摘要
一研究目的:
为探索一种新的免疫毒素治疗胰腺癌的方法,通过SPDP化学偶联法,我们将眼镜蛇毒细胞毒素(CTX)与抗IL-4R单克隆抗体(MAIL4R)构建成免疫毒素MAIL4R-CTX,观察其是否具有导向性杀伤胰腺癌细胞的作用。
二材料与方法
1.眼镜蛇毒细胞毒素的分离纯化:先采用SP-Sephadex C-25离子交换柱层析、再用Superdex 75凝胶过滤及Phenyl- Sepharose High Performance疏水层析两步从舟山眼镜蛇粗毒中精细纯化分离CTX。
2.免疫毒素的制备:应用SPDP偶联法,在室温下,先将CTX与SPDP反应生成CTX-PDP;再将MAIL4R与SPDP反应生成MAIL4R-PDP,后者在DTT的作用下还原为MAIL4R-SH;最后CTX-PDP与MAIL4R-SH在室温下充分反应至少24小时后,生成抗白介素4-受体单抗-细胞毒素免疫毒素(MAIL4R-CTX)。
3.采用SDS-聚丙烯酰胺凝胶电泳、双向免疫扩散和免疫斑点试验检测免疫毒素的组成。
4.采用免疫组织化学染色检测胰腺癌组织中IL-4R的表达。采用免疫细胞化学染色检测体外培养人胰腺癌细胞株PANC-1、BxPC-3中IL-4R的表达。
5.采用DAB法检测免疫毒素MAIL4R-CTX对高表达IL-4R细胞BxPC-3、PANC-1和不表达IL-4R人肺腺癌细胞H1299的结合能力。
6.采用MTT法分别测定CTX、MAIL4R及MAIL4R-CTX对体外培养胰腺癌细胞和肺癌细胞的作用。
三结果
1.眼镜蛇毒粗毒的SP-Sephadex C-25柱层析分离共得14个蛋白峰;组分XIII鉴定为细胞毒素,再经Superdex 75凝胶过滤和Phenyl-Sepharose HP疏水层析获得单一对称蛋白峰,测其分子量为:77.381kDa。将其命名为CTX。
2.CTX与SPDP反应物经Superdex30凝胶柱后获得的第一峰为CTX-PDP;MAIL4R与SPDP反应物经Superdex30凝胶柱获得的第一峰为MAIL4R-PDP;MAIL4R-PDP经DTT还原后再经凝胶柱获得的第一峰为MAIL4R-SH;最后将过量的CTX-PDP与MAIL4R-SH反应物经Superdex30凝胶过滤,获得2个蛋白峰,第一峰即为免疫毒素MAIL4R-CTX。
3.SDS-聚丙烯酰胺凝胶电泳、双向免疫扩散和免疫斑点试验显示该免疫毒素分子中既含有CTX也含有MAIL4R。
4.免疫组织化学染色显示在胰腺癌细胞中,细胞质呈现均匀着色的棕黄色颗粒,而正常胰腺组织细胞均为阴性着色。免疫细胞化学显示在胰腺癌PANC-1、BxPC-3细胞中,细胞质呈现棕黄色颗粒着色,而H1299细胞为阴性着色。
5.高表达IL-4R的BXPC-3和PANC-1细胞DAB染色为棕色,而不表达IL-4R细胞H1299则未被染色。
6.采用MTT法观察到,MAIL4R-CTX在1.2 ug/ml时即明显抑制PANC-1、BxPC-3细胞的生长,在浓度为18.75ug/ml作用4h时PANC-1和BxPC-3细胞分别有86.4%和95.2%被杀伤,H1299细胞仅为26.8%;CTX对PANC-1,BxPC-3和H1299细胞均有明显抑制作用,在浓度为8ug/ml时对三株细胞的抑制率分别达到88.5%,87.2%和90%,但三者无明显差别;MAIL4R对PANC-1、BxPC-3和H1299细胞均无明显抑制作用。
四结论
1.采用SP-Sephadex C-25阳离子交换柱层析、Superdex 75凝胶过滤及Phenyl-Sepharose HP疏水层析三步分离纯化可得到低毒高效的眼镜蛇毒细胞毒素(CTX)纯品。
2.以SPDP法可以成功地将抗白介素-4受体单克隆抗体(MAIL4R)与CTX构建成免疫毒素MAIL4R-CTX。
3.胰腺癌组织和胰腺癌细胞高表达IL-4R,正常胰腺组织不表达IL-4R。免疫毒素MAIL4R-CTX对高表达IL-4R的胰腺癌细胞具有选择性杀伤作用。
1. Objective
To explore an effective new approach that against pancreatic cancer with a novel immunotoxin ,we purify a effective Cobra venom cytotoxin(CTX) from cobra (Naja Naja atra)venom. Then CTX was selected as a "warheads" and anti-IL4-R monoclonal antibody (MAIL4R) as a carrier . With heterotypic bifunctional coupling agent SPDP method, MAIL4R and CTX was connected together and the immunotoxin MAIL4R-CTX was obtained. The selective killing effects on pancreatic carcinoma cells which has high expression of IL-4R was observed and compared so as to provide experimental data for new treatment method of pancreatic cancer.
