喜泊分介导的光动力对大鼠泌乳素瘤MMQ细胞作用的研究
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摘要
目的观察喜泊分介导的光动力对大鼠泌乳素瘤MMQ细胞的作用。方法不同浓度(0、5、10、20μg/mL)的喜泊分与细胞孵育3 h后,先用405 nm波长紫外光照射观察细胞对喜泊分的吸收情况,然后用630 nm波长红光照射不同时间(0、100、500、1 000 s),采用MTT比色法测定细胞的增殖情况并计算抑制率;应用最大抑制率的参数光动力作用后,在倒置显微镜下观察细胞形态的变化,台盼蓝染色观察细胞的存活情况,Hoechst33342染色观察细胞凋亡或坏死的情况,Annexin/PI双染色流式细胞术分析细胞的死亡形式。结果喜泊分与MMQ细胞孵育3 h后,用紫外光照射细胞,细胞激发出红光,说明MMQ细胞对喜泊分吸收良好;随着喜泊分浓度的增加与光照时间的延长,喜泊分-光动力对MMQ细胞的抑制率逐渐升高,当喜泊分浓度为20μg/mL、光照时间为1 000 s时,抑制率达到最高,为84%;应用最大抑制率的参数光动力作用后,细胞开始游离、细胞膜破裂,并出现很多细胞碎片;台盼蓝染色显示光动力导致MMQ细胞大量死亡;Hoechst33342染色显示喜泊分-光动力组MMQ细胞蓝色荧光强度明显增加,光动力导致细胞核固缩,细胞质凝集;Annexin/PI双染色流式细胞术结果分析显示,在喜泊分浓度为20μg/mL、光照时间为1 000 s时,死亡细胞主要是坏死细胞,达到92.3%。结论喜泊分介导的光动力对大鼠泌乳素瘤MMQ细胞具有光动力杀伤效应,可以引起显著的死亡。
Objective To investigate the effect of xibofen-mediated photodynamic therapy on rat prolactinoma cell line MMQ. Methods MMQ cells were treated with different concentrations of xibofen(0, 5, 10, 20 μg / mL) for three hours, and were irradiated with 405 nm wavelength purple light to observe the absorption of MMQ cells for xibofen. Then the cells were irradi- ated with 630 nm wavelength red light for different time(0, 100, 500, 1 000 s) and the cell proliferation and inhibition were mea- sured by MTT assay. Next the cells were treated with photodynamic therapy, which has the highest inhibition on the cells. The mor- phological changes of the cells were observed with inverted microscope; the cell viability was observed by trypan blue assay; the cell apoptosis or necrosis was observed by Hoechst 33342 assay; the characteristics of cell death were determined by Annexin / PI double staining flow cytometry. Results After the treatment with xibofen for three hours, the cells could transmit red light under the irradiation of purple light, which demonstrated that the MMQ cells had fine absorptions for xibofen. The inhibition of xibofen- mediated photodynamic therapy on MMQ cells went up along with the increasement of xibofen concentration and irradiation time. It reached the highest inhibition of 83.64% when the xibofen concentration was 20 μg / mL and irradiation time was 1 000 s. After the treatment with photodynamic therapy of the highest inhibition, the cells became free, the cell membrane fractured and there were much debris. Trypan blue assay showed that xibofen-mediated photodynamic therapy induced MMQ cells death. Hoechst33342 assay showed that the blue fluorescence intensity increased obviously, while the nuclei and cytoplasm shrank. Annexin / PI double staining flow cytometry showed that the dead cells mainly consisted of necrosis cells and the death ratio reached 92.3% when the xibofen concentration was 20 μg / mL and irradiation time was 1000s. Conclusion Xibofen-mediated photodynamic therapy has a photody- namic effect and could induce obvious death on rat prolactinoma cell line MMQ.
Objective To investigate the effect of xibofen-mediated photodynamic therapy on rat prolactinoma cell line MMQ. Methods MMQ cells were treated with different concentrations of xibofen(0, 5, 10, 20 μg / mL) for three hours, and were irradiated with 405 nm wavelength purple light to observe the absorption of MMQ cells for xibofen. Then the cells were irradi- ated with 630 nm wavelength red light for different time(0, 100, 500, 1 000 s) and the cell proliferation and inhibition were mea- sured by MTT assay. Next the cells were treated with photodynamic therapy, which has the highest inhibition on the cells. The mor- phological changes of the cells were observed with inverted microscope; the cell viability was observed by trypan blue assay; the cell apoptosis or necrosis was observed by Hoechst 33342 assay; the characteristics of cell death were determined by Annexin / PI double staining flow cytometry. Results After the treatment with xibofen for three hours, the cells could transmit red light under the irradiation of purple light, which demonstrated that the MMQ cells had fine absorptions for xibofen. The inhibition of xibofen- mediated photodynamic therapy on MMQ cells went up along with the increasement of xibofen concentration and irradiation time. It reached the highest inhibition of 83.64% when the xibofen concentration was 20 μg / mL and irradiation time was 1 000 s. After the treatment with photodynamic therapy of the highest inhibition, the cells became free, the cell membrane fractured and there were much debris. Trypan blue assay showed that xibofen-mediated photodynamic therapy induced MMQ cells death. Hoechst33342 assay showed that the blue fluorescence intensity increased obviously, while the nuclei and cytoplasm shrank. Annexin / PI double staining flow cytometry showed that the dead cells mainly consisted of necrosis cells and the death ratio reached 92.3% when the xibofen concentration was 20 μg / mL and irradiation time was 1000s. Conclusion Xibofen-mediated photodynamic therapy has a photody- namic effect and could induce obvious death on rat prolactinoma cell line MMQ.
