目的 研究丹參酮II_A對(duì)人食管癌細(xì)胞放療敏感性的影響，并基于磷脂酰肌醇3激酶/蛋白激酶B/哺乳動(dòng)物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信號(hào)通路探討其潛在機(jī)制。方法 以人食管癌Eca-109細(xì)胞為受試細(xì)胞，分別采用不同濃度丹參酮II_A和不同放射劑量處理Eca-109細(xì)胞，48 h后MTT法檢測(cè)細(xì)胞增殖活力，計(jì)算丹參酮II_A半數(shù)抑制濃度（IC₅₀）和放射的IC₅₀，分別作為后續(xù)實(shí)驗(yàn)丹參酮II_A濃度和放射劑量。取對(duì)數(shù)生長(zhǎng)期Eca-109細(xì)胞，設(shè)對(duì)照組、丹參酮II_A組、放射組、丹參酮II_A＋放射組、丹參酮II_A＋放射＋PI3K抑制劑（LY294002）組。通過平板克隆實(shí)驗(yàn)、MTT法、流式細(xì)胞術(shù)、熒光質(zhì)粒轉(zhuǎn)染法檢測(cè)細(xì)胞克隆形成能力、增殖活力、凋亡率和自噬狀況，RT-PCR、Western blotting法檢測(cè)細(xì)胞中PI3K/Akt/mTOR信號(hào)通路相關(guān)mRNA和蛋白表達(dá)。結(jié)果 丹參酮Ⅱ_A和放射對(duì)人食管癌Eca-109細(xì)胞增殖活力的抑制作用均呈現(xiàn)劑量相關(guān)性，丹參酮II_A IC₅₀為8.75 mmol/L，放射量IC₅₀為4.63 Gy。與對(duì)照組相比，丹參酮II_A組、放射組、丹參酮II_A＋放射組、丹參酮II_A＋放射＋LY294002組細(xì)胞克隆形成能力和增殖活力明顯降低，凋亡率和自噬體數(shù)量明顯升高（P＜0.05）；PI3K、Akt、mTOR mRNA和蛋白表達(dá)量明顯降低（P＜0.05）；Bcl-2、Bax、cleaved Caspase-3、LC3-I、LC3-II蛋白表達(dá)量及Bax/Bcl-2、Cleaved Caspase-3/Caspase-3、LC3-II/LC3-I明顯升高（P＜0.05）。與丹參酮II_A組或放射組相比，丹參酮II_A＋放射組和丹參酮II_A＋放射＋LY294002組對(duì)各檢測(cè)指標(biāo)的調(diào)控作用明顯增強(qiáng)（P＜0.05）。與丹參酮II_A＋放射組相比，丹參酮II_A＋放射＋LY294002組對(duì)各指標(biāo)的調(diào)控作用明顯增強(qiáng)（P＜0.05）。結(jié)論 丹參酮II_A可能通過下調(diào)PI3K/Akt/mTOR信號(hào)通路，抑制細(xì)胞增殖并促進(jìn)其凋亡與自噬，進(jìn)而增強(qiáng)人食管癌細(xì)胞放療敏感性。

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

邯鄲市科學(xué)技術(shù)研究與發(fā)展計(jì)劃項(xiàng)目（22422083056ZC）