眼镜蛇毒细胞毒素PLGA微球的制备及其抗肝癌作用研究
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摘要
目的
1.从眼镜蛇粗毒中分离纯化细胞毒素,并鉴定其理化性质和生物学活性;
2.制备眼镜蛇毒细胞毒素PLGA微球,研究其表征及体外释药性质;
3.评价眼镜蛇毒细胞毒素PLGA微球瘤体内注射治疗肝癌的药理学作用及安全性;
4.探讨眼镜蛇毒细胞毒素PLGA微球抗肝癌作用的相关机制。
方法
1.眼镜蛇毒细胞毒素的分离纯化、鉴定与生物学活性测定通过凝胶过滤、离子交换等方法从眼镜蛇粗毒中分离纯化眼镜蛇毒细胞毒素;采用SDS-PAGE法测定分离纯化得到的细胞毒素的纯度和分子量,MTT比色法测定纯化的眼镜蛇毒细胞毒素对体外培养细胞的毒性。
2.眼镜蛇毒细胞毒素PLGA微球的制备、表征与体外释放行为采用复乳-溶剂挥发法制备眼镜蛇毒细胞毒素PLGA缓释微球,光学显微镜及扫描电镜观察微球表面形态、激光粒度分析仪测定微球大小和分布;BCA法测定载药微球的包封率、载药率、体外释放行为。
3.眼镜蛇毒细胞毒素PLGA微球瘤体内注射治疗肝癌作用眼镜蛇毒细胞毒素缓释微球注射到荷人肝癌裸鼠皮下移植肿瘤内,超声定期测量裸鼠肝癌体积,26天后剥离小鼠肝癌肿瘤组织,称质量计算抑瘤率,切除肿瘤组织行病理学检查,评价其抗肿瘤作用。
4.眼镜蛇毒细胞毒素PLGA微球抗肝癌作用的相关机制切除肿瘤组织行透射电镜检查,观察凋亡细胞的形态学改变;通过TUNEL法检测细胞凋亡率;免疫印迹法检测眼镜蛇毒细胞毒素对细胞凋亡相关蛋白细胞色素C、caspase-3、-9表达的影响;比色法检测caspase-3、-9的活性;免疫组化法检测PCNA抗原的表达,观察肿瘤细胞增殖抗原的表达。
结果
1.分离纯化的眼镜蛇毒细胞毒素,SDS-PAGE法证实为均一蛋白区带,分子量约7300u,对体外培养的肝癌细胞具有强烈的细胞毒作用。
2.眼镜蛇毒细胞毒素PLGA缓释微球粒径为(34.45±9.85)μm,包封率达(78.13±8.92)%,体外30天累积释放眼镜蛇毒细胞毒素达84.3%。
3.眼镜蛇毒细胞毒素PLGA缓释微球瘤体内注射,可显著抑制裸鼠皮下人肝癌移植瘤的生长,肿瘤生长抑制率达52.36%。
4.眼镜蛇毒细胞毒素PLGA缓释微球具有抑制人肝癌细胞的增殖,又可诱导人肝癌细胞凋亡及坏死作用。免疫组化法检测PCNA抗原的表达减少,从而抑制肝癌细胞的增殖。光学显微镜和电镜可见细胞凋亡及坏死的典型形态学改变;TUNEL法检测CTX-PLGA微球组细胞凋亡率明显增加;免疫印迹可见CTX-PLGA微球促进细胞色素C从线粒体释放到胞浆,且caspase-3、-9表达量增加;比色法可见caspase-3、-9酶活性增强。
结论
1.从眼镜蛇粗毒中分离纯化获得的细胞毒素,具有强烈的细胞毒作用;
2. PLGA微球是眼镜蛇毒细胞毒素局部注射给药较理想的缓释载体;
3.眼镜蛇毒细胞毒素PLGA微球瘤体内注射具有显著的抗肝癌作用;
4.眼镜蛇毒细胞毒素PLGA微球通过抑制人肝癌细胞增殖和促进人肝癌细胞凋亡而抑制肿瘤的生长。
Objective
1. To separate and purify cytotoxin from crude cobra venom, and identify its physicochemical properties and biologic activity;
2. To prepare PLGA microspheres of cobra venom cytotoxin and study its characterization and characteristics of in vitro release;
3. To evaluate the pharmacological effects and safety of intratumoral injection of PLGA microspheres of cobra venom cytotoxin to treat hepatoma;