2. Materials and methods
1. Isolation and Purification of CTX: In this study, the crude venom of cobra venom(Naja atra) was first isolated on SP-Sephadex C-25 cation-exchange column, The effect of tenth～thirteenth protein peaks to the rat isolated heart perfuse preparations and the rat isolated phrenic nerve-diaphragm preparations then be observed; protein peak XIII were further purified by Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography once each; SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by Coomassie brilliant blue stains be used to assess the purity of peak XIII and its mol. wt of them were calculated.
2. Immunotoxin MAIL4R-CTX preparation: In room temperature, the cytotoxin of Naja naja atra reacted with SPDP to form CTX-PDP. Then we made the anti-interleukin4-receptor monoclonal antibody and SPDP have the same reaction to form anti-IL-4-receptor monoclonal antibody-PDP, and it was reduced to monoclonal antibody-SH by DTT. In the end, the monoclonal antibody-SH was thoroughly mixed with CTX-PDP in room temperature for at least 24 hours to produce a new type immunotoxin MAIL4R-CTX.
3. Identified the composition of immunotoxin MAIL4R-CTX by the means of SDS-PAGE polyacrylamide gel electrophoresis and double immunodiffusion and dotimmunobindingassay.
4. Immunohistochemical technique was used to detect the expression of IL-4R in normal and pancreatic cancer specimens and immunocytochemical technique was used to detect the expression of IL-4R in human pancreatic cancer cell lines (PANC-1 and BxPC-3) and human NSCLC cells.
5. Binding ability of the conjugate to BxPC-3 and PANC-1 cells with highly expressed IL-4R and H1299 cells without IL-4R expression was measured by DAB method.
8. Using MTT method, Cytotoxic activity of AIL-4R ,CTX and CTX-AIL-4R were tested in PANC-1,BXPC-3 and H1299 cell lines in vitro respectively.
3. Results
1. Isolation and Purification of CTX: The crude venom of cobra venom(Naja atra) was isolated on SP-Sephadex C-25 cation-exchange column and fractionated into 14 protein peaks; The protein peak XIII could produce the rat isolated Langendorffs preparations to decrease their contracture heights, ultimately leading to complete cardiac arrest. Peak XIII were further purified by Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography once each and a single protein peak be obtained. It migrated as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by Coomassie brilliant blue stains which showed that it was homogeneous with one band. Its mol. wt was calculated to be 7.381 kDa orderly by Image Master VDS. The results showed that the protein peak XIII was a CTX and then we named it CTX in this paper.
2. Gel filtration chromatography of immunotoxin: The reaction products of CTX and SPDP were loaded on Superdex30 gel column and three major protein peaks were obtained, in which the first was CTX-PDP with large molecular weight. The reaction products of MAIL4R and SPDP were loaded on Superdex30 gel column, and 2 peaks were obtained, in which the first peak was MAIL4R-PDP; After reduction of MAIL4R-PDP by DTT, two main peaks were obtained after filtration on the column, in which the first peak was MAIL4R-SH. Finally, filtration of excessive reaction products of CTX-PDP and MAIL4R-SH were carried out on Superdex30 gel column with two protein peaks obtained, in which the first peak was the immunotoxin MAIL4R-CTX, and the second peak was unbound CTX-PDP.
3. In reduced SDS-PAGE polyacrylamide gel electrophoresis, MAIL4R-CTX showed two zones of minor molecular weight protein. Then antivenomous serum made immunoprecipitation among MAIL4R-CTX, CTX, CTX-PDP and peakⅡof MAIL4R-CTX, and eventually the first three samples formed the lines of sedimentation. It is found in Dotimmunobindingassay of the immunotoxin that, immunotoxin MAIL4R-CTX not only had strong antigen-antibody response with anti-cobra venom antibody, but also could directly react with goat anti-mouse antibody, suggesting that MAIL4R-CTX contained both the cobra venom cytotoxin and mouse antibody (ie MAIL4R)
4. Normal pancreas tissue samples did not stain or showed weak positive staining for IL-4R. In contrast, all 26 pancreatic cancer specimens overexpress IL-4R; Both human pancreatic cancer cell lines BxPC-3and PANC-1 were stained brown, while human lung cancer cells H1299 who did not express IL-4R were not stained.
5. BxPC-3 and PANC-1 cells have high expression of IL-4R were stained brown and H1299 cells that do not express IL-4R were not stained in DAB method.
6. By MTT method, we observed that MAIL4R-CTX significantly inhibited growth of BxPC-3, PANC-1 cells that have high expression of IL-4R at the concentration of 1.2 ug / ml, while for H1299 cells with no or low expression of IL-4R, inhibitory effect appeared when the concentration was more than 5 ug / ml. At the concentration of 18.75 ug / ml, the cell killing rate of MAIL4R-CTX on PANC-1 and BxPC-3 cells was 86.4% and 95.2% respectively, while it was only 26.8% on H1299 cells; CTX had rapid killing effects on PANC-1, BxPC-3 and H1299 cells. After 4h’s action,the inhibition rate of CTX in the concentration of 8 ug / ml on three cell strains was 88.5%, 87.2% and 90% respectively; Monoclonal antibody of interleukin-4 receptor had no obvious killing effect on the above cells.
