目的 研究TRAIL基因修饰的脐带间充质干细胞（TRAIL-MSCs）联合化疗药5-氟尿嘧啶（5-FU）对人脑胶质瘤细胞U-87MG、人肺癌细胞A549和人宫颈癌细胞HeLa的杀伤作用。方法 通过基因工程手段结合慢病毒转染体系构建高表达十二聚体TRAIL蛋白（dTRAIL）的TRAIL-MSCs，应用流式细胞技术检测TRAIL-MSCs的生物学特性；通过ELISA技术检测TRAIL-MSCs分泌的TRAIL量来评估转染效果；CCK-8法检测低浓度（0、1、2、4、8、16 μg/mL）5-FU对U-87MG、A549、HeLa细胞的增殖抑制率，并计算其对各细胞的半数抑制浓度（IC₅₀）；CCK-8法检测5-FU（IC₅₀）、UC-MSCs培养上清、TRAIL-MSCs培养上清、5-FU（IC₅₀）＋UC-MSCs培养上清和5-FU（1/4 IC₅₀、1/2 IC₅₀和IC₅₀）＋TRAIL-MSCs培养上清对U-87MG、A549、HeLa细胞的增殖抑制率，计算5-FU和TRAIL-MSCs培养上清的相互作用系数（CDI）；Westernblotting法检测5-FU对U-87MG、A549、HeLa细胞死亡受体DR4、DR5蛋白表达的影响；台盼蓝染色法观察5-FU、UC-MSCs、TRAIL-MSCs、5-FU＋UC-MSCs和5-FU＋TRAIL-MSCs对U-87MG、A549、HeLa细胞的杀伤作用。结果 生物学检测结果表明TRAIL-MSCs在维持UC-MSCs生物学特性的同时能够高表达TRAIL蛋白。5-FU对U-87MG、A549、HeLa细胞均有增殖抑制作用，抑制作用由高到底依次为HeLa细胞（IC₅₀为9.15 μg/mL） >A549细胞（IC₅₀为10.62 μg/mL） >U-87MG细胞（IC₅₀为22.37 μg/mL）。与对照组比较，除A549细胞UC-MSCs培养上清组外，各处理组细胞增殖抑制率均显著升高（P<0.01、0.001）；与相应各5-FUIC₅₀及TRAIL-MSCs培养上清组比较，U-87MG、HeLa细胞5-FU （1/4 IC₅₀、1/2 IC₅₀和IC₅₀） ＋TRAIL-MSCs培养上清组细胞增殖抑制率均显著升高（P<0.001），A549细胞5-FU （IC₅₀） ＋TRAIL-MSCs培养上清组细胞增殖抑制率显著升高（P<0.01、0.001）；5-FU （1/2 IC₅₀） ＋TRAIL-MSCs培养上清组对U-87MG的细胞增殖抑制协同效应最显著（CDI<0.7且P<0.001）； 5-FU （1/4 IC₅₀） ＋TRAIL-MSCs培养上清组对A549的细胞增殖抑制具有协同作用，但其协同效果并不显著； 5-FU （1/4 IC₅₀） ＋TRAIL-MSCs培养上清组对HeLa细胞增殖抑制的协同效果最显著（CDI<0.7且P<0.01）。经5-FU处理后U-87MG、A549、HeLa细胞DR4和DR5的蛋白表达明显升高，其中DR5的表达水平高于DR4，与对照组比较差异均具有统计学意义（P<0.001）。与对照组比较，5-FU、TRAIL-MSCs和5-FU＋TRAIL-MSCs组对于肿瘤细胞U-87MG、A549、HeLa均具显著的杀伤作用（P<0.001）；与5-FU或TRAIL-MSCs组比较，5-FU＋TRAIL-MSCs组的杀伤效果更显著（P<0.001）。结论 TRAIL-MSCs联合低浓度的5-FU对肿瘤细胞U-87MG、A549和HeLa具有显著的细胞杀伤作用，二者联用协同效果显著，机制可能与5-FU提高DR4、DR5蛋白表达、提高肿瘤细胞对TRAIL-MSCs的敏感性相关。