4. To approach the relevant mechanism of the anti-hepatoma effect of PLGA microspheres of cobra venom cytotoxin.
Methods
1. Separation, purification, identification and biologic activity determination of cobra venom cytotoxin
Cobra venom cytotoxin is separated and purified from the crude cobra venom with methods such as gel filtration and ion exchange; the purity and molecular weight of cytotoxin separated and purified are determined with SDS-PAGE method; and the toxicity of purified cobra venom cytotoxin on cells cultured in vitro are determined with MTT colorimetric method.
2. The preparation, characterization and in vitro release of PLGA microspheres of cobra venom cytotoxin
PLGA delayed-release microspheres of cobra venom cytotoxin are prepared with double emulsion - solvent volatilization method; the surface morphology of the microspheres is observed with a light microscope or a scanning electron microscope; the size and distribution of microspheres are determined with a laser particle size analyzer; and the encapsulation rate, drug loading and in vitro release of drug loaded microspheres are determined with BCA method.
3.The effect of intratumoral injection of PLGA microspheres of cobra venom cytotoxin to treat hepatoma
Delayed-release preparation of cobra venom cytotoxin is injected into the subcutaneously transplanted tumors of human hepatoma-bearing nude mice; the hepatoma volume of nude mice is determined with ultrasound regularly; 26 days later, the hepatoma tumor tissue of mice is dissected, its weight is determined, the inhibitory rate of tumor is calculated, the tumor tissue is dissected and the pathological examination is carried out ,and then the anti-tumor effect of cytotoxin is evaluated.
4.Relevant mechanism of the anti-hepatoma effect of PLGA microspheres of cobra venom cytotoxin.
The tumor tissue is dissected, and transmission electron microscope examination is carried out to observe the morphological changes of apoptotic cells; the apoptosis rate is determined with TUNEL method; the influence of cobra venom cytotoxin on the expression of apoptosis - related proteins cytochrome C, caspase-3 and caspase -9 is determined with Western blotting; the activity of caspase-3 and caspase-9 is determined with colorimetric method; the expression of PCNA antigen is determined with immunohistochemical method and the expression of tumor cell proliferation antigen is observed.
Results
1.It is confirmed with SDS-PAGE method that the cobra venom cytotoxin separated and purified is a uniform protein band with a molecular weight of about 7300u. It has intense cytotoxic effect on hepatoma carcinoma cells cultured in vitro.
2. The diameter of PLGA delayed-release microspheres of cobra venom cytotoxin is (34.45±9.85)μm, the encapsulation rate is up to (78.13±8.92) %, and the cumulative in vitro release amount of cobra venom cytotoxin during 30days is up to 84.3%.
3. Intratumoral injection of PLGA delayed release microspheres of cobra venom cytotoxin can significantly inhibit the growth of the subcutaneously transplanted human hepatoma in nude mice, and the inhibition rate of tumor growth is up to 52.36%.
4. PLGA delayed-release microspheres of cobra venom cytotoxin have the effect of inhibiting proliferation of hepatoma carcinoma cells, and it can also induce apoptosis and necrosis of human hepatoma carcinoma cells. It can be seen with immunohistochemical method that the expression of PCNA antigen decreases, and thus the proliferation of hepatoma carcinoma cells is inhibited. Under a light microscope or an electron microscope the typical morphological changes of cell apoptosis and necrosis can be seen; TUNEL examination shows that the apoptosis rate of the CTX-PLGA microspheres group significantly increases; it can be seen in Western blotting that CTX-PLGA microspheres can promote cytochrome C releases from mitochondrion to cytoplasm and the expression of caspase-3 and caspase-9 increases; it can be seen with colorimetric method that the activity of caspase-3 and caspase-9 increases.
