目的 研究肿瘤坏死因子相关的凋亡配体（TRAIL）基因修饰间充质干细胞（TRAIL-MSCs）对神经胶质瘤细胞的杀伤作用。方法 复苏冻存的脐带间充质干细胞（UC-MSCs）种子库细胞，通过慢病毒转染的方法制备TRAIL-MSCs。ELISA法检测UC-MSCs、TRAIL-MSCs细胞上清中TRAIL的含量；采用CCK-8试剂盒法检测重组TRAIL蛋白（rTRAIL，0、25、50、100、200、400 ng/mL）对U87MG、U251细胞的增殖抑制情况；实时荧光定量PCR（qRT-PCR）法检测U87MG、U251细胞中死亡受体DR4、DR5 mRNA表达水平，比较2种细胞对TRAIL的敏感性；将U87MG、U251细胞分别分为对照组、UC-MSCs培养上清（阴性对照）组、TRAIL-MSCs培养上清（含TRAIL约100 ng/mL）组和rTRAIL（100 ng/mL）组，CCK-8法检测细胞增殖抑制率；Annexin V-FITC/PI法检测细胞凋亡率；qRT-PCR法检测DR4、DR5 mRNA表达水平。结果 TRAIL-MSCs培养上清中TRAIL表达量显著高于UC-MSCs（P<0.01）；rTRAIL对U87MG细胞的半数抑制浓度（IC₅₀）为162 ng/mL，而对U251细胞的IC₅₀大于800 ng/mL，U87MG对TRAIL的敏感性更高，差异显著（P<0.01）；U87MG细胞DR4、DR5 mRNA相对表达量均显著高于U251细胞（P<0.01）；与对照组比较，TRAIL-MSCs培养上清、rTRAIL对U87MG、U251细胞均有显著增殖抑制及促进凋亡的作用（P<0.05、0.01） ，TRAIL-MSCs培养上清作用效果显著优于rTRAIL（P<0.01）；与U251相比，U87MG对TRAIL-MSCs培养上清的敏感性更强；TRAIL-MSCs培养上清处理的U87MG细胞DR4、DR5 mRNA表达量显著降低（P<0.01）。结论 TRAIL-MSCs对神经胶质瘤细胞有显著增殖抑制和杀伤作用，其功能可能与其受体DR4及DR5有关。

Objective To study the killing effect of tumor necrosis factor related apoptosis ligand (TRAIL) gene modified mesenchymal stem cells (TRAIL-MSCs) on glioma cells. Methods The cryopreserved umbilical cord mesenchymal stem cells (UC MSCs) seed bank cells were resuscitated and TRAIL-MSCs were prepared by lentivirus transfection. The content of TRAIL in the supernatant of UC-MSCs and TRAIL-MSCs was detected by ELISA. CCK-8 kit was used to detect the inhibition of recombinant TRAIL protein (rTRAIL, 0, 25, 50, 100, 200, 400 ng/mL) on the proliferation of U87MG and U251 cells. The mRNA expression levels of death receptors DR4 and DR5 in U87MG and U251 cells were detected by real-time fluorescence quantitative PCR (qRTPCR). U87MG and U251 cells were divided into control group, UC-MSCs culture supernatant (negative control) group, TRAILMSCs culture supernatant (containing TRAIL about 100 ng/mL) group and rTRAIL (100 ng/mL) group respectively. The inhibition rate of cell proliferation was detected by CCK-8 method; Annexin V-FITC/PI method was used to detect apoptosis. The mRNA expression levels of DR4 and DR5 were detected by qRT-PCR. Results The expression of TRAIL in the supernatant of TRAILMSCs was significantly higher than that of UC-MSCs (P<0.01). The IC₅₀ of rTRAIL on U87MG cells was about 162 ng/mL, while that on U251 cells was more than 800 ng/mL. U87MG was more sensitive to trail (P<0.01). The relative expressions of DR4 and DR5 mRNA in U87MG cells were significantly higher than those in U251 cells (P<0.01). Compared with control group, TRAIL-MSCs culture supernatant and rTRAIL significantly inhibited the proliferation and promoted apoptosis of U87MG and U251 cells (P<0.05, 0.01). The effect of TRAIL-MSCs culture supernatant was significantly better than that of rTRAIL (P<0.01). U87MG was more sensitive to the supernatant of TRAIL-MSCs than U251. The expression of DR4 and DR5 mRNA in U87MG cells treated with TRAIL-MSCs culture supernatant decreased significantly (P<0.01). Conclusion TRAIL-MSCs can significantly inhibit the proliferation and kill glioma cells, and its function may be related to DR4 and DR5.

R965

北京市科技计划课题（Z211100002521006）