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1 生物工程学报 Chin J Biotech 2009, September 25; 25(9): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 生物技术与方法 SMMC-7721 王源, 蔡春尔, 李柏林, 刘承初, 何培民, : 用 3-(4, 5- 二甲基噻唑 -2)-2, 5- 二苯基四氮唑溴盐 (MTT) 法测定提取自条斑紫菜的 R- 藻红蛋白 (R-PE) 和 C- 藻蓝蛋白 (C-PC) 经激光介导对人肝癌 SMMC-7721 细胞的杀伤率, 并检测细胞凋亡率结果显示在 120 mg/l 浓度下, R-PE 在氩离子激光器 100 J/cm 2 辐照下对应的细胞存活率为 27%, C-PC 在 He-Ne 激光器 35 J/cm 2 辐照下对应的细胞存活率为 47%; 单用 2 种激光器辐照对应的细胞存活率分别为 65% 和 70%; 单用 2 种蛋白处理细胞在 72 h 后出现对细胞生长的显著抑制, 最大抑制率分别为 31%(R-PE, 120 mg/l) 和 27%(C-PC, 250 mg/l) 120 mg/l 的 2 种藻胆蛋白在对应激光辐照下, 于照后 8 h 达到细胞凋亡率最大值, 分别为 31.54%(R-PE) 和 32.54%(C-PC) 本实验证明条斑紫菜 R-PE 和 C-PC 具有可开发成为光敏剂的应用前景 : 条斑紫菜, R- 藻红蛋白, C- 藻蓝蛋白, 光敏作用, 细胞凋亡, 肝癌细胞 Photodynamic effect of two kinds of phycobiliproteins on human liver cancer cell line SMMC-7721 in vitro Yuan Wang, Chuner Cai, Bailin Li, Chengchu Liu, and Peimin He Shanghai Ocean University, Shanghai , China Abstract: We studied the effect of photodynamic therapy with phycobiliproteins on human liver cancer cells in vitro. With 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay), we used two phycobiliproteins, R-phycoerythrin (R-PE) and C-phycocyanin (C-PC) prepared from Porphyra yezoensis, to determine the killing rates and apoptosis rates of human liver cancer cells (SMMC-7721) mediated by laser. When the concentration of R-PE was 120 mg/l, the survival rate of human liver cancer cells was 27% after treated by Argon laser with 100 J/cm 2 doses, while the survival rate in the control group (without adding R-PE) was 65%. When the C-PC concentration was 120 mg/l, the survival cell rate was 47% after treated by He-Ne laser with 35 J/cm 2 dose, while the survival rate in the control group (without adding C-PC) was 70%. After handled only with these two kinds of phycobiliproteins for 72 h, the growth of cancer cells presented significant inhibition. The maximal inhibition rates reached up to 31% with R-PE (120 mg/l concentration) and 27% with C-PC (250 mg/l concentration) respectively. After irradiated by laser for 8 h, the maximal cell apoptosis rates were 31.54% with R-PE and 32.54% with C-PC, respectively. It indicated that R-PE and C-PC Received: April 22, 2009; Accepted: July 16, 2009 Supported by: National High Technology Research and Development Program of China (863 Program) (Nos. 2006AA10Z323, 2007AA09Z406), Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education (No. KFT ), Shanghai Education Committee of China (No. S30701). Corresponding author: Peimin He. Tel: ; pmhe@shou.edu.cn 国家高技术研究发展计划 (863 计划 )(Nos. 2006AA10Z323, 2007AA09Z406), 教育部水产种质资源发掘与利用省部共建重点开放实验室 (No. KFT ), 上海市教委优势 ( 重点 ) 学科项目 (No. S30701) 资助