4. Conclusions
1. A purified CTX isoforms from the Naja atra venom was obtained by SP-Sephadex C-25 cation-exchange chromatography, Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography step by step.
2. With heterotypic bifunctional coupling agent SPDP method, MAIL4R and CTX can be successful connected together to be a novel immunotoxin MAIL4R-CTX.
3. IL-4R overexpress in pancreatic cancer specimens and cells but not in normal pancreas. Immunotoxin MAIL4R-CTX had selective killing effect on pancreatic cancer cells which have high expression of IL-4R.
引文
1. Lowenfels AB, M aisonneuve P. Epidemiology and prevention of pancreatic cancer. Jpn J Clin Oncol, 2004, 349(5):238-244.
2. 赵玉沛.胰腺癌的诊治现状与展望[J].医学临床研究,2005,22(10): 1353-1355.
3. 赵蓉,游云,高敏新,等.蛇毒对人及小鼠传代细胞的杀伤作用 .福建医学院学报,1988,22(3):204-209.
4. Brganca BM, Pater NT, Badrinath PG. Isolation and properties of a cobra venom factor selectively cytotoxic to yoshida sarcoma. [J].Biochem Biophys Acta, 1967, 136:508-520.
5. Lipps BV.Novel snake venom proteins cytotoxin to cancer cells in vitro and in vivo systems[J].J Venom Animo Toxins, 1999I5(2)l 172-183.
6. Chaim MA,Borkow G,Ovadia M.Isolationand characterization of a cytotoxin P4 from the venomof Naja nigricollis prefer entially active on tumour cells. [J]. Biochem Int,1991,24:415—421.
7. 许云禄,张明芳.舟山眼镜蛇毒细胞毒索一 F 对人鼻咽癌细胞和乳鼠心肌细胞的抑制作用[J].福建医科大学学报,2004,38(2):121—124.
8. 左长清,林振桃,孔天翰.中华眼镜蛇毒组分 CⅡ诱导多药耐药白血病细胞K562/A02 凋亡[J].广州医学院学报, 2004;32:26—30.
9. Fawzia.Treatment tfials of E.A.C with crude N.NigrieoUis ven. om and its fraction III fmice][A1.In:Kornalik F.Mebs D.Proeeedingof the 7th European symposium on an imal,plan t an dmicrobial toxins[C].1986.107
10. 罗伯诚,王存川,杨冠群,等.口服眼镜蛇毒抑制小鼠肿瘤的研究 [J].中华实验外科杂志,1993,1:31～32.
11. 许云禄,吴国土,林建忠.舟山眼镜蛇毒细胞毒素-F 的体内抗肿瘤作用[J].福建医科大学学报, 2003,37 (3): 298-300.
12. 许云禄.张明芳,刘广芬.舟山眼镜蛇毒细胞毒索的分离纯化及其体外抗肿瘤活性[J].中国生化药物杂志,2003,24(3):127—13O.
13. 李钦章, 黄沿中, 杨冠群.眼镜蛇膜眼镜蛇膜毒素与单克隆抗体偶联物对鼻咽癌细胞的特异性杀伤作用[J].暨南大学学报(自然科学版),1998,19(3):94-99.
14. 嘉佑,张和君,杨爱德等.中华眼镜蛇膜毒素与 McAb 偶联物对白血病细胞的杀伤作用[J].中国医学科学院学报,1992,14(3):179-183.
15. Obiri NI, Siegel JP, Varricchio F, et a1.Expression of high-affinity IL-4 receptors on human melanoma, ovarian and breast carcinoma cells. [J].Clin. Exp. Immunol , 1994,95: 148–155.
16. Topp MS, Koenigsmann M, Mire-Sluis A, et al. Recombinant Recombinant human interleukin-4 inhibits growth of some human lung tumor cell lines in vitro and in vivo. [J].Blood, 1993,82: 2837–2844.
17. Murata T, Noguchi PD, and Puri RK. Receptors for interleukin (IL)-4 do notassociate with the common-chain, and IL-4 induces the phosphorylation of JAK2 tyrosine kinase in human colon carcinoma cells. [J].Biol. Chem, 1995,270: 30829–30836.
18. Rand R. W Kreitman, RJ, Patronas, N, et a1. Intratumoral administration of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-grade glioma. [J].Clin. Cancer Res. 2000, 6: 2157–216.
19. Husain SR, Gill P, Kreitman RJ, et a1. Interleukin-4 receptor expression on AIDS-associated Kaposi’s sarcoma cells and their targeting by a chimeric protein comprised of circularly permuted interleukin-4 and Pseudomonas exotoxin. [J].Mol. Med., 1997, 3: 327–328.
20. Kawakami, K, Leland, P, and Puri, RK. Structure, function, and targeting of interleukin 4 receptors on human head and neck cancer cells. [J].Cancer Res, 2000,60: 2981–2987.
