TRAIL基因修饰间充质干细胞对神经胶质瘤细胞杀伤作用研究

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。

Killing effect of TRAIL gene modified mesenchymal stem cells on glioma cells

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.