生物工程学报 Chin J Biotech 2008, January 25; 24(1): 68-73 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn 2008 Institute of Microbiology, CAS & CSM, All rights reserved. 研究报告 王淑静 1,2,3, 刘兴汉 4, 季宇彬 1, 陈宁 3 1, 150076 2, 150030 3, 150076 4 -, 150081 : 肿瘤抑素抗肿瘤相关肽 -19 肽是由肿瘤抑素 185~203 位氨基酸组成, 具有直接抑制黑色素瘤细胞生长作用, 但其对肝癌细胞增殖和凋亡是否有影响, 对肝癌是否具有治疗作用还需进一步研究 本研究中采用基因工程技术将合成 19 肽基因与载体 ptyb2 重组后进行蛋白表达 纯化获得 19 肽 通过 MTT 法 生长曲线观察 19 肽对人肝癌细胞生长抑制作用 ; TUNEL 标记法 流式细胞仪细胞周期检测法 透射电镜观察 19 肽对肝癌细胞凋亡的影响 ; 小鼠 H22 腹水型转移型肝癌实体瘤抑瘤实验证明其体内的抑瘤作用 MTT 实验和生长曲线实验表明随着 19 肽浓度的增加肝癌细胞的存活率下降 在相同 19 肽浓度下, 随着作用时间延长存活细胞逐渐减少 电镜观察治疗组细胞出现明显凋亡, 流式细胞仪可检测到前 G1 峰, TUNEL 标记法也证实治疗组可见明显的凋亡细胞, 体内 19 肽作用的小鼠 H22 腹水型转移型肝癌的抑瘤率达 48.46% 可见, 肿瘤抑素 19 肽可抑制肝癌细胞生长, 促进肝癌细胞凋亡, 对肝癌具有一定的治疗作用 : 肿瘤抑素, 细胞增殖, 细胞凋亡, 肿瘤治疗 Effect of Tumstatin Anti-tumor Peptide on Proliferation and Apoptosis of Hepatocarcinoma Cell Shujing Wang 1,2,3, Xinghan Liu 4, Yubin Ji 1, and Ning Chen 3 1 Institute of Pharmacology, Harbin Commercial University, Harbin 150076, China 2 Postdoctoral Station of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China 3 Pharmacological College, Harbin Commercial University, Harbin 150076, China 4 Bio-pharmaceutical Key Laboratory of Heilongjiang Province Incubator of State Key Laboratory, Harbin Medical University, Harbin 150086, China Abstract: In this study, the basic sequences of 19peptide were synthesized and inserted into vector ptyb2. After being expressed and purified, the soluble 19peptide was obtained. The inhibiting effect on hepatocarcinoma cell was selected by 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay and cell growth curve. The apoptosis index was observed using TdT-mediated dutp nick end labeling (TUNEL), flow cytometry and transmission electron microscope (TEM). Its anti-tumor activity in vivo was verified in H22 ascitic fluid transfevent hepatocarcinoma of mice. 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-di- Received: March 26, 2007; Accepted: September 17, 2007. Supported by: the National Natural Science Foundation (No.30472035), Natural Science Foundation of Heilongjiang Province (No.TD2005-21) and the Research project from Department of Education of Heilongjiang Province (No. 11511104). Corresponding author: Shujing Wang. Tel: +86-451-84838207; E-mail: misswsj@163.com 国家自然基金资助项目 (No.30472035), 黑龙江省自然科学基金资助项目 (No.TD2005-21), 黑龙江省教育厅自然基金资助项目 (No. 11511104)
: 69 phenyl-2h-tetrazolium bromide (MTT) assay and cell growth curve showed cell viability decrease with the 19peptide.Survival hepatocarcinoma cell decreased with time. In the treatment group, obvious change of apoptosis, the appearance of sub-g1 phase and dyed brown cells were seen by transmission electron microscope (TEM), flow cytometry and TdT-mediated dutp nick end labeling (TUNEL), The inhibition rate of mouse H22 ascitic fluid transfevent hepatocarcinoma growth was 48.46%. As a whole, 19peptide could directly inhibit hepatocarcinoma cell proliferation and promote hepatocarcinoma cell apoptosis. Keywords: Tumstatin, cell proliferation, cell apoptosis, tumor therapy (Tumstatin) IVα3, 80 - [1] 2000, Kamphaus, [2] C 185~203 19, N 74~98 T7 (25 ),,,,, (28 kd), -,, 19,,,,, [3],,,,, 19,, 1 材料和方法 1.1 材料 1.1.1 JM109 BL-21(DE3) Promega, ptyb2 NEB 1.1.2 H22 ; 1.1.3 Nde I Sma I Xho I NEB ; RPMI1640 Sigma ; ; TUNEL 1.2 方法 1.2.1 19 185~203,, 19 19 ptyb2 BL-21(DE3), 0.1 mmol/l IPTG, 28 6 h, 19, [4,5] 19 : 5 T ATG GCT AGC CCT TTC CTA GAA TGT CAT GGA AGA GGA ACG TGC AAC TAC TAC TCA AAC TCC 3 3 AC CGA TCG GGA AAG GAT CTT ACA GTA CCT TCT CCT TGC ACG TTG ATG ATG AGT TTG AGG 5 1.2.2 MTT 1640, 0.25%,, 5 10 4 /ml, 100 μl 96, 24 h 19, 6 PBS 19 0 μg /ml, 44 μg/ml, 88 μg/ml, 132 μg/ml, 176 μg/ml, 220 μg/ml 48 h, 5 mg/ml MTT 20 μl, 4 h, 100 μl DMSO, 10~20 min, 490 nm, (A ), 19 = / 100% 1.2.3,