21. Atkins MB, Vachino G, Tilg H, et a1.Phase I evaluation of thrice daily intravenous bolus interleukin-4 in patients with refractory malignancy. [J].Clin. Oncol, 1992,10:1802–1809.
22. Volpert, OV, Fong T, Koch AE, et a1. Inhibition of angiogenesis by interleukin 4. [J].Exp. Med. 1998,188: 1039–1046.
23. Mariko K, Koji K, Mitomu K, et a1. Hodgkin lymphoma therapy with interleukin-4 receptor–directed cytotoxin in an infiltrating animal model [J].Blood, 2005, 105(9):3707-3713.
24. Debinski W,Hass GP,Husain SR,el al.Targeting of interleukin-13receptor on human renal-cll carcinoma-cells by a recombinant chimericprotein composed of interleukin-13 and a truncated form of pseudomonasexotoxin-A(PE38QQR). [J].Blood,1996,2(3):350-353.
25. Koji K,Mariko K,Syed R,et al.Targeting interleukin-4 receptors for effective pancreatic cancer therapy. [J].Cancer Res,2002,62(1): 3575-3580.
26. 欧阳晶,王健伟,脱厚珍,等.白喉毒素类免疫毒素研究进展 [J].中国生物工程杂志,2003,23 (4):36-42.
1. Staff of the department of Pharmacology University of Edinburgh 1970, Pharmacological experiments on isolated preparations:P116-117.
2. Bulbring E. Observation on the isolated phrenic nerve-diaphragm of the rat. Br.J.Pharmac,1946,1,38
3. 张明芳,许云禄.眼镜蛇毒细胞毒素的体内外抗肿瘤活性及毒性比较[J].福建医科大学学报, 2005,39(3): 243-246.
4. Jayaraman-G,Kumar-T-K,Tsai-C-C,et a1. Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra(Naja naja atra) — identification of structural features importantfor the lethal action of snake venom cardiotoxins [J].Protein Sci,2000,9(4):637—646.
5. Brganca BM, Pater NT, Badrinath PG. Isolation and properties of a cobra venom factor selectively cytotoxic to yoshida sarcoma. [J].Biochem Biophys Acta, 1967, 136:508-520.
6. 赵蓉,游云,高敏新,等.蛇毒对人及小鼠传代细胞的杀伤作用 .福建医学院学报,1988,22(3):204-209.
7. Rand RW,Kreitman RJ,Patronas N,et al.Intratumoral administration of recombinant circularly permuted interleukin-4-pseudomonas exotoxin in patients with high-grade glioma.[J].Clin Cancer Res,2OOO,6(6): 2l57-2l65.
8. 许云禄.张明芳,刘广芬.舟山眼镜蛇毒细胞毒索的分离纯化及其体外抗肿瘤活性[J].中国生化药物杂志,2003,24(3):127—13O.
9. 李浩淼,杨惠玲,李爱民,等.中华眼镜蛇毒对荷 S180 小鼠抑瘤作用及其机制初探[J].河南中医,2001,19 (2):19—22.
10. 许云禄,吴国土,林建忠,等.舟山眼镜蛇毒细胞毒素-F 的体内抗肿瘤作用[J].福建医科大学学报, 2003,37(3): 298-300.
11. Dufton MJ, Hider RC. Structure and pharmacology of Elapid cytotoxins [J].Pharmac Ther. 1988, 36: 31-40.
12. Perry WLM . Drugs and the perfused rabbit heart[A] . Pharmacological experiments on the isolated preparations[C]. 2nd ed,Hongkong:Common Wealth Printing Press Ltd,1970,117-119.
13. Abranham IL,Fritz HC.The preparation of phospholipase A2-free cardiotoxin from snake venom by hydrophobic interaction chromatography. [J].Toxicon,1979,17:193—197.
1. 张玲,李官成,朱兆玲,等.抗 CD3、CD7 单抗-PAPS 免疫毒素的构建及联合杀伤效应[J].中山大学学报(医学科学报),2003,24(3):39-41.
2. 张玲,冯炼强,林学颜.抗 CD4 单抗-PAP 免疫毒素对 T 淋巴母细胞样 CEM细胞的体外效应[J]. 中山大学学报(医学科学报),2004,25(5):441-444.
3. Hinman CL,Tang HP.A membrane lytic immunocon-jugate selective for human tumor T lymphocytes[J].Int J Immunopha,1998,20,467--468.
4. Sambrook J,Frisch E F,Maniatis T. Techniques in immuology.In:Molecular Cloning:A laboratory Manual 2nded [M].New York:Cold Spring Harbor Laboratory Press,1989.324-898
5. 苗毅,喻春钊,高文涛.胰腺癌生物治疗的现状、问题及展望[J].腹部外科,2005,18(2):80-82.
6. 孔丽君,王清明,陈惠鹏.肿瘤导向治疗研究进展[J].生物技术通讯,2002,13(6):459-461.
7. Ehrlich P.The collected paper of Paul Ehrlich[J].1956,596-618.
8. Duvic M,Kuzel T,Olsen E,et a1.Quality of life is significantly improveed CTCL patients who responded to DAB389IL-2(ONKAT) fusion proteinBlood,1998, 92(1):2572-2576.