Conclusion
1. The cytotoxin separated and purified from crude cobra venom has intense cytotoxic effect;
2. PLGA microsphere is an ideal delayed-release carrier of cobra venom cytotoxin for local injection administration;
3. Intratumoral injection of PLGA microspheres of cobra venom cytotoxin has significant anti-hepatoma effect;
4. PLGA microspheres of cobra venom cytotoxin inhibit the growth of tumors through inhibiting proliferation of human hepatoma carcinoma cells and promoting apoptosis of human hepatoma carcinoma cells.
1.从眼镜蛇粗毒中分离纯化细胞毒素,并鉴定其理化性质和生物学活性;
2.制备眼镜蛇毒细胞毒素PLGA微球,研究其表征及体外释药性质;
3.评价眼镜蛇毒细胞毒素PLGA微球瘤体内注射治疗肝癌的药理学作用及安全性;
4.探讨眼镜蛇毒细胞毒素PLGA微球抗肝癌作用的相关机制。
方法
1.眼镜蛇毒细胞毒素的分离纯化、鉴定与生物学活性测定通过凝胶过滤、离子交换等方法从眼镜蛇粗毒中分离纯化眼镜蛇毒细胞毒素;采用SDS-PAGE法测定分离纯化得到的细胞毒素的纯度和分子量,MTT比色法测定纯化的眼镜蛇毒细胞毒素对体外培养细胞的毒性。
2.眼镜蛇毒细胞毒素PLGA微球的制备、表征与体外释放行为采用复乳-溶剂挥发法制备眼镜蛇毒细胞毒素PLGA缓释微球,光学显微镜及扫描电镜观察微球表面形态、激光粒度分析仪测定微球大小和分布;BCA法测定载药微球的包封率、载药率、体外释放行为。
3.眼镜蛇毒细胞毒素PLGA微球瘤体内注射治疗肝癌作用眼镜蛇毒细胞毒素缓释微球注射到荷人肝癌裸鼠皮下移植肿瘤内,超声定期测量裸鼠肝癌体积,26天后剥离小鼠肝癌肿瘤组织,称质量计算抑瘤率,切除肿瘤组织行病理学检查,评价其抗肿瘤作用。
4.眼镜蛇毒细胞毒素PLGA微球抗肝癌作用的相关机制切除肿瘤组织行透射电镜检查,观察凋亡细胞的形态学改变;通过TUNEL法检测细胞凋亡率;免疫印迹法检测眼镜蛇毒细胞毒素对细胞凋亡相关蛋白细胞色素C、caspase-3、-9表达的影响;比色法检测caspase-3、-9的活性;免疫组化法检测PCNA抗原的表达,观察肿瘤细胞增殖抗原的表达。
结果
1.分离纯化的眼镜蛇毒细胞毒素,SDS-PAGE法证实为均一蛋白区带,分子量约7300u,对体外培养的肝癌细胞具有强烈的细胞毒作用。
2.眼镜蛇毒细胞毒素PLGA缓释微球粒径为(34.45±9.85)μm,包封率达(78.13±8.92)%,体外30天累积释放眼镜蛇毒细胞毒素达84.3%。
3.眼镜蛇毒细胞毒素PLGA缓释微球瘤体内注射,可显著抑制裸鼠皮下人肝癌移植瘤的生长,肿瘤生长抑制率达52.36%。
4.眼镜蛇毒细胞毒素PLGA缓释微球具有抑制人肝癌细胞的增殖,又可诱导人肝癌细胞凋亡及坏死作用。免疫组化法检测PCNA抗原的表达减少,从而抑制肝癌细胞的增殖。光学显微镜和电镜可见细胞凋亡及坏死的典型形态学改变;TUNEL法检测CTX-PLGA微球组细胞凋亡率明显增加;免疫印迹可见CTX-PLGA微球促进细胞色素C从线粒体释放到胞浆,且caspase-3、-9表达量增加;比色法可见caspase-3、-9酶活性增强。
结论
1.从眼镜蛇粗毒中分离纯化获得的细胞毒素,具有强烈的细胞毒作用;
2. PLGA微球是眼镜蛇毒细胞毒素局部注射给药较理想的缓释载体;
3.眼镜蛇毒细胞毒素PLGA微球瘤体内注射具有显著的抗肝癌作用;
4.眼镜蛇毒细胞毒素PLGA微球通过抑制人肝癌细胞增殖和促进人肝癌细胞凋亡而抑制肿瘤的生长。
Objective
1. To separate and purify cytotoxin from crude cobra venom, and identify its physicochemical properties and biologic activity;
2. To prepare PLGA microspheres of cobra venom cytotoxin and study its characterization and characteristics of in vitro release;
3. To evaluate the pharmacological effects and safety of intratumoral injection of PLGA microspheres of cobra venom cytotoxin to treat hepatoma;
