摘要目的 观察5-氨基酮戊酸(5-aminolevulinic acid,5-ALA)介导的光动力治疗对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)的体内外作用并探讨其机制,为临床OSCC治疗提供参考.方法 将SCC25细胞分为对照组(5-ALA质量浓度为0 mg/L)和实验组(5-ALA浓度分别为10、25、50、100与150 mg/L),各组依次孵育2、4、8、12、24h后收取细胞,用流式细胞术检测OSCC细胞SCC25与5-ALA共孵育后原卟啉Ⅸ(protoporphyrinⅨ,PpⅨ)在胞内的生成水平.将SCC25细胞分为对照组(0 mg/L 5-ALA)、单独激光照射组、单独5-ALA组(100 mg/L)和5-ALA结合激光照射组(5-ALA质量浓度分别为5、10、25、50与100 mg/L),分别用甲基噻唑基四唑(methyl thiazolyltetrazolium,MTT)法(每组依次孵育4、8、12h)、DCFH-DA荧光探针法(每组孵育12h)和膜联蛋白V-异硫氰酸荧光素腆化丙啶(annexin V-fluorescein isothiocyanate/propidiumiodide,Annexin V-FITC/PI)双染色法(每组孵育12 h)检测5-ALA结合激光照射(波长635 nm,功率87 mW/cm2,能量密度10.4 J/cm2)对SCC25细胞生长的抑制作用、胞内活性氧生成水平及细胞凋亡的诱导效应.构建SCC25细胞移植瘤裸鼠模型,将裸鼠分为对照组(只注射生理盐水)、5-ALA给药组(50 mg/kg)及5-ALA结合激光照射组(给药剂量分别为10、25和50mg/kg),考察肿瘤局部注射5-ALA后行肿瘤局部激光照射(波长635 nm,照射功率158 mW/cm2,能量密度94.8 J/cm2)对体内肿瘤生长的抑制作用.结果 SCC25细胞内PpⅨ生成水平与5-ALA浓度(0～150mg/L)及培养时间(0～24h)呈正相关;当5-ALA质量浓度增至100 mg/L后,胞内PpⅨ生成水平趋于相对恒定.在SCC25细胞中,与5、10、25、50、100 mg/L的5-ALA孵育12h结合激光照射后细胞存活率及晚期凋亡率[分别为(82.3±5.2)％、(3.13±0.38)％;(74.6±9.3)％、(5.38±0.55)％;(38.3±9.7)％、(17.97±2.72)％;(9.2±3.8)％、(24.47±3.37)％;(7.2±0.8)％、(43.01±5.96)％]与对照组[(96.3±6.0)％、(0.35±0.13)％]相比,均表现出显著的细胞生长抑制和凋亡诱导效应(P＜0.05).与对照组(0.96±0.15)相比,5、10、25、50、100 mg/L 5-ALA结合激光照射后各组荧光强度分别为(1.46±0.12)×104、(2.16±0.30)×104、(3.57±0.34)×104、(81.70±13.05)×104及(113.00±7.35)×104,SCC25细胞中活性氧均大量生成(P＜0.05),其生成水平与胞内PpⅨ含量呈正相关.在荷瘤小鼠体内,各浓度5-ALA结合激光照射治疗均高效抑制了SCC25肿瘤的生长,各5-ALA结合激光治疗组的肿瘤体积均显著小于对照组(P＜0.05).结论 5-ALA介导的光动力治疗可以触发OSCC细胞中活性氧的大量生成,产生显著的细胞毒性与细胞凋亡诱导作用,进而有效抑制肿瘤的体内外生长.

abstractsObjective To investigate the in vitro and in vivo effects of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy against oral squamous cell carcinoma (OSCC) and preliminarily explore the possible mechanisms.Methods SCC25 cells were divided into the control group (5-ALA of 0 mg/L) and the experimental group (5-ALA of 10,25,50,100 and 150 mg/L).The production ofprotoporphyrin Ⅸ (PpⅨ) induced by 5-ALA in SCC25 cells was detected using the flow cytometry.SCC25 ceils were divided into the control group (5-ALA of 0 mg/L),lazer alone group,5-ALA alone group (5-ALA of 100 mg/L) and the 5-ALA combined with laser irradiation group (5-ALA of 5,10,25,50 and 100 mg/L),the cytotoxicity of 5-ALA combined with laser irradiation (wave length 635 nm,power density 87 mW/cm2 and laser dose 10.4 J/cm2) was evaluated in SCC25 cells using the methyl thiazolyltetrazolium assay (incubation times of 4,8 and 12 h in each group) and the induction effect of combination treatment on the cell apoptosis was assessed by the flow cytometry (incubation time of 12 h in each group).The intracellular production of reactive oxygen species (ROS) triggered by 5-ALA combined with laser irradiation was determined using a fluorescence probe method (incubation time of 12 h in each group).A mouse OSCC xenograft model bearing SCC25 tumor was built,and the mice were divided into control group (saline),5-ALA group (5-ALA of 50 mg/kg)and 5-ALA combined with laser irradiation group (5-ALA of 10,25 and 50 mg/kg).Antitumor effect of 5-ALA combined with laser irradiation (wave length 635 nm,power density 158 mW/cm2 and laser dose 94.8 J/cm2) was further measured.Results 5-ALA induced the production of PpⅨ in SCC25 cells in a drug concentration (0-150 mg/L)-and incubation time (0-24 h)-dependent manner.When the 5-ALA concentration was 100 mg/L,the intracellular PpⅨ production was in a relatively stable state.Cell viability and apoptosis rate of 5,10,25,50,100 mg/L 5-ALA combined with laser irradiation are,respectively,(82.3±5.2)％,(3.13±0.38)％;(74.6±9.3)％,(5.38±0.55)％;(38.3±9.7)％,(17.97±2.72)％;(9.2±3.8)％,(24.47±3.37)％;(7.2±0.8)％,(43.01±5.96)％,which indicated that 5-ALA combined with laser irradiation notably inhibited the growth of SCC25 cells and also induced significant cell apoptosis compared with the control group [(96.3± 6.0)％,(0.35±0.13)％,P＜0.05].After combination treatment (5-ALA of 5,10,25,50 and 100 mg/L combined with laser irradiation,the mean fluorescence intensity of dichlorofluorescein is (1.46±0.12)× 104,(2.16±0.30)× 104,(3.57±0.34)× 104,(81.70± 13.05)× 104,(113.00±7.35)× 104,respectively,a large amount of ROS was produced in SCC25 cells compared with the control group [(0.96± 0.15) × 104,P＜0.05],which was in positive correlation with the intracellular PpⅨ content.5-ALA (concentration of 10,25 and 50 mg/kg) combined with laser irradiation greatly suppressed the tumor growth in SCC25 tumor-bearing mice compared to the control group (P＜0.05).Conclusions 5-ALA-mediated photodynamic therapy can trigger the generation of intracellular ROS that has significant cytotoxicity and apoptosis induction effect,and thus inhibit the tumor growth both in vitro and in vivo.