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1 生物工程学报 Chin J Biotech 2009, August 25; 25(8): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 医学与免疫生物技术 1, 张海燕 2, 李艳 1, 王喆 1, 张必良 1, 2 1, , : 本研究旨在建立可用于活体成像的小鼠肺癌移植瘤模型利用脂质体将荧光素酶表达载体 pgl4.17(luc2/neo) 转染至人非小细胞肺癌细胞株 A549, 经 G418 筛选获得稳定表达荧光素酶的细胞克隆根据体外生物发光情况及细胞的生长特性, 从中挑选合适克隆, 进行裸鼠皮下接种, SCID 鼠尾静脉接种, 建立肺癌移植瘤模型利用活体成像系统监测肿瘤的生长转移情况, 并用切片 HE 染色进一步验证小鼠模型移植瘤的原位成瘤和转移能力实验结果表明 : 本研究成功地构建了可用于活体成像的小鼠肺癌移植瘤模型, 模型稳定可靠直观灵敏, 为肿瘤生长转移机制的研究及抗肿瘤药物的研发提供了重要工具 : 荧光素酶, A549 细胞, 动物模型, 移植瘤, 活体成像 Establishment of xenograft mouse models to study human lung cancer by using in vivo imaging system Haiyan Zhang 1, 2, Yan Li 1, Zhe Wang 1, and Biliang Zhang 1, 2 1 State Key Laboratory for Respiratory Diseases, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou , China 2 University of Science and Technology of China, Hefei , China Abstract: We established xenograft mouse models for studying human lung cancer by using an in vivo imaging system. We first transfected pgl4.17 (luc2/neo) plasmid into human non-small lung cancer A549 cells and screened cell lines stably expressing a luciferase reporter gene with G418. Then we analyzed the correlation of luciferase activity and cells number by in vitro bioluminescence. Furthermore, we compared cell growth characteristics by cell counting. We selected suitable clones and inoculated subcutaneously into nude mice or intravenously into SCID mice to construct lung cancer xenograft models. Using an in vivo imaging system, we monitored the growth and metastasis of the tumors. Finally, we verified the extents of tumorigenesis and metastasis by tissue sections with Hematoxylin and Eosin (HE) staining. In our study, we successfully established the xenograft mouse models for in vivo imaging with luciferase expressed lung cancer cells. These models provided convenient, sensitive, intuitive and stable tools for studying the mechanisms of lung cancer progression and development of anticancer drug. Keywords: luciferase, A549 cell, animal model, xenograft, in vivo imaging system Received: April 3, 2009; Accepted: May 20, 2009 Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2006AA02Z191), Bureau of Science and Technology of Guangzhou Municipality (No. 2007Z1-E4041), Science and Technology Development Funds of Guangzhou Development District (No. 2007G-P029). Corresponding author: Biliang Zhang. Tel: ; zhang_biliang@gibh.ac.cn 国家高技术研究发展计划 (863 计划 ) (No. 2006AA02Z191), 广州市科技攻关重大项目 (No. 2007Z1-E4041), 广州经济开发区科技项目 (No. 2007G-P029) 资助

2 : 1205 [1],, 2010, 3,, [2],,,, [3-4] (Luciferase),, [5-7] pgl4.17[luc2/neo] A549, A549-luc,, 1 材料和方法 1.1 材料 A549 ; pgl4.17[luc2/neo] Promega, (Luciferase) G418, 20 o C Lipofectamine TM 2000 Invitrogen ; G418 ; (SCXK( ) ), SCID (SCXK( ) ), SPF 1.2 方法 G418 A549 10% 1640, /ml, 100 μl 96, 24 h, G418, 0~1 mg/ml 10, 96, 6, 10~14 d G418, A A549, 1 6, 24 h 70%~90% Lipofectamine TM G h, 1:10 24,, G418,,, 96, 24 3, A549-luc A549-luc /ml /ml /ml /ml /ml /ml, 100 μl 96, μl 30 mg/ml, 37 o C 10 min, A549 A549-luc, 24, / 1~7 d, 3,

3 1206 ISSN CN /Q Chin J Biotech August 25, 2009 Vol.25 No.8, 1.2.5, 6~7, (20±2) g, A549 A549-luc, PBS /ml, 200 μl, 3 (IVIS 200) 150 μl 30 mg/ml, 10~25 min 1, SCID SCID, 6~7, (20±2) g, A549 A549-luc, PBS /ml, 200 μl SCID, 3 (IVIS 200) 150 μl 30 mg/ml, 10~25 min 3 1, A549 A549-luc A549 A549-luc 26 d,, ; 30 d, SCID,, 3 μm, HE 1.3 统计学处理 (means SD), SPSS 结果 2.1 稳定表达荧光素酶的 A549 细胞系 (A549-luc) 的建立及鉴定 A549 G μg/ml, pgl4.17(luc2/neo) G418, 9,, 1A A549-luc clone1, A549-luc clone2 A549-luc clone7, A549-luc clone5 A549-luc clone6 A549-luc clone8 A549-luc clone9, A549-luc clone1 A549-luc clone2 A549-luc clone7,,, A549-luc clone1 A549-luc clone2 R , A549-luc clone7 R 0.847,,, A549-luc clone1 A549-luc clone2 A549-luc clone7 图 1 A549-luc 的荧光素酶鉴定 Fig. 1 Identification of luciferase activity in A549-luc cells. (A) In vitro bioluminescence of A549-luc cells. (B) The luciferase activity of different concentration of A549-luc cells.