4. To approach the relevant mechanism of the anti-hepatoma effect of PLGA microspheres of cobra venom cytotoxin.
Methods
1. Separation, purification, identification and biologic activity determination of cobra venom cytotoxin
Cobra venom cytotoxin is separated and purified from the crude cobra venom with methods such as gel filtration and ion exchange; the purity and molecular weight of cytotoxin separated and purified are determined with SDS-PAGE method; and the toxicity of purified cobra venom cytotoxin on cells cultured in vitro are determined with MTT colorimetric method.
2. The preparation, characterization and in vitro release of PLGA microspheres of cobra venom cytotoxin
PLGA delayed-release microspheres of cobra venom cytotoxin are prepared with double emulsion - solvent volatilization method; the surface morphology of the microspheres is observed with a light microscope or a scanning electron microscope; the size and distribution of microspheres are determined with a laser particle size analyzer; and the encapsulation rate, drug loading and in vitro release of drug loaded microspheres are determined with BCA method.
3.The effect of intratumoral injection of PLGA microspheres of cobra venom cytotoxin to treat hepatoma
Delayed-release preparation of cobra venom cytotoxin is injected into the subcutaneously transplanted tumors of human hepatoma-bearing nude mice; the hepatoma volume of nude mice is determined with ultrasound regularly; 26 days later, the hepatoma tumor tissue of mice is dissected, its weight is determined, the inhibitory rate of tumor is calculated, the tumor tissue is dissected and the pathological examination is carried out ,and then the anti-tumor effect of cytotoxin is evaluated.
4.Relevant mechanism of the anti-hepatoma effect of PLGA microspheres of cobra venom cytotoxin.
The tumor tissue is dissected, and transmission electron microscope examination is carried out to observe the morphological changes of apoptotic cells; the apoptosis rate is determined with TUNEL method; the influence of cobra venom cytotoxin on the expression of apoptosis - related proteins cytochrome C, caspase-3 and caspase -9 is determined with Western blotting; the activity of caspase-3 and caspase-9 is determined with colorimetric method; the expression of PCNA antigen is determined with immunohistochemical method and the expression of tumor cell proliferation antigen is observed.
Results
1.It is confirmed with SDS-PAGE method that the cobra venom cytotoxin separated and purified is a uniform protein band with a molecular weight of about 7300u. It has intense cytotoxic effect on hepatoma carcinoma cells cultured in vitro.
2. The diameter of PLGA delayed-release microspheres of cobra venom cytotoxin is (34.45±9.85)μm, the encapsulation rate is up to (78.13±8.92) %, and the cumulative in vitro release amount of cobra venom cytotoxin during 30days is up to 84.3%.
3. Intratumoral injection of PLGA delayed release microspheres of cobra venom cytotoxin can significantly inhibit the growth of the subcutaneously transplanted human hepatoma in nude mice, and the inhibition rate of tumor growth is up to 52.36%.
