灵杆菌素分离纯化及其抑制肿瘤细胞增殖的研究
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摘要
本文研究了粘质沙雷氏菌中灵杆菌素的提取方法,采用热酚水法提取灵杆菌素以及对提取出的脂多糖粗制品进行了纯化,并通过MTT法、电镜、PT-PCR和DNALadder、流式等方法对灵杆菌素的抗肿瘤增殖作用进行初步研究。发现灵杆菌素对结肠癌LS174t细胞有明显的抑制作用并呈浓度时间依赖关系;其诱导LS174t细胞出现典型的细胞凋亡超微结构改变;RT-PCR检测发现经不同浓度(5、10、25、50、100μg·ml~(-1))的灵杆菌素处理后细胞中bcl-2的表达下调,bax的表达上调;DNA电泳出现凋亡特有的“梯子”条带;流式细胞仪检测发现灵杆菌素可引起LS174t细胞凋亡并阻滞于S期。
表明灵杆菌素能有效地通过诱导凋亡抑制LS174t细胞生长,其机制可能与抑制抗凋亡因子bcl-2的表达,增强凋亡因子bax的表达有关。
This paper studies on the prodigiosin was prepared from Serratia marcescens with hot phenol water method and purified by ultracentrifugafion.Preliminary study by MTT assay,electron microscopy,PT-PCR,DNA Ladder and flow cytometfic inhibited tumor cell proliferation with prodigiosin.Prodigiosin inhibited growth and proliferation of colon cancer cells LS174t in dose and time dependent manner.Prodigiosin induced colon cancer cells LS174t to undergo typically apoptotic.RT-PCR showed that after treatment with prodigiosin for different concentrations(5、10、25、50、100μg.ml~(-1)),expression lever of Bcl-2 was reduced,Bax was increased.Agarose gel eletrophoresis of DNA revealed the apoptosis characteristic.Prodigiosin arrested LS174t cells in S-phase.
The results showed that prodigiosin can induce the apoptosis of LS174t and may be mediated by down-regulation of Bcl-2 and up-regulation of Bax.
表明灵杆菌素能有效地通过诱导凋亡抑制LS174t细胞生长,其机制可能与抑制抗凋亡因子bcl-2的表达,增强凋亡因子bax的表达有关。
This paper studies on the prodigiosin was prepared from Serratia marcescens with hot phenol water method and purified by ultracentrifugafion.Preliminary study by MTT assay,electron microscopy,PT-PCR,DNA Ladder and flow cytometfic inhibited tumor cell proliferation with prodigiosin.Prodigiosin inhibited growth and proliferation of colon cancer cells LS174t in dose and time dependent manner.Prodigiosin induced colon cancer cells LS174t to undergo typically apoptotic.RT-PCR showed that after treatment with prodigiosin for different concentrations(5、10、25、50、100μg.ml~(-1)),expression lever of Bcl-2 was reduced,Bax was increased.Agarose gel eletrophoresis of DNA revealed the apoptosis characteristic.Prodigiosin arrested LS174t cells in S-phase.
The results showed that prodigiosin can induce the apoptosis of LS174t and may be mediated by down-regulation of Bcl-2 and up-regulation of Bax.