4 : 稳定表达荧光素酶的 A549 细胞系的生长特性 A549 A549-luc clone1 A549-luc clone2 A549-luc clone7, 2 4, A549 A549-luc clone1 A549-luc clone2 A549-luc clone7 ; 5 6, A549-luc clone2 A549; 7, A549-luc clone2 A549, 3 A549-luc A549,,,,, [8], A549-luc clone7, 19,,, A549-luc clone7 G418, A549-luc clone7, A549-luc A549-luc clone1 A549-luc clone2 A549-luc clone7 2.3 裸鼠皮下移植瘤模型的建立 A549 A549-luc clone1 A549-luc clone2 A549-luc clone7,, 3 A549, ( ), 5 d,, A549-luc clone1, A549-luc clone7, A549-luc clone1, 12, 26, 图 2 Fig. 2 A549-luc 细胞的生长曲线 Growth curve of A549-luc cells. 图 3 Fig. 3 活体成像监测皮下接种的 A549-luc clone 在裸鼠体内生长情况 (n=3) In vivo imaging of subcutaneous tumor growth of A549-luc clone cell in nude mice (n=3).

1208 ISSN1000-3061 CN11-1998/Q Chin J Biotech August 25, 2009 Vol.25 No.8 图 4 活体成像监测尾静脉接种的 A549-luc clone 在 SCID 鼠体内的生长情况 (n=3) Fig.

(B) In vivo imaging of intravenous tumor growth of A549-luc clone2 cell. clone2,, 2.

5 1208 ISSN CN /Q Chin J Biotech August 25, 2009 Vol.25 No.8 图 4 活体成像监测尾静脉接种的 A549-luc clone 在 SCID 鼠体内的生长情况 (n=3) Fig. 4 In vivo imaging of intravenous tumor growth of A549-luc clone cell in SCID mice (n=3). (A) In vivo imaging of intravenous tumor growth of A549-luc clone1 cell. (B) In vivo imaging of intravenous tumor growth of A549-luc clone2 cell. clone2,, 2.4 SCID 鼠尾静脉移植瘤模型的建立 A549-luc clone1 A549-luc clone2 SCID, 4,, A549-luc clone 1, A549-luc clone2 SCID,,,,, 4, A549-luc clone1 SCID, ( 4A) SCID A549-luc clone1 24,,, SCID A549-luc clone2 15, 30, SCID,, A549-luc clone2 ( 4B), A549-luc clone2 SCID, 2.5 A549 及 A549-luc 细胞原位成瘤及转移能力的病理形态学观察 A549 A549-luc, 26 d,,, 5, A549-luc clone1,, A549 A549-luc clone2 A549-luc clone7, A549 A549-luc clone1 图 5 皮下移植瘤模型的 HE 切片 (20 ) Fig. 5 HE staining of xenograft in subcutaneous implanted tumor models (20 ).

6 : 1209 图 6 尾静脉接种模型脏器的 HE 切片 (20 ) Fig. 6 HE staining of organs in intravenous models (20 ). A549-luc clone2 SCID, 30 d SCID,, HE, 6 3,, A549-luc clone2, A549 A549-luc clone1 2, A549-luc clone2 A549 A549-luc clone1, 3 讨论,, [9],, [10],,,, pgl4.17[luc2/neo] A549, 3,, A549-luc clone2, A549-luc clone1

7 1210 ISSN CN /Q Chin J Biotech August 25, 2009 Vol.25 No.8 A549-luc clone2 SCID,, A549-luc clone2, A549 A549-luc, A549-luc clone2,, :, ;, ;, ;,,,, A549-luc, REFERENCES [1] Jemal A, Siegel R, Ward E, et al. Cancer statistics, CA Cancer J Clin, 2006, 56(2): [2] Rembrink K, Romijn JC, vanderkwast TH, et al. Orthotopic implantation of human prostate cancer cell lines: a clinically relevant animal model for metastatic prostate cancer. Prostate, 1997, 31(3): [3] Licha K, Olbrich C. Optical imaging in drug discovery and diagnostic applications. Adv Drug Del Rev, 2005, 57(8): [4] Ray P, Wu AM, Gambhir SS. Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice. Cancer Res, 2003, 63(6): [5] Caceres G, Zankina R, Zhu X, et al. Determination of chemotherapeutic activity in vivo by luminescent imaging of luciferase-transfected human tumors. Anticancer Drugs, 2003, 14(7): [6] Jenkins DE, Oei Y, Hornig YS, et al. Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clin Exp Metastasis, 2003, 20(8): [7] Zeamari S, Rumping G, Floot B, et al. In vivo bioluminescence imaging of locally disseminated colon carcinoma in rats. Br J Cancer, 2004, 90(6): [8] Brutkiewicz S, Mendonca M, Stantz K, et al. The expression level of luciferase within tumor cells can alter tumor growth upon in vivo bioluminescence imaging. Luminescence, 2007, 22(3): [9] Yu YA, Timiryasova T, Zhang Q, et al. Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals. Anal Bioanal Chem, 2003, 377(6): [10] Caceres G, Zhu XY, Jiao JA, et al. Imaging of luciferase and GFP-transfected human tumors in nude mice. Luminescence, 2003, 18(4):

眉题 1205 Keywords: luciferase, A549 cell, animal model, xenograft, in vivo imaging system 癌症极大地威胁着人类的健康 [1], 世界卫生组荧光素酶 (Luciferase) 的编码基因和 G418 的抗性基织

眉题 1205 Keywords: luciferase, A549 cell, animal model, xenograft, in vivo imaging system 癌症极大地威胁着人类的健康 [1], 世界卫生组荧光素酶 (Luciferase) 的编码基因和 G418 的抗性基织生物工程学报 Chin J Biotech 2009, August 25; 25(8): 1204-1210 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 医学与免疫生物技术