Objective Investigation on the therapeutic effects of genetically modified umbilical cord mesenchymal stem cells (TRAIL-MSCs) combined with chemotherapy drugs 5-fluorouracil (5-FU) in tumor cells U-87MG, A549 and HeLa. Methods The TRAIL-MSCs were constructed by genetic engineering combined with lentivirus transfection system. The biological characteristics of TRAIL-MSCs were detected by flow cytometry. The expression of TRAIL was detected by ELISA technique to evaluate the transfection effect. CCK-8 method was used to detect the proliferation inhibition rate of U-87MG, A549 and HeLa cells with low concentration (0, 1, 2, 4, 8, 16 μg/mL) 5-Fu, and calculate the IC₅₀ of 5-FU to each cell. CCK-8 assay was used to detect 5-FU (IC₅₀), UC-MSCs supernatant, TRAIL-MSCs supernatant, 5-FU (IC₅₀) + UC-MSCs supernatant and 5-FU (1/4 IC₅₀, 1/2 IC₅₀ and IC₅₀) + TRAIL-MSCs culture supernatant on the proliferation inhibition rate of U-87MG, A549, HeLa cells, the interaction coefficient (CDI) between 5-FU and TRAIL-MSCs culture supernatant was calculated. Western blotting was used to detect the effects of 5-FU on the expression of U-87MG, A549 and HeLa cell death receptors DR4 and DR5. Trypan blue staining was used to observe the killing effect of 5-FU, UC-MSCs, TRAIL-MSCs, 5-Fu + UC-MSCs and 5-Fu + TRAIL-MSCs on U-87MG, A549 and HeLa cells. Results The biological detection results showed that TRAIL-MSCs could maintain the biological characteristics of UC-MSCs and at the same time express TRAIL protein. 5-FU could inhibit the proliferation of U-87MG, A549 and HeLa cells, and the order of inhibition was HeLa cells (IC₅₀ 9.15 μg/mL) > A549 cells (IC₅₀ 10.62 μg/mL) > U-87mg cells (IC₅₀ 22.37 μg/mL). Compared with control group, the inhibition rate of cell proliferation was significantly increased in all treatment groups except the UC-MSCs culture supernatant group of A549 cell (P<0.01, 0.001). Compared with the corresponding 5-FU IC₅₀ and TRAIL-MSCs supernatant groups, the proliferation inhibition rate of U-87MG, 5-FU (1/4 IC₅₀, 1/2 IC₅₀and IC₅₀) + TRAIL-MSCs supernatant group was significantly increased (P<0.001). The proliferation inhibition rate of A549 cells cultured with 5-FU (IC₅₀) + TRAIL-MSCs supernatant was significantly increased (P<0.01, 0.001). The synergistic effect of 5-FU (1/2 IC₅₀) + TRAIL-MSCs culture supernatant on proliferation inhibition of U-87MG cells was the most significant (CDI < 0.7 and P<0.001). The 5-FU (1/4 IC₅₀) + TRAIL-MSCs supernatant group had a synergistic effect on the proliferation inhibition of A549 cells, but the synergistic effect was not significant. The synergistic effect of 5-FU (1/4 IC₅₀) + TRAIL-MSCs culture supernatant on proliferation inhibition of HeLa cells was the most significant (CDI < 0.7 and P<0.01). After 5-FU treatment, the protein expression of DR4 and DR5 in U-87MG, A549 and HeLa cells was significantly increased, and the expression level of DR5 was higher than that of DR4, with statistically significant differences compared with control group (P<0.001). Compared with the control group, 5-FU, TRAIL-MSCs and 5-Fu + TRAIL-MSCs had significant killing effect on tumor cells U-87MG, A549 and HeLa (P<0.001). Compared with 5-FU or TRAILMSCs group, the killing effect of 5-FU+ TRAIL-MSCs group was more significant (P<0.001). Conclusion TRAIL-MSCs combined with low concentration of 5-FU had significant cytotoxic effect on tumor cells U-87MG, A549 and HeLa, and the synergistic effect of the two was significant. The mechanism may be related to the improvement of DR4 and DR5 protein expression and sensitivity of tumor cells to TRAIL-MSCs by 5-FU.

R965

北京市科技计划课题（Z211100002521006）