引文
[1]吴淑燕,周正宇.细菌感染诱导巨噬细胞凋亡机制.国外医学流行病学传染病学分册,2002,29(4):236-239
[2]余濒.医学微生物学.北京:人民卫生出版社,1983:23-30
[3]Woodward H M M,Clarke K B.A case of infection in man by the bacterium prodigiosum.Lancet,1913,2(7):314-315
[4]Wheat R P,Zuckerman A,Rantzl A.Infection due to chromobacteria:report of eleven cases.Arch Intern Med,1951,88(3):461-466
[5]Endoh M,Okuno R,Mukaigawa J,et al.Two nosocomial outbreaks of sepsis caused by Serratia marcescens,which occurred in July 1999 and January 2002 Tokyo.Kansenshogaku Zasshi,2004,78(4):295-304
[6]Garcia F R,Paz R C,Gonzalez R S,et al.Cutaneous infection caused by Serratia marcescens in a child.J Am Acad Dermatol,2006,55(2):357-364
[7]关世斌.国外神灵杆菌脂多糖的研究概况(Ⅰ).灵菌素的发酵,提取,理化性质,结构与功能部分.微生物通报,1978,7(2):81-86
[8]朱建国.临床常见细菌鉴定手册.北京医科大学&中国协和医科大学联合出版社,1993,3(9):112-114
[9]Gales A C,Biedenbach D J,Winokur P,et al Diagnostic Microbiology and Infectious disease,2001,39(2):125-127
[10]张瑞斌,愈嘉林.灵菌素LPS的抑瘤作用研究.中华微生物学和免疫学杂志,2001,21(4):450-453
[11]焦炳华,赵虎,周凯等.灵杆菌细胞壁成分CWF—Ⅰ的提取纯化以及体外抗肿瘤作用.第二军医大学学报,1995,16(5):429-433
[12]潘劲草,黄志成,余文炳,等.肠出血性大肠杆菌O157H7脂多糖抗原的制备及鉴定.中国人兽共患病杂志,1999,15(3):59-61
[13]张艳红,杜元钊,吴延功,等.肠炎沙门氏菌脂多糖的提取与制备.动物医学进展,2001,22(3):79-80
[14]陶金莉,沈亚领,魏东芝,等.产灵菌红素沙雷氏菌的诱变育种.微生物学通报,2004,31(2):45-48
[15]Jawetz E,Melnick J I,Adelberg E A著.医学微生物学.邓瑞麟译.北京:人民卫生出版社,1983,9(2):270-272
[16]邱世翠,曲云,胡涛英,等内毒素致热作用对家兔体温、外周血细胞的影响.滨州医学院学报,1992,15(3):211-212
[17]潘颖,尹华,张子东,等.生白素防治介入化疗引起白细胞减少症的临床观察.内 蒙古科技与经济,2001,5(4):140-143
[18]梅贤良,张旭红.内毒素与多系统器官功能衰竭.新医学,1992,23(4):216-217
[19]张艳红,杜元钊,吴延功.等.肠炎沙门氏菌脂多糖的提取与制备.动物医学进展,2001,22(3):79-80
[20]杨益大,孙永良,何南祥,等.内毒素致病机理研究进展.国外医学流行病学传染病学分册,1991,18(2):73-76
[21]何坎,刘怀琼.内毒素对血小板的作用.生理科学进展,1991,22(1):69-71
[22]余晓峰,黄策,汪美先,等.国外医学微生物分册,1993,16(5):217-219
[23]全竹富,黎介寿.国外医学外科学分册,1995,22(5):284-286
[24]焦炳华,余庆.内毒素的结构及其与功能的关系.国外医学分子生物学分册,1987,9(4):168-170.
[25]梁前进.内毒素及其生物学效应.生物学通报,1997,32(9):18-19
[26]Starnes C O.Coley's toxins in perspective.Nature,1992,3(5):11-12
[27]Onier N,Hilpert S,Arnould L,et al.Cure of colon cancer metastasis in rats with the new lipid A OM 174.Apoptosis of tumor cells and immunization of rats.Clin Exp Metastasis,1999,17(4):299-306
[28]Baveye S,Elass E,Fernig D G,et al.Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules,E-selectin and ICAM-1,induced by the CD14-lipopolysaccharide complex.Infect Immun,2000,68(12):6519-6525
[29]Sakuta T,Matsushita K,Yamaguchi N,et al.Enhanced production of vascular endothelial growth factor by human monocytic cells stimulated with endotoxin through transcription factor SP-1.J Med Microbiol,2001,50(3):233-237
[30]Nativ O,Medalia O,Mor Y,et al.Treatment of experimental mouse bladder tumor by LPS-induced epithelial cell shedding.Br J Cancer,1996,74(4):603-605
[31]Chicoine MR,Won EK,Zahner MC.Intratumoral injection of lipopolysaccharide causes regression of subcutaneously implanted mouse glioblastoma multiforme.Neurosurgery,2001,48(3):607-614.
