目的 研究TRAIL基因修飾的臍帶間充質(zhì)干細胞（TRAIL-MSCs）聯(lián)合化療藥5-氟尿嘧啶（5-FU）對人腦膠質(zhì)瘤細胞U-87MG、人肺癌細胞A549和人宮頸癌細胞HeLa的殺傷作用。方法 通過基因工程手段結(jié)合慢病毒轉(zhuǎn)染體系構(gòu)建高表達十二聚體TRAIL蛋白（dTRAIL）的TRAIL-MSCs，應(yīng)用流式細胞技術(shù)檢測TRAIL-MSCs的生物學(xué)特性；通過ELISA技術(shù)檢測TRAIL-MSCs分泌的TRAIL量來評估轉(zhuǎn)染效果；CCK-8法檢測低濃度（0、1、2、4、8、16 μg/mL）5-FU對U-87MG、A549、HeLa細胞的增殖抑制率，并計算其對各細胞的半數(shù)抑制濃度（IC₅₀）；CCK-8法檢測5-FU（IC₅₀）、UC-MSCs培養(yǎng)上清、TRAIL-MSCs培養(yǎng)上清、5-FU（IC₅₀）＋UC-MSCs培養(yǎng)上清和5-FU（1/4 IC₅₀、1/2 IC₅₀和IC₅₀）＋TRAIL-MSCs培養(yǎng)上清對U-87MG、A549、HeLa細胞的增殖抑制率，計算5-FU和TRAIL-MSCs培養(yǎng)上清的相互作用系數(shù)（CDI）；Westernblotting法檢測5-FU對U-87MG、A549、HeLa細胞死亡受體DR4、DR5蛋白表達的影響；臺盼藍染色法觀察5-FU、UC-MSCs、TRAIL-MSCs、5-FU＋UC-MSCs和5-FU＋TRAIL-MSCs對U-87MG、A549、HeLa細胞的殺傷作用。結(jié)果 生物學(xué)檢測結(jié)果表明TRAIL-MSCs在維持UC-MSCs生物學(xué)特性的同時能夠高表達TRAIL蛋白。5-FU對U-87MG、A549、HeLa細胞均有增殖抑制作用，抑制作用由高到底依次為HeLa細胞（IC₅₀為9.15 μg/mL） >A549細胞（IC₅₀為10.62 μg/mL） >U-87MG細胞（IC₅₀為22.37 μg/mL）。與對照組比較，除A549細胞UC-MSCs培養(yǎng)上清組外，各處理組細胞增殖抑制率均顯著升高（P<0.01、0.001）；與相應(yīng)各5-FUIC₅₀及TRAIL-MSCs培養(yǎng)上清組比較，U-87MG、HeLa細胞5-FU （1/4 IC₅₀、1/2 IC₅₀和IC₅₀） ＋TRAIL-MSCs培養(yǎng)上清組細胞增殖抑制率均顯著升高（P<0.001），A549細胞5-FU （IC₅₀） ＋TRAIL-MSCs培養(yǎng)上清組細胞增殖抑制率顯著升高（P<0.01、0.001）；5-FU （1/2 IC₅₀） ＋TRAIL-MSCs培養(yǎng)上清組對U-87MG的細胞增殖抑制協(xié)同效應(yīng)最顯著（CDI<0.7且P<0.001）； 5-FU （1/4 IC₅₀） ＋TRAIL-MSCs培養(yǎng)上清組對A549的細胞增殖抑制具有協(xié)同作用，但其協(xié)同效果并不顯著； 5-FU （1/4 IC₅₀） ＋TRAIL-MSCs培養(yǎng)上清組對HeLa細胞增殖抑制的協(xié)同效果最顯著（CDI<0.7且P<0.01）。經(jīng)5-FU處理后U-87MG、A549、HeLa細胞DR4和DR5的蛋白表達明顯升高，其中DR5的表達水平高于DR4，與對照組比較差異均具有統(tǒng)計學(xué)意義（P<0.001）。與對照組比較，5-FU、TRAIL-MSCs和5-FU＋TRAIL-MSCs組對于腫瘤細胞U-87MG、A549、HeLa均具顯著的殺傷作用（P<0.001）；與5-FU或TRAIL-MSCs組比較，5-FU＋TRAIL-MSCs組的殺傷效果更顯著（P<0.001）。結(jié)論 TRAIL-MSCs聯(lián)合低濃度的5-FU對腫瘤細胞U-87MG、A549和HeLa具有顯著的細胞殺傷作用，二者聯(lián)用協(xié)同效果顯著，機制可能與5-FU提高DR4、DR5蛋白表達、提高腫瘤細胞對TRAIL-MSCs的敏感性相關(guān)。

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

北京市科技計劃課題（Z211100002521006）