2 1418 ISSN CN /Q Chin J Biotech September 25, 2009 Vol.25 No.9 extracted from Porphyra yezoensis could develop to new photosensitizers for cancer photodynamic therapy. Keywords: Porphyra yezoensis, R-phycoerythrin, C-phycocyanin, photodynamic therapy, cell apoptosis, liver cancer cell (WHO), 650, 430, 2,,,, [1] (Photodynamic therapy, PDT) 20 [2], [3] 1993, Photofrin( Porfimer sodium, ) [4] 20 [5] [6] (HP),, 5- (Delta-aminolevulinic acid, ALA) [7] [8],,,, [9-10] 1988 Morcos [11] 0.25 g/l,,,,,,, [12] R-PE C-PC Hep-2 [13] He-Ne, R-PE C-PC SMMC-7721, PDT, 1 材料与方法 1.1 实验材料 (Porphyra yezoensis), SMMC-7721 ; Vivaflow 200 超滤膜包 Vivascience 公司 ; RPMI-1640 Gibco ; Napco 6500 Thermo ; A30 Motic ; 630 Biorad ; INNOVR 400 Coherent ; He-Ne (QHP ) 1.2 藻胆蛋白提取与光谱分析 [14],,, R-PE C-PC,, 4 o C [15] : R-PE =OD 565 /OD 280 ; C-PC =OD 615 /OD 肿瘤细胞培养方法 SMMC-7721 [12], EDTA, 10 9 /L, L 藻胆蛋白经激光激发对 SMMC-7721 细胞生存率的影响 ( mg/l)

3 : SMMC SMMC-7721, 4 h, 514 nm R-PE, 100 mw, 15 min, 100 J/cm nm He-Ne C-PC, 30 mw, 20 min, 35 J/cm 2 24 h, MTT 12, : =( / ) 100% 2 结果 2.1 藻胆蛋白光谱分析及纯度鉴定 R-PE C-PC, R-PE 498 nm 565 nm, 565 nm, 540 nm 1, R-PE [16], C-PC 615 nm,, C-PC [17] ( 1 2) R-PE C-PC 5.5(OD 564 /OD 280 ) 4.0(OD 615 /OD 280 ) SPSS12.0 t 1.5 激光单独辐照对 SMMC-7721 细胞生长的影响,,, J/cm 2, He-Ne J/cm 2 12,, 1.6 藻胆蛋白本身对 SMMC-7721 细胞生长的影响, 图 1 藻红蛋白吸收光谱 Fig. 1 Absorption of R-Phycoerythrin mg/l,, h 12,, 1.7 藻胆蛋白经激光激发对 SMMC-7721 细胞凋亡率的测定 ( 100 J/cm 2 (PE) 35 J/cm 2 (PC), 120 mg/l) ( h) SMMC-7721, PBS 2000 r/min, 5 min L Carnoy ( : =3:l), (120 mg/l) 图 2 藻蓝蛋白吸收光谱 Fig. 2 Absorption of C-Phycocyanin. 2.2 R-PE 和 C-PC 经激光激发对 SMMC-7721 细胞的杀伤效果 R-PE 60 mg/l 40%, 250 mg/l 20% C-PC 35 J/cm 2 59%(250 mg/l)( 1)

4 1420 ISSN CN /Q Chin J Biotech September 25, 2009 Vol.25 No.9 表 1 藻胆蛋白光动力杀伤对 SMMC-7721 细胞存活率的影响 Table 1 Survival rates of SMMC-7721 under phycobiliproteins PDT Concentration of PB (mg/l) 0 (Control) Laser only Survival rates of R-PE group (%)( X ±S) 100% 65±6.8 * 59±5.0* 40±6.1** 27±6.4** 20±5.6** Survival rates of C-PC group (%)( X±S) 100% 70±4.5* 73±6.1* 65±4.8* 47±6.7** 41±7.1** t-test, *P<0.05, **P< 激光辐照能量对 SMMC-7721 细胞生长的影响 514 nm SMMC-7721, J/cm 2 65%, He-Ne nm, J/cm 2 8 h 9.55% 9.91%( 3), 8.0%(R-PE) 10.9%(C-PC) ( 4) 120 mg/l R-PE C-PC 100 J/cm 2, % ( 2 3) 2.4 R-PE 和 C-PC 对 SMMC-7721 细胞生长的影响 24 h, ; 48 h, C-PC250 mg/l ; 72 h, ( mg/l), 31% (PE, 120 mg/l) 27% (250 mg/l)( 4) 2.5 R-PE 和 C-PC 经激光激发对 SMMC-7721 细胞凋亡率的影响 120 mg/l, R-PE C-PC 图 3 单用 120 mg/l 藻胆蛋白处理细胞凋亡率 Fig. 3 Apotosis ratio of cells 8 h later treated only by 120 mg/l phycobiliproteins. C-PC is above, R-PE is below, same below. 表 nm 氩离子激光器单独辐照对 SMMC-7721 细胞生存率的影响 Table 2 Survival rates of SMMC-7721 under different irradiation of 514 nm Argon laser alone Accumulated radiation dose (J/cm 2 ) 0 (Control) Survival rates(%)( X±S) 100% 66±3.8* 65±6.8* 66±3.5* t-test, *P<0.05. 表 nmhe-ne 激光单独辐照对 SMMC-7721 细胞生存率的影响 Table 3 Survival rates of SMMC-7721 under different irradiation of nm He-Ne laser alone Accumulated radiation dose (J/cm 2 ) 0 (Control) Survival rates (%) ( X±S) 100% 73±5.2* 70±4.5* 68±3.8* t-test, * P<0.05. 表 4 藻胆蛋白本身对 SMMC-7721 细胞存活率的影响 Table 4 Survival rates of SMMC-7721 under different concentration of phycobiliproteins alone Concentration of PB (mg/l) 0 (Control) Survival rates of R-PE group after 24 h (%) 100% 99±1.2 96±2.1 92±0.9 89±1.9 Survival rates of R-PE group after 48 h (%) 100% 90±2.7 91±2.1 85±1.4 85±3.5 Survival rates of R-PE group after 72 h (%) 100% 84±4.1 80±4.2* 69±2.7** 75±6.7** Survival rates of C-PC group after 24 h (%) 100% 98±1.3 98±1.8 95±2.1 86±2.4 Survival rates of C-PC group after 48 h (%) 100% 97±3.1 91±2.6 88±2.6 82±3.7* Survival rates of C-PC group after 72 h (%) 100% 87±5.3 75±3.6* 78±7.0* 73±6.3** t-test, *P<0.05, **P<0.01.