4. PLGA delayed-release microspheres of cobra venom cytotoxin have the effect of inhibiting proliferation of hepatoma carcinoma cells, and it can also induce apoptosis and necrosis of human hepatoma carcinoma cells. It can be seen with immunohistochemical method that the expression of PCNA antigen decreases, and thus the proliferation of hepatoma carcinoma cells is inhibited. Under a light microscope or an electron microscope the typical morphological changes of cell apoptosis and necrosis can be seen; TUNEL examination shows that the apoptosis rate of the CTX-PLGA microspheres group significantly increases; it can be seen in Western blotting that CTX-PLGA microspheres can promote cytochrome C releases from mitochondrion to cytoplasm and the expression of caspase-3 and caspase-9 increases; it can be seen with colorimetric method that the activity of caspase-3 and caspase-9 increases.
Conclusion
1. The cytotoxin separated and purified from crude cobra venom has intense cytotoxic effect;
2. PLGA microsphere is an ideal delayed-release carrier of cobra venom cytotoxin for local injection administration;
3. Intratumoral injection of PLGA microspheres of cobra venom cytotoxin has significant anti-hepatoma effect;
4. PLGA microspheres of cobra venom cytotoxin inhibit the growth of tumors through inhibiting proliferation of human hepatoma carcinoma cells and promoting apoptosis of human hepatoma carcinoma cells.
引文
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42 Roos G. ,Christensson PI,el Hag IA,et al.Degradable starch microsph- eres in cytostatic treatment of a liver carcinoma;experimental studies in rats with 5-fluorouracil, tauromustine,carmustine, doxorubicin and RSU-1069.Anticancer Res,1993,13(3):635-641.
43徐蔚,张纪。碳铂壳聚糖缓解释的制备。军医进修学院学报,2000,21(2):92-94。
44 Liang LS,Jackson J,Min W,et al.Methotrexate loaded poly(L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint. J Pharm Sci,2004,93(4):943-956.
45 Ertl B,Platzer P,Wirth M,et al.Poly(D,L-lactic-co-glycolic acid) microspheres for sustained delivery and stabilization of camptothecin.J Control Release,1999,61(3):305-317.
46 Sinha VR,Trehan A.Biodegradable microspheres for protein delivery.J Control Release,2003,31(3):261-280.
47 Bartus RT,Tracy MA,Emerich DF,et al. Sustained delivery of proteins for novel therapeutic agents.Science,1998,281(5380):1161-1162.
48 Ravivarapu HB,Burton K,DeLuca PP.Polymer and microsphere blending to alter the release of a peptide from PLGA microspheres.Eur J Pharm Biopharm,2000,50(2):263-270.
49 Aubert-Pouessel A,Venier-Julienne MC,Clavreul A,et al.In vitro study of GDNF release from biodegradable PLGA microspheres.J Control Release, 2004,95(3):463-475.
50陈英杰,许向阳,周建平。乳酸-羟基乙酸共聚物微球的研究进展。中国药科大学学报,2007,38(2):186-189。
51吴细丕,钱林法,主编。实验动物与肿瘤研究。北京:中国医药科技出版社,2000:112-113。
52 Ain JF, Gouillat C, Bertrand S,et al. Human hepatocellular carcinoma transplanted in nude mice: a relevant experimental model to assess tumoral destruction by alcoholization. J Surg Res,1994,57(3):366- 372.
53 Zajchowski DA,Biroc SL,Liu HL,et al.Anti-tumor efficacy of the nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-β-Darabinofuranosyl) cytosine(4′-thio-FAC)in human pancreatic and ovarian tumor xenograft models.Int J Cancer,2005,114(6):1002-1009.
54金岩,李海波,关洪全。中药诱导肿瘤细胞凋亡机制的研究进展。中医研究,2005,18(3):52-57。
55 Oron U,Chaim-Matyas A,Ovadia M.Histopathological change in WHEI-3B leukemia cells following intoxication by cytotoxin P4 from Naja nigricollis nigricollis venom.Toxicon,1992,32(9):1122-1126.