[32]Sourek J,Oravec C.Antitumor ativity of bacterial endotoxins and their subunit in in vitro test.Cent Eur J Public Health,1994,2(1):62-65
[33]Engelhardt R,Mackensen A,galanos C.Phase Ⅰ trial of intravenously administered endotoxin(Salmonella abortus equi) in cancer patients.Cancer Research,1991,51(2):2524-2530
[34]Otto F,Schmid P,Macksen A,et al.Phase Ⅱ trial of intrvenous endotoxin in patients with colorectal and non-small cell lung cancer.Eur J Cancer,1996,32A(10):1712-1718
[35]Hennemann B,Beckmann G,Eichelmann A,et al.Phase Ⅰ trial of adoptive immunotherapy of cancer patients using monocyte-derived macrophages activated with intertfen gamma and lipopolysaccharide(LPS).Cancer Immunol Immunother,1998,45(5):250-256
[36]张大志,任红,张定凤.内毒素致HepG2细胞凋亡的研究.现代医药卫生,2004,20(8):63-67
[37]郑军.革兰阴性杆菌内毒素诱导肝癌腹水瘤细胞系Hca-F25/cL-163.凋亡作用的研究.中国微生态学,2002,14(2):86-92
[38]关世斌,谷才恩,朱杰,等.灵菌素的研究Ⅰ、发酵,提取与理化性质.中国抗生素杂志,1979,(6):15-21
[39]齐洁琳,牛作兴,田禾,等.灵杆菌素对肿瘤化疗所致血细胞下降的疗效观察.山东医药,2000,40(9):24-25
[40]朱长义,魏然,徐新,等.灵菌素激发小鼠免疫功能研究.泰山医学院学报,1989,10(4):270-272
[41]贾钰铭,庞军,鲁子平,等.灵杆菌素、惠尔血治疗化疗病人白细胞下降的成本、效果分析.中国药房,2001,12(2):91-92
[42]陈艳才,古至洲,蒋荣,等.灵杆菌素对恶性肿瘤化疗后白细胞减少症的治疗.肿瘤研究与临床,1999,11(5):340-341
[43]陈艳才,古至洲,蒋荣,等.灵杆菌素和GM-CSF对恶性肿瘤化疗后白细胞减少症治疗效果对比.医学理论与实践,1999,12(11):644
[44]辛华主编.细胞生物学实验.北京:科学出版社,2001:91-92
[45]章静波,黄东阳,方瑾主编.细胞生物学实验技术.北京:化学工业出版社,2006:31-42
[46]Montaner B,Pe'rez-Toma's R.Prodigiosin-induced apoptosis in human colon cancer cells.Life Sci,2001,68(2):2025-2036
[47]翟中和主编.细胞生物学.北京:高等教育出版社,1995:35-42
[48]Fulda S,Debatin K M.Resveratrol-mediated sensitization to TRAIL-induced apoptosis depends on death receptor and mitochondrial signaling[J].Eur Ⅰ cancer,2005,41(5):786-798.
[49]Atten M I,Godoy-Romero F,Attar B M,et al.Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells[J].Invest New Prug,2005,23(2):111-119.
[50]Liontas A,Yeger H.Curcumin and resveratrol induce apoptosis and nuclear translocation and activation of P53 in human neuroblastoma[J].Anticancer Res,2004,24(2B):987-998.
[51]LiY,Bhuiyan M,Mohammad R M,et al.Induction of apoptosisin Breast cancer cells by TPA.Oncogene,1998,17(2):2915-2920.