5 : SMMC h, 25.6% 31.5% 31.1% 27.6% 32.5% 29.6( 5~7) R-PE C-PC 3 讨论 100 mg/l 12 J/cm 2 HR % [18], 28.8 J/cm 2 He-Ne 图 4 单用 100 J/cm 2 激光处理的细胞凋亡率 Fig. 4 Apotosis ratio of cells 8 h later after 100 J/cm 2 illumination of laser. Bcap-3 72% [19] 100 mg/l 25.6 J/cm 2 S180 76% [10], 图 5 Fig. 5 图 6 Fig. 6 图 7 Fig. 7 PDT 处理 4 h 后的细胞凋亡率 Apotosis ratio of cells 4 h later after PDT treatment. PDT 处理 8 h 后的细胞凋亡率 Apotosis ratio of cells 8 h later after PDT treatment. PDT 处理 12 h 后的细胞凋亡率 Apotosis ratio of cells 12 h later after PDT treatment % [9] 100 mg/l He-Ne Bcap-3 85% 79% [19] He-Ne 120 mg/l SMMC % 53%, 250 mg/l 80% 59%, 28.8 J/cm 2 S180 79%~88% [20], S180 69%~80%, 46%~81% [21] 4 h S180 31%~65% [20], 4 h 28%~68% [21],, 30 mg/l, SMMC-7721,, 120 mg/l 24 h 8% 5%, 4 h,,,

