目的 研究腫瘤壞死因子相關(guān)的凋亡配體（TRAIL）基因修飾間充質(zhì)干細胞（TRAIL-MSCs）對神經(jīng)膠質(zhì)瘤細胞的殺傷作用。方法 復(fù)蘇凍存的臍帶間充質(zhì)干細胞（UC-MSCs）種子庫細胞，通過慢病毒轉(zhuǎn)染的方法制備TRAIL-MSCs。ELISA法檢測UC-MSCs、TRAIL-MSCs細胞上清中TRAIL的含量；采用CCK-8試劑盒法檢測重組TRAIL蛋白（rTRAIL，0、25、50、100、200、400 ng/mL）對U87MG、U251細胞的增殖抑制情況；實時熒光定量PCR（qRT-PCR）法檢測U87MG、U251細胞中死亡受體DR4、DR5 mRNA表達水平，比較2種細胞對TRAIL的敏感性；將U87MG、U251細胞分別分為對照組、UC-MSCs培養(yǎng)上清（陰性對照）組、TRAIL-MSCs培養(yǎng)上清（含TRAIL約100 ng/mL）組和rTRAIL（100 ng/mL）組，CCK-8法檢測細胞增殖抑制率；Annexin V-FITC/PI法檢測細胞凋亡率；qRT-PCR法檢測DR4、DR5 mRNA表達水平。結(jié)果 TRAIL-MSCs培養(yǎng)上清中TRAIL表達量顯著高于UC-MSCs（P<0.01）；rTRAIL對U87MG細胞的半數(shù)抑制濃度（IC₅₀）為162 ng/mL，而對U251細胞的IC₅₀大于800 ng/mL，U87MG對TRAIL的敏感性更高，差異顯著（P<0.01）；U87MG細胞DR4、DR5 mRNA相對表達量均顯著高于U251細胞（P<0.01）；與對照組比較，TRAIL-MSCs培養(yǎng)上清、rTRAIL對U87MG、U251細胞均有顯著增殖抑制及促進凋亡的作用（P<0.05、0.01） ，TRAIL-MSCs培養(yǎng)上清作用效果顯著優(yōu)于rTRAIL（P<0.01）；與U251相比，U87MG對TRAIL-MSCs培養(yǎng)上清的敏感性更強；TRAIL-MSCs培養(yǎng)上清處理的U87MG細胞DR4、DR5 mRNA表達量顯著降低（P<0.01）。結(jié)論 TRAIL-MSCs對神經(jīng)膠質(zhì)瘤細胞有顯著增殖抑制和殺傷作用，其功能可能與其受體DR4及DR5有關(guān)。

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

北京市科技計劃課題（Z211100002521006）