56 Lipps BV.Novel snake venom proteins cytotoxin to cancer cells in vitro and in vivo systems.J Venom Animo Toxins,1999,5(2):172-183.
57 Strizhkov BN,Blishchenko EYu,Satpaev DK,et al.Both neurotoxinⅡfrom venom of Naja naja oxiana and its endogenous analogue induce apoptosis in tumor cells.FEBS Lett,1994,340(1-2):22-24.
58 Araki S,Ishida T,Yamamoto T,et al.Induction of apoptosis by hemorrhagic snake venom in vascular endothelial cells.Biochem Biophys Res Commun,1993,190(1):148-153.
59 Suhr SM,Kim DS.Identification of the snake venom substance that induces apoptosis.Biochem Biophys Res Commun,1996,224(1):134-139.
60 Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br J Cancer, 1972,26(4):239-257.
61姜泊,主编。细胞凋亡基础与临床。第一版。北京:人民军医出版社,1999,26。
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63 Zhang Y,Bhavnani BR.Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor(AIF) and this process is inhibited by equine estrogens.BMC Neurosci,2006,7:49.
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66 Hong SJ,Dawson TM,Dawson VL.Nuclear and mitochondrial conversations in cell death:PARP-1 and AIF signaling.Trends Pharmacol Sci, 2004,25(5):259-264.
67 Yu W,Sanders BG,Kline K.RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells involves Bax translocation to motochondria.Cancer Res,2003,63(10):2483-2491.
68 Zou H,Henzel WJ,Liu X,et al.Apaf-1,a human protein homologous to C.elegans CED-4,participates in cytochrome c-dependent activation of caspase-3.Cell,1997,90(3):389-390.
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40 Novotny J,Zinek K.Application of epirubicin containing albumin microspheres in the experimental therapy of breast cancer. Neoplasma,1994,41(4):201-204.
41姚瑶,陶昱,丁燕飞,等。依托泊苷明胶微球的制备。中国现代应用药学杂志,2005,22(3):219-221。
42 Roos G. ,Christensson PI,el Hag IA,et al.Degradable starch microsph- eres in cytostatic treatment of a liver carcinoma;experimental studies in rats with 5-fluorouracil, tauromustine,carmustine, doxorubicin and RSU-1069.Anticancer Res,1993,13(3):635-641.
43徐蔚,张纪。碳铂壳聚糖缓解释的制备。军医进修学院学报,2000,21(2):92-94。
44 Liang LS,Jackson J,Min W,et al.Methotrexate loaded poly(L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint. J Pharm Sci,2004,93(4):943-956.
45 Ertl B,Platzer P,Wirth M,et al.Poly(D,L-lactic-co-glycolic acid) microspheres for sustained delivery and stabilization of camptothecin.J Control Release,1999,61(3):305-317.
46 Sinha VR,Trehan A.Biodegradable microspheres for protein delivery.J Control Release,2003,31(3):261-280.
47 Bartus RT,Tracy MA,Emerich DF,et al. Sustained delivery of proteins for novel therapeutic agents.Science,1998,281(5380):1161-1162.
48 Ravivarapu HB,Burton K,DeLuca PP.Polymer and microsphere blending to alter the release of a peptide from PLGA microspheres.Eur J Pharm Biopharm,2000,50(2):263-270.
49 Aubert-Pouessel A,Venier-Julienne MC,Clavreul A,et al.In vitro study of GDNF release from biodegradable PLGA microspheres.J Control Release, 2004,95(3):463-475.
50陈英杰,许向阳,周建平。乳酸-羟基乙酸共聚物微球的研究进展。中国药科大学学报,2007,38(2):186-189。
51吴细丕,钱林法,主编。实验动物与肿瘤研究。北京:中国医药科技出版社,2000:112-113。
52 Ain JF, Gouillat C, Bertrand S,et al. Human hepatocellular carcinoma transplanted in nude mice: a relevant experimental model to assess tumoral destruction by alcoholization. J Surg Res,1994,57(3):366- 372.