[52]胡野,凌志强,单小云.细胞凋亡的分子医学.北京:军事医学科学出版社,2002:31-42
[2]余濒.医学微生物学.北京:人民卫生出版社,1983:23-30
[3]Woodward H M M,Clarke K B.A case of infection in man by the bacterium prodigiosum.Lancet,1913,2(7):314-315
[4]Wheat R P,Zuckerman A,Rantzl A.Infection due to chromobacteria:report of eleven cases.Arch Intern Med,1951,88(3):461-466
[5]Endoh M,Okuno R,Mukaigawa J,et al.Two nosocomial outbreaks of sepsis caused by Serratia marcescens,which occurred in July 1999 and January 2002 Tokyo.Kansenshogaku Zasshi,2004,78(4):295-304
[6]Garcia F R,Paz R C,Gonzalez R S,et al.Cutaneous infection caused by Serratia marcescens in a child.J Am Acad Dermatol,2006,55(2):357-364
[7]关世斌.国外神灵杆菌脂多糖的研究概况(Ⅰ).灵菌素的发酵,提取,理化性质,结构与功能部分.微生物通报,1978,7(2):81-86
[8]朱建国.临床常见细菌鉴定手册.北京医科大学&中国协和医科大学联合出版社,1993,3(9):112-114
[9]Gales A C,Biedenbach D J,Winokur P,et al Diagnostic Microbiology and Infectious disease,2001,39(2):125-127
[10]张瑞斌,愈嘉林.灵菌素LPS的抑瘤作用研究.中华微生物学和免疫学杂志,2001,21(4):450-453
[11]焦炳华,赵虎,周凯等.灵杆菌细胞壁成分CWF—Ⅰ的提取纯化以及体外抗肿瘤作用.第二军医大学学报,1995,16(5):429-433
[12]潘劲草,黄志成,余文炳,等.肠出血性大肠杆菌O157H7脂多糖抗原的制备及鉴定.中国人兽共患病杂志,1999,15(3):59-61
[13]张艳红,杜元钊,吴延功,等.肠炎沙门氏菌脂多糖的提取与制备.动物医学进展,2001,22(3):79-80
[14]陶金莉,沈亚领,魏东芝,等.产灵菌红素沙雷氏菌的诱变育种.微生物学通报,2004,31(2):45-48
[15]Jawetz E,Melnick J I,Adelberg E A著.医学微生物学.邓瑞麟译.北京:人民卫生出版社,1983,9(2):270-272
[16]邱世翠,曲云,胡涛英,等内毒素致热作用对家兔体温、外周血细胞的影响.滨州医学院学报,1992,15(3):211-212
[17]潘颖,尹华,张子东,等.生白素防治介入化疗引起白细胞减少症的临床观察.内 蒙古科技与经济,2001,5(4):140-143
[18]梅贤良,张旭红.内毒素与多系统器官功能衰竭.新医学,1992,23(4):216-217
[19]张艳红,杜元钊,吴延功.等.肠炎沙门氏菌脂多糖的提取与制备.动物医学进展,2001,22(3):79-80
[20]杨益大,孙永良,何南祥,等.内毒素致病机理研究进展.国外医学流行病学传染病学分册,1991,18(2):73-76
[21]何坎,刘怀琼.内毒素对血小板的作用.生理科学进展,1991,22(1):69-71
[22]余晓峰,黄策,汪美先,等.国外医学微生物分册,1993,16(5):217-219
[23]全竹富,黎介寿.国外医学外科学分册,1995,22(5):284-286
[24]焦炳华,余庆.内毒素的结构及其与功能的关系.国外医学分子生物学分册,1987,9(4):168-170.
[25]梁前进.内毒素及其生物学效应.生物学通报,1997,32(9):18-19
[26]Starnes C O.Coley's toxins in perspective.Nature,1992,3(5):11-12
[27]Onier N,Hilpert S,Arnould L,et al.Cure of colon cancer metastasis in rats with the new lipid A OM 174.Apoptosis of tumor cells and immunization of rats.Clin Exp Metastasis,1999,17(4):299-306
[28]Baveye S,Elass E,Fernig D G,et al.Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules,E-selectin and ICAM-1,induced by the CD14-lipopolysaccharide complex.Infect Immun,2000,68(12):6519-6525
[29]Sakuta T,Matsushita K,Yamaguchi N,et al.Enhanced production of vascular endothelial growth factor by human monocytic cells stimulated with endotoxin through transcription factor SP-1.J Med Microbiol,2001,50(3):233-237
[30]Nativ O,Medalia O,Mor Y,et al.Treatment of experimental mouse bladder tumor by LPS-induced epithelial cell shedding.Br J Cancer,1996,74(4):603-605
[31]Chicoine MR,Won EK,Zahner MC.Intratumoral injection of lipopolysaccharide causes regression of subcutaneously implanted mouse glioblastoma multiforme.Neurosurgery,2001,48(3):607-614.