70 ISSN1000-3061 CN11-1998/Q Chin J Biotech January 25, 2008 Vol.24 No.1, 5 10 4 /ml 24 400 μl, 24 h, 400 μl 19 44 μg/ml 19 PBS 3,,,,,, 6 d - 1.2.4 (TUNEL),,, 5 10 4 /ml 6, 6 1 ml, 24 h 19 44 μg/ml 19, PBS, 24 h, 4% 20~30 min, 50 μl TUNEL, 50 μl PDB 50~100 μl DAB,,, 10, 1.2.5, 2 10 5 /ml, PBS, 19 44 μg/ml 37 24 h, PBS 2,, 0.25%,, 1000 g 10 min,, PBS 2, 1~1.5 ml70%, 0.5 10 6 ~1.0 10 6 /ml,,, 4 24 h Cycle TEST TM PLUS DNA Reagent Kit 1.2.6, 2 10 5 /ml, PBS, 19 44 μg/ml 24 h, 0.25%, 1500 g, 15 min,, PBS, 4 24 h, (50~80 nm) 1.2.7 18~22 g 24 2, 19 100 μl ; 19 4.88 μg/kg 19, 1, 7 d,, H.E,, 2 结果 2.1 19 肽获得 19 ptyb2,, 19 BL21(DE3), 19, 90% [5] 2.2 细胞活性检测 2.2.1 MTT MTT ( 1),, 19, 19,, 19, (P < 0. 05) 图 1 19 肽对人肝癌细胞生长的抑制作用 Fig. 1 The inhibitory effect of 19peptide on human hepatocarcinoma cell
: 71 2.2.2 2.2.3, 19, 19,,,, 19 0, ( 2), 19 TUNEL,,,, 10, 33.9% 3.48%( 3) 2.2.4 19 G1, 10.07% 1.75% G0-G1 G2-M, S ( P < 0.05) 图 2 人肝癌细胞生长曲线 19 G0/G1, Fig. 2 The growth curve of human hepatocarcinoma cell, 图 3 凋亡细胞原位末端标记法观察人肝癌细胞形态改变 Fig. 3 Morphological observation of human hepatocarcinoma cell using TUENEL assay (A) Experiment under fluorescent microscopy; (B) Control under optical microscopy; (C) Experiment under optical microscopy Fig. 4 图 4 流式细胞仪细胞周期实验检测 19 肽对肝癌细胞周期的影响 The effect of 19peptide on cell circle of human hepatocarcinoma cell using FAC
72 ISSN1000-3061 CN11-1998/Q Chin J Biotech January 25, 2008 Vol.24 No.1 2.2.5,,,, 19,,,,,,,, 2.3 动物活性检测 2.3.1 19, 19 t, 19, P 0.05 48.46% ( 1) 2.3.2 (x20), 19 ( 6) (A) Control (B) Experiment Fig. 5 图 5 透射电镜检测肝癌细胞形态结构 Morphological changes of human hepatocarcinoma cell using TEM Table 1 表 1 19 肽对 H22 小鼠腹水转移性肝癌的影响 Effect of 19peptide on H22 ascitic fluid transfevent hepatoma of mice growth( x±s, n=12) x i /g x i /g IR/% Control 19 peptide 1.0152 3.1523 2.7645 2.9969 2.5083 2.6511 2.4325 3.0718 2.8947 1.7968 2.0356 2.8568 1.4224 1.7016 0.6547 2.1896 0.8616 0.9472 1.3080 0.8116 1.7822 1.8980 0.7315 1.2459 2.5147±0.6247 1.2962±0.5095 48.4551 Fig. 6 图 6 19 肽对小鼠腹水型转移性肝癌中癌组织的影响 The effect of 19peptide on tumor tissue in murine H22 ascitic fluid transfevent hepatocarcinoma 3 分析与讨论,,,,,,,
: 73, 19,,,,, 19, SNS [6] 19 [5],, [7],,,,, MTT 19 19 19,,, 19 44 μg/ml,, 2~3 d,, 19, 19 TUNEL 19,,,,,,,, TUNEL 19, G0/G1, 19, 19,,,, 19, 48.46%,, 19 -,,, 19 REFERENCES [1] Saus J, Wieslander J, Langeveld J P, et al. Identification of the goodpasture antigen as the alpha 3(IV) chain of collagen IV. J Biol Chem, 1988, 263(26): 13374 13380. [2] Maeshima Y, Colorado P C, Torre A, et al. Distinct antitumor properties of a type IV collagen domain derived from basement membrane. J Biol Chem, 2000, 275(28): 21340 21348. [3] Floquet N, Pasco S, Ramont L, et al.. The antitumor properties of the alpha3(iv)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent.j Biol Chem, 2004, 279(3): 2091 2100. [4] Yu Q, Zhou LY, Lin XS, et al. Expression,purification and activity of recombinant human osteogenic growth peptide (rhogp). Chin J Biochem Mol Biol, 2004, 20(4): 467 472.,,,.., 2004, 20(4): 467 472. [5] Wang SJ, Liu Y, Lin XS, et al. Cloning and biological activity of anti-tumor peptide of Tumstatin. Chin J Biochem Mol Biol, 2005, 21(3): 322 328.,,,.., 2005, 21(3): 322 328. [6] Maeshima Y, Colorado P C, Kalluri R. Two RGD-independent alpha v beta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties. J Biol Chem, 2000, 275(31): 23745 23750. [7] Maeshima Y, Yerramalla UL, Dhanabal M, et al. Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis. J Biol Chem, 2001, 276(34): 31959 31968.