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

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（dTRAIL）。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（25 μmol/L）、DEX（25 μmol/L）＋UC-MSCs组和DEX（25 μmol/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。

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

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 mesenchymalstem 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 was 85%, 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. Conclusion DEX can significantly improve the efficacy of TRAIL gene modified umbilical cord mesenchymal stem cells in the treatment of acute Blymphocytic leukemia, which may become a potential new strategy for hematologic tumors. The mechanism was related to the significantly increased expression of DR4 and DR5 by DEX and enhanced sensitivity of NALM-6 cells to TRAIL protein.