[32]Sourek J,Oravec C.Antitumor ativity of bacterial endotoxins and their subunit in in vitro test.Cent Eur J Public Health,1994,2(1):62-65
[33]Engelhardt R,Mackensen A,galanos C.Phase Ⅰ trial of intravenously administered endotoxin(Salmonella abortus equi) in cancer patients.Cancer Research,1991,51(2):2524-2530
[34]Otto F,Schmid P,Macksen A,et al.Phase Ⅱ trial of intrvenous endotoxin in patients with colorectal and non-small cell lung cancer.Eur J Cancer,1996,32A(10):1712-1718
[35]Hennemann B,Beckmann G,Eichelmann A,et al.Phase Ⅰ trial of adoptive immunotherapy of cancer patients using monocyte-derived macrophages activated with intertfen gamma and lipopolysaccharide(LPS).Cancer Immunol Immunother,1998,45(5):250-256
[36]张大志,任红,张定凤.内毒素致HepG2细胞凋亡的研究.现代医药卫生,2004,20(8):63-67
[37]郑军.革兰阴性杆菌内毒素诱导肝癌腹水瘤细胞系Hca-F25/cL-163.凋亡作用的研究.中国微生态学,2002,14(2):86-92
[38]关世斌,谷才恩,朱杰,等.灵菌素的研究Ⅰ、发酵,提取与理化性质.中国抗生素杂志,1979,(6):15-21
[39]齐洁琳,牛作兴,田禾,等.灵杆菌素对肿瘤化疗所致血细胞下降的疗效观察.山东医药,2000,40(9):24-25
[40]朱长义,魏然,徐新,等.灵菌素激发小鼠免疫功能研究.泰山医学院学报,1989,10(4):270-272
[41]贾钰铭,庞军,鲁子平,等.灵杆菌素、惠尔血治疗化疗病人白细胞下降的成本、效果分析.中国药房,2001,12(2):91-92
[42]陈艳才,古至洲,蒋荣,等.灵杆菌素对恶性肿瘤化疗后白细胞减少症的治疗.肿瘤研究与临床,1999,11(5):340-341
[43]陈艳才,古至洲,蒋荣,等.灵杆菌素和GM-CSF对恶性肿瘤化疗后白细胞减少症治疗效果对比.医学理论与实践,1999,12(11):644
[44]辛华主编.细胞生物学实验.北京:科学出版社,2001:91-92
[45]章静波,黄东阳,方瑾主编.细胞生物学实验技术.北京:化学工业出版社,2006:31-42
[46]Montaner B,Pe'rez-Toma's R.Prodigiosin-induced apoptosis in human colon cancer cells.Life Sci,2001,68(2):2025-2036
[47]翟中和主编.细胞生物学.北京:高等教育出版社,1995:35-42
[48]Fulda S,Debatin K M.Resveratrol-mediated sensitization to TRAIL-induced apoptosis depends on death receptor and mitochondrial signaling[J].Eur Ⅰ cancer,2005,41(5):786-798.
[49]Atten M I,Godoy-Romero F,Attar B M,et al.Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells[J].Invest New Prug,2005,23(2):111-119.
[50]Liontas A,Yeger H.Curcumin and resveratrol induce apoptosis and nuclear translocation and activation of P53 in human neuroblastoma[J].Anticancer Res,2004,24(2B):987-998.
[51]LiY,Bhuiyan M,Mohammad R M,et al.Induction of apoptosisin Breast cancer cells by TPA.Oncogene,1998,17(2):2915-2920.
[52]胡野,凌志强,单小云.细胞凋亡的分子医学.北京:军事医学科学出版社,2002:31-42