地塞米松提高TRAIL基因修饰脐带间充质干细胞对急性T淋巴细胞白血病细胞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 on acute T lymphoblastic leukemia

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T淋巴细胞白血病细胞系(Jurkat)的影响。方法通过慢病毒载体将SPD-TRAIL基因转染脐带间充质干细胞(UC-MSCs)构建TRAIL-MSCs,使其能够表达十二聚体TRAIL蛋白(dTRAIL)。通过CCK-8法、流式细胞术检测UC-MSCs、TRAIL-MSCs、DEX(200μmol/L)、DEX(200μmol/L)+UC-MSCs和DEX(200μmol/L)+TRAIL-MSCs对Jurkat细胞增殖、凋亡的影响;显微镜光镜下观察细胞形态及密度;通过实时荧光定量PCR及Western blotting法检测各组Jurkat细胞表面死亡受体DR4、DR5 mRNA和蛋白表达水平。结果CCK-8结果显示,各处理组均可以抑制Jurkat细胞的增殖,与对照组比较有显著性差异(P<0.01);同时,DEX可以提高TRAIL-MSCs(抑制率约20%)对Jurkat细胞的增殖抑制作用,抑制率提高至60%,与TRAIL-MSCs组比较差异显著(P<0.01)。显微镜观察结果发现,DEX与TRAILMSCs联合组对肿瘤细胞Jurkat的杀伤作用最明显;流式细胞术结果显示,TRAIL-MSCs对Jurkat细胞有促进凋亡的作用,凋亡率达10%,单独使用DEX后,凋亡率约20%,与对照组比较差异显著(P<0.01);DEX联合TRAIL-MSCs作用于Jurkat细胞48 h后,细胞凋亡率为38%,较TRAIL-MSCs组显著提高(P<0.01)。TRAIL-MSCs组DR4、DR5的mRNA、蛋白水平较对照组显著降低(P<0.01),DEX组DR4、DR5的mRNA、蛋白水平较对照组显著升高(P<0.01),DEX与TRAIL-MSCs联合作用后,DR4、DR5的mRNA、蛋白水平较DEX组显著降低(P<0.01)。结论DEX可以提高肿瘤细胞Jurkat对TRAILMSCs的敏感性,增强TRAIL-MSCs对肿瘤细胞的杀伤作用,可能与提高DR4、DR5表达有关。

Objective Genetically modified mesenchymal stem cells(TRAIL-MSCs)expressing TRAIL were constructed and combined with dexamethasone(DEX)to detect the effect on acute T lymphoblastic leukemia cell line(Jurkat).Methods The proliferation inhibition,apoptosis,cell density and morphology of Jurkat cells induced by DEX(200μmol/L)combined with TRAILMSCs were detected by CCK-8,flow cytometry and microscope observation.At the same time,qRT-PCR and Western blotting techniques were used to detect the expression of DR4 and DR5 in Jurkat cells at nucleic acid level and protein level.Results The results of CCK-8 showed that TRAIL-MSCs could inhibit the proliferation of tumor cells,with an inhibition rate of about 20%,which was significantly different from that of the control group(P<0.01).DEX could improve the inhibitory effect of TRAILMSCs on the proliferation of tumor cell Jurkat,and the inhibition rate was increased to 60%.Compared with TRAIL-MSCs group,the difference was significant(P<0.01).The results of microscopic observation showed that the killing effect of DEX combined with TRAIL-MSCs on tumor cell Jurkat was the most obvious.The results of apoptosis detected by flow cytometry showed that TRAIL-MSCs could promote the apoptosis of tumor cell Jurkat,but after the combination of DEX and TRAIL-MSCs,the apoptosis rate increased from 10%to 38%,which was significantly different from that of the control group and DEX group(P<0.01).The effects of different treatments on the nucleic acid level and protein level of DR4 and DR5 in Jurkat cells were detected by qPCR and WB respectively.The results showed that the expression of DR4 and DR5 in TRAIL-MSCs group was significantly lower than that in control group(P<0.01).DEX can promote the expression of DR4 and DR5,which is significantly different from that of the control group(P<0.01).After the treatment of DEX and TRAIL-MSCs,the expression of DR4 and DR5 was significantly lower than that of DEX group(P<0.01).Conclusion DEX can enhance the sensitivity of tumor cell Jurkat to TRAIL-MSCs and enhance the killing effect of TRAIL-MSCs on tumor cells,which may be related to the increase of DR4 and DR5 expression in tumor cells by DEX.