9. 李俊植.免疫毒素研究及临床应用进展(二)[J].中国生物制品学杂志,2004,
17(2):119-122.
10.Kreitman, R. J. Immunotoxins in cancer therapy. [J].Curr. Opin. Immunol., 1999, 11: 570–578.
11. Jerry Thompson, Huaizhong Hu, Joshua Scharff, et a1.An Anti-CD3 Single-chain Immunotoxin with a Truncated Diphtheria Toxin Avoids Inhibition by Pre-existing Antibodies in Human Blood[J].1995,270(47): 28037–28041.
12. Hi M, Kreitman RJ, Onda M, et a1.In vitro antibody evolution targeting germline hot spots to increase activity of all anti-CD22 immuneotoxin[J].Journal of Biological Chemistry 2005,280(1):607-617.
13. Carol A. Vater, Laura M. Bartle, John D. Leszyk, , et a1. Ricin A Chain Can Be Chemically Cross-linked to the Mammalian Ribosomal Proteins L9 and L10 [J].1995, 270: 12933 – 12940.
14. Tsai LC, Chen YL, Lee C,et al.Growth suppression of human aolorectalcarcinoma in nude mice by monoclonal antibody C27一abrin A chain conjugate [J].Dis Colon Rectum, 1995,38(10): 1067-1069.
15. 李钦章, 黄沿中, 杨冠群.眼镜蛇膜眼镜蛇膜毒素与单克隆抗体偶联物对鼻咽癌细胞的特异性杀伤作用[J].暨南大学学报(自然科学版),1998,19(3):94-99.
16. 嘉佑,张和君,杨爱德等.中华眼镜蛇膜毒素与 McAb 偶联物对白血病细胞的杀伤作用[J].中国医学科学院学报,1992,14(3):179-183.
17. 仲晓燕,刘广芬,王晴川,中华眼镜蛇毒细胞毒素14的纯化及抗癌活性[J].中国药理学报,1993,14:279-282。
18. Dubovskli PV,Lesovoy DM,Arseniev AS,et al.Interaction of three-finger toxins with phospholipid membranes:comparison of S-and P-type cytotoxins. Biochem [J] 2005, 387(3):807-815.
19. Braganca B.M.,Pater N.T,Badrinath P.G.,Isolation and properties of a cobra venom factor selectively cytotoxin to yoshida sarcoma.Biochem Biophys Acta[J].1967,136:508-520.
20. Harvey,A.L. Cardiotoxins from cobra venom.IN:Reptile Venoms and Toxins, pp.85-106. New York: Marcel Dekker.
21. Chaim MA,Borkow G,Ovadia M. Isolation and characterization of a cytotoxin P4 from the venom of Naja nigricollis nigricollis preferentially active on tumor cells.Bichem Int[J].1991,24(3):415-421.
22. 杜苍雨.中华眼镜蛇膜毒素的体外溶吉田肉瘤作用[J].生物化学与生物物理学报,1980,12(5):3495
23. 许云禄,杨丽娟,刘广芬,舟山眼镜蛇毒细胞毒素的分离纯化及其抗肿瘤活性[J].中国生化药物杂志,2003,24(3):127-130.
24. 甄永苏.抗体药物与肿瘤靶向治疗[J].医学研究杂志,2007,32(2):1-2.
25. Schmidt M,Reiser P,Hills D,et a1.Expression of an oncoenic mutant EGF receptor markedly increases the sensitivity of cells to an EGF-receptor-specific antibody-toxin[J].Int J Cancer,1998,75 (6):878—884.
26. Obiri NI, Siegel JP, Varricchio F, et a1.Expression of high-affinity IL-4 receptorson human melanoma, ovarian and breast carcinoma cells. [J].Clin. Exp. Immunol , 1994,95: 148–155.
27. Topp MS, Koenigsmann M, Mire-Sluis A, et al. Recombinant Recombinant human interleukin-4 inhibits growth of some human lung tumor cell lines in vitro and in vivo. [J].Blood, 1993,82: 2837–2844.
28. Murata T, Noguchi PD, and Puri RK. Receptors for interleukin (IL)-4 do not associate with the common-chain, and IL-4 induces the phosphorylation of JAK2 tyrosine kinase in human colon carcinoma cells. [J].Biol. Chem, 1995,270: 30829–30836.
29. Rand R. W Kreitman, RJ, Patronas, N, et a1. Intratumoral administration of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-grade glioma. [J].Clin. Cancer Res. 2000, 6: 2157–216.
30. Husain SR, Gill P, Kreitman RJ, et a1. Interleukin-4 receptor expression on AIDS-associated Kaposi’s sarcoma cells and their targeting by a chimeric protein comprised of circularly permuted interleukin-4 and Pseudomonas exotoxin. [J].Mol. Med., 1997, 3: 327–328.
31. Kawakami, K, Leland, P, and Puri, RK. Structure, function, and targeting of interleukin 4 receptors on human head and neck cancer cells. [J].Cancer Res, 2000,60: 2981–2987.
