5-氨基酮戊酸介导的光动力治疗对口腔鳞状细胞癌体内外作用的研究  

In vitro and in vivo effects of 5-aminolevulinic acid-mediated photodynamic therapy against oral squamous cell carcinoma

作　　者：朱梦琪 史澍睿 万国运 王银松[3] 王悦[1] 张连云[2] 赵艳红[1] Zhu Mengqi;Shi Shurui;Wan Guoyun;Wang Yinsong;Wang Yue;Zhang Lianyun;Zhao Yanhong(Department of Orthodontics, Stomatological Hospital, Tianjin Medical University, Tianjin 300070,China;Department of Prosthodontics, Stomatological Hospital, Tianjin Medical University, Tianjin 300070, China;Departmert of Pharmaceutics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China)

机构地区：[1]天津医科大学口腔医院正畸科,300070 [2]天津医科大学口腔医院修复科,300070 [3]天津医科大学药学院药剂学系,300070

出　　处：《中华口腔医学杂志》2019年第3期176-182,共7页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81572655、81573005).

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、24 h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(protoporphyrin Ⅸ,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、12 h)、DCFH-DA荧光探针法(每组孵育12 h)和膜联蛋白V-异硫氰酸荧光素/碘化丙啶(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/cm^2)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50 mg/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/cm^2,能量密度94.8 J/cm^2)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(0~150 mg/L)及培养时间(0~24 h)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12 h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�Objective 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 of protoporphyrin Ⅸ(PpⅨ) induced by 5-ALA in SCC25 cells was detected using the flow cytometry. SCC25 cells 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/cm^2) 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/cm^2 and laser dose 94.8 J/cm^2) 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

关 键 词：光化学疗法 癌 鳞状细胞 5-氨基酮戊酸 细胞凋亡 

分 类 号：R739.8[医药卫生—肿瘤]