6 1422 ISSN CN /Q Chin J Biotech September 25, 2009 Vol.25 No.9 24 h, R-PE< 25 mg/l, R-PC C-PC<50 mg/l, Bcap-37, R-PE 25 mg/l, R-PC C-PC 50 mg/l, [19] S180 [10] 2 SMMC % 32.54%, (Apoptosis) [22], : [23] [24] 100 mg/l R-PE PDT h 56.2% 82.2% 98.1% 3,,,,,,, HpD, ph,,,, 1~2 d, HpD [25] ph 5( ph ) ph 7( ph ) [26],, REFERENCES [1] Chakraborty M, Gelbard A, Carrasquillo JA, et al. Use of radiolabeled monoclonal antibody to enhance vaccine-mediated antitumor effects. Cancer Immunol Immun, 2008, 57: [2] Zhang JP, Xie J, Zhang SP. Photosensitive properties of two kinds of phycobiliprotein on photodynamic reaction. Chin Sci Bull, 1999, 4(5): ,,,.., 1999, 4(5): [3] Dougherty T. Photodynamic therapy-new approaches. Semin Surg Oncol, 1989, 5: [4] Cai CE, Wu QL, Xu CH, et al. Advance of phycoerythrin as a photosensitize. Lett Biotechnol, 2004, 15(6): ,,,.., 2004, 15(6): [5] Dickson EFG, Goyan RL, Pottier RH. New directions in photodynamic therapy. Cell Mol Biol, 2002, 48(8): [6] Sharman WM, Allen CM, van Lier JE. Photodynamic therapeutics: basic principles and clinical application. Drug Discov Today, 1994, 4(11): [7] Bunke A, Zerbe O, Schmid H, et al. Degradation mechanism and stability of 5-aminolevulinic acid. J Pharm Sci, 2000, 89(10): [8] Thomas JV, Katrin Eichler, Martin GM, et al. Interstitial photodynamic laser therapy in interventional oncology. Eur Radiol, 2004, 14(12): [9] Li GW, Wang GC, Lu L, et al. Effect of the photo sensitivity of phycoerythrin on survival rate of human tumor cell line. Acta Laser Biol Sin, 1997, 6(3): ,,,. R-., 1997, 6(3): [10] Li GW, Wang GC, Wen BG, et al. Phycoerythrin as a novel photosensltlzer applied in the photodynamic therapy of cancer. Chin J Can Prev Treat, 2001, 8(4): ,,,.., 2001, 8(4): [11] Morcos NC, Berns M, Henry WL. Phycocyanin: laser activation, cytotoxic effect, and uptake in human atherosclerotic plaque. Laser Surg Med, 1988, 8(1): [12] He PM, Xu SN, Zhang HY, et al. Bioremediation efficiency in the removal of dissolved inorganic nutrients by the red seaweed Porphyra yezoensis, cultivated in the open sea. Water Res, 2008, 42(4): [13] Wang Y, Zhang LX, Cai CE, et al. Photodynamic effect of two kinds of phycobiliproteins on human laryngeal cancer cell line Hep-2. Biotechnol Bull, 2008, 195(4): ,,,. Hep-2., 2008, 195(4):

7 : SMMC [14] Cai CE, Wu QL, He PM. Extraction and purification of R-phycoerythrin in Porphyra yezoensis. Lett Biotechnol, 2005, 16(5): ,,. R-., 2005, 16(5): [15] Galland-Irmouli AV, Pons L, Lucon M, et al. One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis. J Chromatogr B: Biomed Sci Appl, 2000, 739(1): [16] Sui ZH, Zhang XC. Review on phycoerythrin research. Marine Sci, 1998, 22(4): ,.., 1998, 22(4): [17] O'hEocha C. Phycobilin In Physiology and biochemistry of algae//lewin RA, ed. New York and London: Acad Press, 1962: [18] Cai XH, He LM, Jiang JL, et al. Study on promotion of laser therapy for carcinoma using phycocyanin from Spirulina. Chin J Mar Drugs, 1995, 14(1): ,,,.., 1995, 14(1): [19] Chen XQ, Gong XG, Liang Q, et al. Photodynamic effect of three kinds of phycobiliproteins on human mammary cancer line Bcap-37. J Zhejiang Univ (Science Edition), 2005, 32(4): ,,,. Bcap-37. ( ), 2005, 32(4): [20] Huang B, Li ZG, Wang GC, et al. The experimental research of R-phycoerythrin and its chromophore polypeptides in the photodynamic therapy of cancer. J Univ Sci Technol Chin, 2001, 31(2): ,,,. R-., 2001, 31(2): [21] Huang B, Wang GC, Li ZG. Antitumor studies of C-phycocyanin chromophore peptides mediated photodynamic therapy. Acta Laser Biol Sin, 2002, 11(3): ,,.., 2002, 11(3): [22] Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Brit J Cancer, 1972, 26 (4): [23] Vaux DL, Stresser A. The molecular biology of apoptosis. Proc Natl Acad Sci USA, 1996, 93(6): [24] Li GW. Application of phycobiliproteins in the photodynamic therapy of cancer. Hefei: University of Science and Technology of China, 1997: :, 1997: [25] Liao WJ. Clinical Photodynamic Therapy on Oncology. Beijing: People's Medical Publishing House, 2006: :, 2006: [26] Li GW, Wang GC, Qi Y, et al. Study of morphological mechanism on photodynamic killing mediated by R-phycoerythrin. J China Univ Sci Technol, 1999, 29(15): ,,,. R-., 1999, 29(15):

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