32. Mariko K, Koji K, Mitomu K, et a1. Hodgkin lymphoma therapy with interleukin-4 receptor–directed cytotoxin in an infiltrating animal model [J].Blood, 2005, 105(9):3707-3713.
33. Obiri NI, Tandon N, and Puri RK. Up-regulation of intercellular adhesion molecule 1 (ICAM-1) on human renal cell carcinomacells by interleukin-4. Int. [J]. Cancer, 61: 635–642, 1995.
34. Paul, WE. Interleukin-4: a prototypic immunoregulatory lymphokine. [J].Blood , 1991,77:1859–1870.
35. Volpert, OV, Fong T, Koch AE, et a1. Inhibition of angiogenesis by interleukin 4. [J].Exp. Med. 1998,188: 1039–1046.
36. Atkins MB, Vachino G, Tilg H, et a1.Phase I evaluation of thrice daily intravenous bolus interleukin-4 in patients with refractory malignancy. [J].Clin. Oncol, 1992,10:1802–1809.
37. Kawakami K, Kawakami M, and Puri RK. Overexpressed cell surface interleukin-4 receptor molecules can be successfully targeted for antitumor cytotoxin therapy. [J].Crit. Rev. Immunol, 2001, 21: 299–310.
38. Debinski W,Hass GP,Husain SR,el al.Targeting of interleukin-13receptor on human renal-cll carcinoma-cells by a recombinant chimericprotein composed of interleukin-13 and a truncated form of pseudomonasexotoxin-A(PE38QQR). [J].Blood,1996,2(3):350-353.
39 Koji K,Mariko K,Syed R,et al.Targeting interleukin-4 receptors for effective pancreatic cancer therapy. [J].Cancer Res,2002,62(1): 3575-3580.
40. Puri RK. Structure and functions of interleukin 4 and its receptor. In: R. Kurzrock, M. Talpaz, (eds.), Cytokines: Interleukins and TheirReceptors. Norwell, MA: Kluwer Academic, 1995, pp. 143–185.
41. Murata T, Obiri NI, Puri RK. Structure of and signaltransduction through interleukin-4 and interleukin-13 receptors. et al.Int. J. Mol. Med., 1998,1: 551–557.
42. Obiri NI, Debinski W, Leonard WJ, et al. Receptorfor interleukin 13: interaction with interleukin 4 by a mechanism that does not involve the common chain shared by receptors for interleukins 2, 4, 7, 9, and 15. [J].Biol. Chem. 1995, 270: 8797–8804.
43. 李俊植.免疫毒素研究及临床应用进展(一)[J].中国生物制品学杂志,2004,17(1):60-63.
44.欧阳晶,王健伟,脱厚珍,等.白喉毒素类免疫毒素研究进展 [J].中国生物工程杂志,2003,23 (4):36-42.
1. Mariko K, Koji K, Vitaly A. et al. Interleukin 4 Receptor on Human Lung Cancer: A Molecular Target for Cytotoxin Therapy. [J]. Clinical Cancer Research, 2002, 8: 3503–3511.
2.凌江红,李家邦,聂海明,等.幽门螺杆菌阳性消化性溃疡患者胃黏膜核因子κB、b-防御素-2表达及其与胃窦炎症的关系 [J].世界华人消化杂志, 2006,14(4):376-381.
3. Sambrook J,Frisch E F,Maniatis T. Techniques in immuology.In:Molecular Cloning:A laboratory Manual 2nd ed [M].New York:Cold Spring Harbor Laboratory Press,1989.324-898.
4. 刘 建 文 主 编 . 药 理 实 验 方 法 学 - 新 技 术 与 新 方 法 [M]. 化 学 工 业 出 版社.2003,25-32.
5.赵玉沛.胰腺癌的诊治现状与展望[J].医学临床研究,2005,22(10): 1353-1355.
6. Sunamura M, Sun L, Lozoschl L. et a1. The antiangiogenesis effect of inter leukin 12 during early growth of human pancreatic cancer in SCID mice [J].Pancreas, 2000,20: 227-235.
7. Staib L, Ijnk KH, Beger HG. Immunotherapy in pancreatic cancer current status and future [J]. 1angenbecks Arch Surg. 1999, 384(4): 396-406.
8. Koji K,Mariko K,Syed R,et al.Targeting interleukin-4 receptors for effective pancreatic cancer therapy. [J].Cancer Res,2002,62(1): 3575-3580.
9. Mariko K, Koji K, Mitomu K, et a1. Hodgkin lymphoma therapy with interleukin-4 receptor–directed cytotoxin in an infiltrating animal model [J].Blood, 2005, 105(9):3707-3713.
10. Kawakami, K, Leland, P, and Puri, RK. Structure, function, and targeting of interleukin 4 receptors on human head and neck cancer cells. [J].Cancer Res, 2000,60: 2981–2987.
11. Koji K, Mariko K, Syed RH, et al. Effect of Interleukin (IL)-4 Cytotoxin on Breast Tumor Growth after in Vivo Gene Transfer of IL-4Receptor Chain. [J].Clinical Cancer Research, 2003, 9: 1826-1836.
