联合TRAIL-MSCs抑制率达85%,与TRAIL-MSCs和DEX组比较差异显著(P<0.01)。显微镜下观察结果显示,不同处理方式对肿瘤细胞Nalm-6均有一定的杀伤作用,其中以DEX与TRAIL-MSCs联合用药组的杀伤作用最明显。TRAIL-MSCs可以促进Nalm-6细胞凋亡,凋亡率达10%,DEX组凋亡率达到15%,与对照组比较有显著差异(P<0.05、0.01);而DEX+TRAIL-MSCs组凋亡率达36%,与DEX和TRAIL-MSCs组比较有显著差异(P<0.01)。与对照组比较,TRAIL-MSCs组DR4、DR5 mRNA表达量显著降低(P<0.01);DEX可以促进DR4、DR5 mRNA表达,与对照组比较差异显著(P<0.01);DEX+TRAIL-MSCs组较DEX组DR4、DR5 mRNA表达均显著降低(P<0.01);各组蛋白检测结果与mRNA结果基本一致。结论DEX可以显著提高TRAIL-MSCs对Nalm-6细胞的杀伤作用,或将成为一种颇具潜力的血液肿瘤治疗新手段;其机制与DEX显著提高DR4、DR5表达,增强Nalm-6细胞对TRAIL蛋白的敏感性相关。Objective Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)gene modified umbilical cord mesenchymal stem cells(TRAIL-MSCs)were constructed,and the effect of TRAIL-MSCs combined with dexamethasone(DEX)on acute B lymphocytic leukemia(Nalm-6)cells was detected.Methods We transfected SPD-TRAIL gene into umbilical cord mesenchymal stem cells by lentiviral vector to enable them to express TRAIL protein.Then,the effects of DEX combined with TRAIL-MSCs on the proliferation and apoptosis of Nalm-6 were detected by CCK-8 and flow cytometry,respectively.The mRNA and protein levels of DR4 and DR5 on the surface of Nalm-6 cells treated with DEX and TRAIL-MSCs were further detected.Results Compared with control group,UC-MSCs and TRAIL-MSCs significantly inhibited the proliferation of Nalm-6 cells(P<0.01),but the inhibition rate was less than 30%,the inhibition rate of DEX was about 43%,and the inhibition rate of DEX combined with TRAIL-MSCs was85%,compared with TRAIL-MSCS and DEX groups,there was significant difference(P<0.01).The results of microscope observation showed that different treatment methods had certain killing effect on Nalm-6 tumor cells,among which the combination of DEX and TRAIL-MSCs showed the most obvious killing effect.TRAIL-MSCs could promote the apoptosis of Nalm-6 cells,the apoptosis rate was 10%,and the apoptosis rate of DEX group was 15%,which was significantly different from that of control group(P<0.05,0.01).The apoptosis rate of DEX+TRAIL-MSCs group was 36%,which was significantly different from that of Dex and TRAIL-MSCs groups(P<0.01).Compared with control group,the mRNA expression levels of DR4 and DR5 in TRAIL-MSCs group were significantly decreased(P<0.01).DEX could promote the mRNA expression of DR4 and DR5,and the difference was significant compared with control group(P<0.01).The mRNA expression of DR4 and DR5 in DEX+TRAIL-MSCs group was significantly lower than that in DEX group(P<0.01).Protein detection results of each group were basically consistent with mRNA results.Conclusio

关 键 词：脐带间充质干细胞 肿瘤坏死因子相关凋亡诱导配体(TRAIL) 急性B淋巴细胞白血病 联合治疗 死亡受体4/5 

分 类 号：R965[医药卫生—药理学]