引文
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Cole CD,Liu JK,Sheng X,et al.Hypericin-mediated photodynamic therapy of pituitary tumors:preclinical study in a GH4C1 rat tumor model.J Neurooncol,2008,87(3):255-261.
丁慧颖,顾瑛.竹红菌素光动力学作用中细胞的凋亡和坏死.中国激光医学杂志,2007,16(5):320-324.
Wyld L,Reed MW,Brown NJ.Differential cell death response to photodynamic therapy is dependent on dose and cell type.Br J Cancer,2001,84(10):1384-1386.
Kamuhabwa AR,Agostinis PM,D'Hallewin MA,et al.Cellular photodestruction induced by hypericin in AY-27rat bladder carcinoma cells.Photochem Photobiol,2001,74(2):126-132.
Ali SM,Olivo M.Bio-distribution and subcellular localization of Hypericin and its role in PDT induced apoptosis in cancer cells.Int J Oncol,2002,21(3):531-540.
王海军,陈明振,张恒,等.神经内镜辅助经蝶显微手术切除侵犯中下斜坡的垂体腺瘤.中华显微外科杂志,2003,26(3):190-191.
Shirasu N,Nam SO,Kuroki M.Tumor-targeted photodynamic therapy.Anticancer Res,2013,33(7):2823-2831.
Bhuvaneswari R,Gan YY,Soo KC,et al.The effect of photodynamic therapy on tumor angiogenesis.Cell Mol Life Sci,2009,66(14):2275-2283.
董振香,葛瑞听,刘兰芳.喜泊分.中国新药杂志,2006,15(21):1895-1896.
Juarranz A,Jaén P,Sanz-Rodríguez F,et al.Photodynamic therapy of cancer.Basic principles and applications.Clin Transl Oncol,2008,10(3):148-154.
Saczko J,Chwi kowska A,Kulbacka J,et al.Photooxidative action in cancer and normal cells induced by the use of photofrin in photodynamic therapy.Folia Biol(Praha),2008,54(1):24-29.
Kim CH,Chung CW,Lee HM,et al.Synergistic effects of 5-aminolevulinic acid based photodynamic therapy and celecoxib via oxidative stress in human cholangiocarcinoma cells.Int J Nanomedicine,2013,8:2173-2185.
Gong HY,Sun MX,Hu S,et al.Benzochloroporphyrin derivative induced cytotoxicity and inhibition of tumor recurrence during photodynamic therapy for osteosarcoma.
Asian Pac J Cancer Prev,2013,14(5):3351-3355.Marks PV.Adjuvant therapy for pituitary adenomas:the possible role of photodynamic therapy.Ann R Coll Surg Engl,1995,77(4):308-312.
Marks PV,Buxton T,Furneaux CE.In vitro study of the effect of photodynamic therapy on pituitary adenomas.Br J Neurosurg,1993,7(4):401-406.
Kirollos RW,Marks PV,Igbaseimokumo U,et al.A preliminary experimental in vivo study of the effect of photodynamic therapy on human pituitary adenoma implanted in mice.Br J Neurosurg,1998,12(2):140-145.
Cole CD,Liu JK,Sheng X,et al.Hypericin-mediated photodynamic therapy of pituitary tumors:preclinical study in a GH4C1 rat tumor model.J Neurooncol,2008,87(3):255-261.
丁慧颖,顾瑛.竹红菌素光动力学作用中细胞的凋亡和坏死.中国激光医学杂志,2007,16(5):320-324.
Wyld L,Reed MW,Brown NJ.Differential cell death response to photodynamic therapy is dependent on dose and cell type.Br J Cancer,2001,84(10):1384-1386.
Kamuhabwa AR,Agostinis PM,D'Hallewin MA,et al.Cellular photodestruction induced by hypericin in AY-27rat bladder carcinoma cells.Photochem Photobiol,2001,74(2):126-132.
Ali SM,Olivo M.Bio-distribution and subcellular localization of Hypericin and its role in PDT induced apoptosis in cancer cells.Int J Oncol,2002,21(3):531-540.