[1] Jayaraman-G,Kumar-T-K,Tsai-C-C,et a1. Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra(Naja naja atra) — identification of structural features importantfor the lethal action of snake venom cardiotoxins [J].Protein-Sci,2000,9(4):637—646.
[2] 许云禄,吴国土,林建忠.舟山眼镜蛇毒细胞毒素-F的体内抗肿瘤作用[J].福建医科大学学报, 2003,37 (3): 298-300.
[3] 谭获,应红光,郝玉书,等.中华眼镜蛇组份抗白血病实验及初步临床应用研究[J].中国现代医学杂志,1999,9(12):22-23.
[4] 梁敏仪,陈家坤,朱蔚云,等.蛇毒心脏毒素对荷瘤小鼠的抗肿瘤作用初步研究[J].蛇志,1998, 1O (2): 1—4.
[5] Vogt W . Factors in cobra venoms affecting thecomplement system [J].Toxicon,1982,20(1): 299
[6] Braganca BM,Pater NT,Badrinath PG.Isolation and properties of a cobra venom factor selectively cytotoxic to yoshida sarcoma[J]. Biochim Biophys Acta, l967,l 36(3):508—520.
[7] 钟吉富.中国蛇协第九次学术会议综述 .医学研究通讯.1993, 22(4):10
[8] Chaim MA,Borkow G,Ovadia M.Isolationand characterization of a cytotoxin P4 from the venomof Naja nigricollis prefer entially active on tumour cells. [J].Biochem Int,1991,24:415—421.
[9] Lipps BV.Novel snake venom proteins cytotoxin to cancer cells in vitro and in vivo systems[J].J Venom Animo Toxins, 1999I5(2)l 172-183.
[10] 许云禄.张明芳,刘广芬.舟山眼镜蛇毒细胞毒索的分离纯化及其体外抗肿瘤活性[J].中国生化药物杂志,2003,24(3):127—13O.
[11] 许云禄,张明芳.舟山眼镜蛇毒细胞毒索一F对人鼻咽癌细胞和乳鼠心肌细胞的抑制作用[J].福建医科大学学报,2004,38(2):121—124.
[12] 任先达,张海伟,叶春玲,等.眼镜蛇毒灌胃后的兔血清对人鼻咽癌CNE一2细胞株的影响[J].暨南大学学报(医学版),2004,25(4): 436-440.
[13] 左长清,林振桃,孔天翰.中华眼镜蛇毒组分CⅡ诱导多药耐药白血病细胞K562/A02凋亡[J].广州医学院学报, 2004;32:26—30
[14] 赵蓉,游云,高敏新,等.蛇毒对人及小鼠传代细胞的杀伤作用 .福建医学院学报,1988,22(3):204
[15] 王锡锋,曹宜生,刘一平.等.眼镜蛇毒细胞毒素的分离、纯化及抗癌活性[J].中国生化药物杂志,1999,20(3):131
[16] 雷丹青,刘绵林,蓝利,等.广西眼镜蛇蛇毒细胞毒素的抗肿瘤作用[J].实用癌症杂志,2004, 19 (2):127—131.
[17] Fawzia.Treatment tfials of E.A.C with crude N.NigrieoUis ven. om and its fraction III fmice][A1.In:Kornalik F.Mebs D.Proeeedingof the 7th European symposium on an imal,plan t an dmicrobial toxins[C].1986.107
[18] 林振桃,赵路宁,植飞,等.眼镜蛇毒组分C对小鼠实验性肝癌细胞生长影响的探讨 [J].中国蛇毒杂志,1997,9(1):3～6.
[19] 罗伯诚,王存川,杨冠群,等.口服眼镜蛇毒抑制小鼠肿瘤的研究 [J].中华实验外科杂志,1993,1:31～32.
[20] 孙鹏,徐辰,任先达 等.口服三种形式眼镜蛇毒的急性毒性及对肝癌移植瘤的抑瘤作用[J].暨南大学学报(医学版),2002,23(4): 1-3.
[21] 杨惠玲,余清声,简志瀚,等.中华眼镜蛇毒对人鼻咽癌等抑瘤作用的实验研究[J].癌症,1999,18(1):27-29.
[22] 李浩淼,杨惠玲,李爱民,等.中华眼镜蛇毒对荷S180小鼠抑瘤作用及其机制初探[J].河南中医,2001,19 (2):19—22.
[23] 朱坤样.眼镜蛇毒抗肝癌作用研究[J].新消化病学杂志,1996, 4(8):436-437.
[24] 罗肖萍,赵路宁,管锦霞,等.眼镜蛇毒及其组分C对小鼠实验性肝癌抗癌作用的病理学研究[J].蛇志,1999, 11(3):11-15.
[25] Plate GJH, Venezuela M.Crotoxin complex as eytotoxie agent[P].United States Patent:5164196,1992.
[26] Costa LA, Miles H, Araujo CE,et al. Tumor regression of advanced carcinomas following intra- and/or peri-tumoral inoculation with VRCTC-310 in humans: preliminary report of two cases[J].Immunopharmacol Immunotoxicol,1998,20(1):15-25.