53 Zajchowski DA,Biroc SL,Liu HL,et al.Anti-tumor efficacy of the nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-β-Darabinofuranosyl) cytosine(4′-thio-FAC)in human pancreatic and ovarian tumor xenograft models.Int J Cancer,2005,114(6):1002-1009.
54金岩,李海波,关洪全。中药诱导肿瘤细胞凋亡机制的研究进展。中医研究,2005,18(3):52-57。
55 Oron U,Chaim-Matyas A,Ovadia M.Histopathological change in WHEI-3B leukemia cells following intoxication by cytotoxin P4 from Naja nigricollis nigricollis venom.Toxicon,1992,32(9):1122-1126.
56 Lipps BV.Novel snake venom proteins cytotoxin to cancer cells in vitro and in vivo systems.J Venom Animo Toxins,1999,5(2):172-183.
57 Strizhkov BN,Blishchenko EYu,Satpaev DK,et al.Both neurotoxinⅡfrom venom of Naja naja oxiana and its endogenous analogue induce apoptosis in tumor cells.FEBS Lett,1994,340(1-2):22-24.
58 Araki S,Ishida T,Yamamoto T,et al.Induction of apoptosis by hemorrhagic snake venom in vascular endothelial cells.Biochem Biophys Res Commun,1993,190(1):148-153.
59 Suhr SM,Kim DS.Identification of the snake venom substance that induces apoptosis.Biochem Biophys Res Commun,1996,224(1):134-139.
60 Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br J Cancer, 1972,26(4):239-257.
61姜泊,主编。细胞凋亡基础与临床。第一版。北京:人民军医出版社,1999,26。
62 Imajoh M,Sugiura H,Oshima S.Morphological changes contribute toapoptotic cell death and are affected by caspase-3 and caspase-6 inhibitors during red sea bream iridovirus permissive replication. Virology,2004,322(2):220-230.
63 Zhang Y,Bhavnani BR.Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor(AIF) and this process is inhibited by equine estrogens.BMC Neurosci,2006,7:49.
64 Cheema ZF,Santillano DR,Wade SB,et al.The extracellular matrix,P53 and estrogen compete to regulate cell-surface Fas/Apo-1 suicide receptor expression in proliferating embryonic cerebral cortical precursors,and reciprocally,Fas-ligand modifies estrogen control of cell-cycle proteins.BMC Neurosci,2004,5:11.
65 Muhlethaler-Mottet A,Balmas K,Auderset K,et al.Restoration of TRAIL-induced apoptosis in a caspase-8-deficient neuroblastoma cell line by stable re-expression of caspase-8.Ann NY Acad Sci, 2003,1010:195-199.
66 Hong SJ,Dawson TM,Dawson VL.Nuclear and mitochondrial conversations in cell death:PARP-1 and AIF signaling.Trends Pharmacol Sci, 2004,25(5):259-264.
67 Yu W,Sanders BG,Kline K.RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells involves Bax translocation to motochondria.Cancer Res,2003,63(10):2483-2491.
68 Zou H,Henzel WJ,Liu X,et al.Apaf-1,a human protein homologous to C.elegans CED-4,participates in cytochrome c-dependent activation of caspase-3.Cell,1997,90(3):389-390.
69 Browm NM,Martin SM,Maurice N,et al.Caspase inhibition blocks cell death and results in cell cycle arrest in cytokine-deprived hematopoietic cells.J Biol Chem,2007,282(4):2144-2155.
70 Eldadah BA,Faden AI.Caspase pathways,neuronal apopotosis,and CNS injury.J Neurotrauma,2000,17(10):811-829.
71张雨平,赵聪敏,奚敏,等。缺氧缺血对新生鼠脑Caspase-3效应蛋白酶及细胞凋亡的影响。中国临床康复杂志,2005,9(3):192-193。
72 Cohen GM . Caspases:the executioners of apopotosis. Biochem J, 1997, 326(part1):l-16.
73 Villa P Kaufmann SH,Earnshaw WC. Caspase and caspase inhibitors. Trends Biochem Sci,1997,22(10):388-393.
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