目的 研究丹参酮II_A对人食管癌细胞放疗敏感性的影响，并基于磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路探讨其潜在机制。方法 以人食管癌Eca-109细胞为受试细胞，分别采用不同浓度丹参酮II_A和不同放射剂量处理Eca-109细胞，48 h后MTT法检测细胞增殖活力，计算丹参酮II_A半数抑制浓度（IC₅₀）和放射的IC₅₀，分别作为后续实验丹参酮II_A浓度和放射剂量。取对数生长期Eca-109细胞，设对照组、丹参酮II_A组、放射组、丹参酮II_A＋放射组、丹参酮II_A＋放射＋PI3K抑制剂（LY294002）组。通过平板克隆实验、MTT法、流式细胞术、荧光质粒转染法检测细胞克隆形成能力、增殖活力、凋亡率和自噬状况，RT-PCR、Western blotting法检测细胞中PI3K/Akt/mTOR信号通路相关mRNA和蛋白表达。结果 丹参酮Ⅱ_A和放射对人食管癌Eca-109细胞增殖活力的抑制作用均呈现剂量相关性，丹参酮II_A IC₅₀为8.75 mmol/L，放射量IC₅₀为4.63 Gy。与对照组相比，丹参酮II_A组、放射组、丹参酮II_A＋放射组、丹参酮II_A＋放射＋LY294002组细胞克隆形成能力和增殖活力明显降低，凋亡率和自噬体数量明显升高（P＜0.05）；PI3K、Akt、mTOR mRNA和蛋白表达量明显降低（P＜0.05）；Bcl-2、Bax、cleaved Caspase-3、LC3-I、LC3-II蛋白表达量及Bax/Bcl-2、Cleaved Caspase-3/Caspase-3、LC3-II/LC3-I明显升高（P＜0.05）。与丹参酮II_A组或放射组相比，丹参酮II_A＋放射组和丹参酮II_A＋放射＋LY294002组对各检测指标的调控作用明显增强（P＜0.05）。与丹参酮II_A＋放射组相比，丹参酮II_A＋放射＋LY294002组对各指标的调控作用明显增强（P＜0.05）。结论 丹参酮II_A可能通过下调PI3K/Akt/mTOR信号通路，抑制细胞增殖并促进其凋亡与自噬，进而增强人食管癌细胞放疗敏感性。

Objective To investigate the effect of tanshinone II_A on radiotherapy sensitivity of human esophageal carcinoma cells, and explore its potential mechanism based on the PI3K/Akt/mTOR signaling pathway. Methods The human esophageal cancer Eca-109 cells was used as test cells, which were treated with different concentrations of tanshinone II_A and different radiation doses respectively. 48 h later, the cell proliferation activity was detected by MTT method, the half-inhibitory concentration of IC₅₀ tanshinone II_A (as the concentration of tanshinone II_A for follow-up experiments) and IC₅₀ radiation (as the radiation dose for follow-up experiments) were calculated respectively. Take the Eca-109 cells in logarithmic growth period and set blank group, tanshinone II_A group, radiation group, tanshinone II_A + radiation group, tanshinone II_A + radiation + PI3K inhibitor (LY294002) group. The cloning ability, proliferation activity, apoptosis rate, and autophagy were detected by plate cloning assay, MTT assay, flow cytometry or fluorescent plasmid transfection. The PI3K/Akt/mTOR signaling pathway related mRNA and protein expression were detected by RT-PCR or Western blotting method. Results Both tanshinone II_A and radiation showed dose-dependent inhibitory effects on the proliferation of human esophageal cancer Eca-109 cells. The IC₅₀ tanshinone II_A was 8.75 mmol/L, and the IC₅₀ radiation was 4.63 Gy. Compared with the control group, the clonogenesis ability and proliferation activity of tanshinone II_A group, radiation group, tanshinone II_A+ radiation group and Tanshinone II_A + radiation + LY294002 group were significantly decreased, the apoptosis rate and autophagosome number were significantly increased (P < 0.05). The mRNA expressions of PI3K, Akt, mTOR were significantly decreased (P < 0.05). The protein expression of PI3K, Akt, mTOR were significantly decreased. The protein expression of Bcl-2, Bax, Cleaved Caspase-3, LC3-I, LC3-II, and the expression ratios of Bax/Bcl-2, Cleaved Caspase-3/Caspase-3, LC3-II/LC3-I were significantly increased (P < 0.05). Compared with tanshinone II_A group or radiation group, the regulation effect of tanshinone II_A + radiation group and tanshinone II_A+ radiation + LY294002 group on the detection indexes was significantly enhanced (P < 0.05). Compared with tanshinone II_A+ radiation group, the regulation effect of tanshinone II_A+ radiation + LY294002 group was significantly enhanced (P < 0.05). Conclusions Tanshinone II_A may inhibit cell proliferation, promote apoptosis and autophagy by down-regulating PI3K/Akt/mTOR signaling pathway, thus enhancing the radiotherapy sensitivity of human esophageal cancer cells.

R285.5

邯郸市科学技术研究与发展计划项目（22422083056ZC）