[27] 吴洪斌.蛇毒胶囊的研制及在癌症治疗中的应用[J].上海医药,1997,4:24.
[28] 李俊德.杨通礼,孙永章主编. 防治肿瘤特技集成 北京:北京科学技术出版杜,1997 I5～I7.
[29] 褚嘉桔,张和君,杨爱德,等.中华眼镜蛇膜毒素与M 偶联物对白血病细胞的杀伤作用[J].中国医学科学院学报,1992,14(3):179-182.
[30] 李钦章,黄沿中,杨冠群.眼镜蛇毒素与单克隆抗体偶联物对鼻咽癌细胞的特异性杀伤作用 . 暨南大学学报(自然科学版), 1998,19(3):94-99.
[31] Zaheer A,Noronha SH,Hospattankar AV,et a1.Inactivation of (Na+-K+)-stimul- ated ATPase by a eytotoxie protein from cobra venom in relation to its lyctic efects on cells[J].Biochem Biophys Aeta,1975,394(2):293-297
[32] Jayara man-G,Krishnaswamy-T,et a1.Binding of nucleotide triphosphates to cardiotoxin analogue II from the Taiwan cobra venom (Naja naja atra). Elucidation of the structural interactions in the dATP-cardiotoxin analogue II complex[J].J-Biol-Chem,1999,274 (25):17869-17875.
[33] Fletcher JE, Jiang MS. Possible mechanisms of action of cobra snake venom cardiotoxin sand bee venom melit-tin[J].Toxicon,1993,31(6):669-675.
[34] Dimafi ST.et a1.The cytotoxins of cobra venoms:Imlation and partial characterization[J].Biochim Biophys Acta,1975;393:320-329.
[35] Lauterwein J.and Wuthrich K.A possible structural basis for the different modes of action of neurotoxins and cardiotoxins from snake venom[J].Febs letters,1978;93:181-187.
[36] Chi-Huey W ,Yee Hsiung C,Mien-Chie H,et a1.Renaturation of a reduced Taiwan Cobra cardiotoxin[J].Biochim Biophys Acta,1978; 533:105-111.
[37] Dufourcq J.Faucx,n JF.Bemard E.Structure function relationships for cardiotoxins interacting with phospholipids [J].Toxin,1982.20(1):165-172.
[38] Stevens TR. Hinman CL. Activities of cobra venom cytotoxins toward heart and leukemic Y-cells depend on localize amino acid differences[J]. Toxicon,1997,35(5): 659-669.
[39] Borkow G. Chaim NA. Ovadia M. Binding of cytotoxinP4 from Naja nigricollis nigricollis to B16 F 10 1melanoma and WEHI-3B leukemia cells[J].FemsMicrobiol 1mmunol, 1992 5(13):139.
[40] Sumandea M,Das S,Sumandea C,et a1.Roles of aromatic residues in high intedacial activity of Naja aaja atra phospholipase A2[J].Biochemistry,1999,38 (49):16290-16297.
[41] 龚海云,乐毅,余清声,等.眼镜蛇毒直接溶解因子对人肝癌细胞株细胞内游离钙动态变化的影响[J].细胞生物学杂志,1998;20(3):137～140.
[42] 龚海云,乐毅,余清声,等.中华眼镜蛇毒心脏毒素对人肝癌细胞株细胞膜的影响[J].中国应用生理学杂志,1999;15(4):354～357.
[43] 谭获,郝玉书,应红光,等、中华眼镜蛇毒组分C诱导白血病细胞凋亡作用的研究[J].中国中西医结合杂志,2000;20(4):272～275.
[44] 苗俊英,荒木聪颜,林博司.血管内皮细胞凋亡过程中几种基因表达的研究[J].山东大学学报(自然科学版),2000;35(3):102～107.
[45] 李红良,任先达,罗英儒,等.中华眼镜蛇毒口服血清制剂对HepG2细胞凋亡的影响[J].中国病理生理杂志,2002;18(2):199～ 200.
[46] 罗汕,胡以则,彭和平.中华眼镜蛇毒组分C对人肝癌细胞株BEL一7402细胞凋亡及端粒酶活性的影响[J].广州医学院学报,2003;31(3): 1-4.
[47] 左长清,林振桃,孔天翰.中华眼镜蛇毒组分CⅡ抗耐药K562/A02细胞作用机制的研究[J].中国临床药理学与治疗学2005;10(9): 1015-1019.
[48] 龚海云,余清声,管锦霞,等.眼镜蛇毒直接溶解因子对肝癌细胞株H7420产生NO和LPO的影响[J].中国病理生理杂志,1998: 14(1):18～21.
[49] 杨惠玲,马志楷,余清声,等.中华眼镜蛇毒对荷人鼻咽癌棵鼠血浆MDA和SOD的影响[J].癌症,1998, 17(4):249—250.
[50] 杨惠玲,杨星,郑芹,等.中华眼镜蛇毒对荷人鼻咽癌棵鼠血过氧化氢酶和GSH-PX活性的影响[J].中国病理生理杂志,2001, 17(10):976—978.