羟基磷灰石纳米粒子对大鼠肝线粒体作用的研究
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摘要
羟基磷灰石纳米粒子对癌细胞的抑制作用是无机纳米粒子的生物学效应,也是生物中心多年来的研究课题。本论文围绕这一课题,在前人研究的基础上,选择“羟基磷灰石纳米粒子对大鼠肝线粒体作用的研究”作为主要研究内容,目的是在细胞器水平上,将纳米粒子与线粒体直接作用,通过检测线粒体琥珀酸脱氢酶活性,探讨纳米粒子对线粒体活性的影响,为进一步研究纳米粒子的抑癌机制提供理论依据。
实验通过传统的组织匀浆和差速离心方法来提取和分离大鼠肝细胞线粒体,选择线粒体的标志酶琥珀酸脱氢酶来检测其活性;探讨了线粒体琥珀酸脱氢酶活力检测条件;最后,将不同浓度纳米HAP粒子悬浮液与线粒体直接作用,以没加纳米HAP粒子作为对照,测定线粒体琥珀酸脱氢酶活力,并对数据进行分析,最后得出结果:
1.测定得出线粒体琥珀酸脱氢酶的比活性为7.376±0.910u/mg蛋白质,线粒体活性满足实验要求;
2.在37℃条件下,两天后线粒体琥珀酸脱氢酶的比活性丧失,而在-70℃条件下,两个月内比活性保持不变;
3.纳米HAP粒子随着浓度的增大和作用时间的延长,对线粒体琥珀酸脱氢酶的比活性抑制作用加强,表明HAP纳米粒子对线粒体琥珀酸脱氢酶的比活性抑制有剂量和时间的依赖性。
本实验改进了琥珀酸脱氢酶试剂盒的检测方法,使检测快速、方便、成本大大降低;并且在传统细胞水平研究上将纳米粒子直接与线粒体作用。
The effect of hydroxyapatite nano-particles on cancer cells is a kind of biological effect of inorganic nano-particles, and it had been studied by Bomedical Materials and Engineering Research Center of Wuhan University of Technology since many years before. On the basis of the former researeh, the author selected "the effect of HAP nano-particles on the mitcochondria of liver rats" as his main research content in the subject. In this study, we have studied HAP nano-particles effect of mitochondria from the perspective of organelles, tested SDH activity of mitochondria, and explored the effect of HAP nano-particles on the mitcochondria. Therefore this study aims to provide the theoretical foundation for further researching inhibitive effect on cancer cell.
Liver mitochondria of rat were isolated and extracted by traditional methods, we obtained active mitochondria by the test of SDH which is a key marker of mitochondria; we investigated the condition of SDH test of mitochondria; last, we put different concentrations of HAP nano-particles to directly treat mitochondria with negative control, then conclusions are made:
1. The SDH activity of mitochondria was 7.376±0.910 u/mg protain, it fit to our experimental request;
2. The SDH activity of mitochondria was higher at 37℃but its activity disappeared after two days, and the same as in two months at -70℃;
3. HAP nano-particles inhibit the SDH activity of mitochondria, and became lower and faster compared with negative control depend on time and dose.
We bettered the test of mitochondrial activity by SDH kit, which made test more quickly, conveniently and cut down its cost. Moreover we put HAP nano-particles to mitochondria directly, which based on the traditional cell level research.
实验通过传统的组织匀浆和差速离心方法来提取和分离大鼠肝细胞线粒体,选择线粒体的标志酶琥珀酸脱氢酶来检测其活性;探讨了线粒体琥珀酸脱氢酶活力检测条件;最后,将不同浓度纳米HAP粒子悬浮液与线粒体直接作用,以没加纳米HAP粒子作为对照,测定线粒体琥珀酸脱氢酶活力,并对数据进行分析,最后得出结果:
1.测定得出线粒体琥珀酸脱氢酶的比活性为7.376±0.910u/mg蛋白质,线粒体活性满足实验要求;
2.在37℃条件下,两天后线粒体琥珀酸脱氢酶的比活性丧失,而在-70℃条件下,两个月内比活性保持不变;
3.纳米HAP粒子随着浓度的增大和作用时间的延长,对线粒体琥珀酸脱氢酶的比活性抑制作用加强,表明HAP纳米粒子对线粒体琥珀酸脱氢酶的比活性抑制有剂量和时间的依赖性。
本实验改进了琥珀酸脱氢酶试剂盒的检测方法,使检测快速、方便、成本大大降低;并且在传统细胞水平研究上将纳米粒子直接与线粒体作用。
The effect of hydroxyapatite nano-particles on cancer cells is a kind of biological effect of inorganic nano-particles, and it had been studied by Bomedical Materials and Engineering Research Center of Wuhan University of Technology since many years before. On the basis of the former researeh, the author selected "the effect of HAP nano-particles on the mitcochondria of liver rats" as his main research content in the subject. In this study, we have studied HAP nano-particles effect of mitochondria from the perspective of organelles, tested SDH activity of mitochondria, and explored the effect of HAP nano-particles on the mitcochondria. Therefore this study aims to provide the theoretical foundation for further researching inhibitive effect on cancer cell.
Liver mitochondria of rat were isolated and extracted by traditional methods, we obtained active mitochondria by the test of SDH which is a key marker of mitochondria; we investigated the condition of SDH test of mitochondria; last, we put different concentrations of HAP nano-particles to directly treat mitochondria with negative control, then conclusions are made:
1. The SDH activity of mitochondria was 7.376±0.910 u/mg protain, it fit to our experimental request;
2. The SDH activity of mitochondria was higher at 37℃but its activity disappeared after two days, and the same as in two months at -70℃;
3. HAP nano-particles inhibit the SDH activity of mitochondria, and became lower and faster compared with negative control depend on time and dose.
We bettered the test of mitochondrial activity by SDH kit, which made test more quickly, conveniently and cut down its cost. Moreover we put HAP nano-particles to mitochondria directly, which based on the traditional cell level research.
引文
[1]国家质量监督检验检疫总局,国家标准化管理委员会.中华人民共和国国家标准(GB /T 19619-2004):纳米材料术语[S].北京:中国标准出版社,2004.12.
[2]张立德,等 纳米材料科学[M]沈阳:辽宁出版社,1994
[3]张立德,等 开拓原子和物质的中间领域-纳米微粒与纳米固体物理,1992.21(3):167-172
[4]郑玉峰 李莉著 生物医用材料 哈尔滨工业[M]大学出版社2005
[5]Bermudez E,Mangum JB,Wong BA,et al.Pulmonary responses of rats,mice and hamsters to subchronic inhalation of ultrafine titaniumdi oxide particles.Toxicol Sci 2004 77(2):347-57
[6]Renwick LC,Donaldson K,Clouter A.Impairment of alveolarmacrophage phagocytosis by ultrafine particles.Toxicol Appl Pharmacol 2001 172:119-27
[7]金一和,孙鹏,张颖花.纳米材料对人体潜在性影响问题[J].自然,2002,23(5):306-7
[8]Birringer R,Gleiter H,Klein HP,et al.N anocr2ystalline meterials:A n app roach to a novel so 12id st ructurew ith gas like diso rder?Phy Let A,1984;102:365-369
[9]Ch ikara Hayash i.U lt rafine part icales.J V alcumScience and Techno logy,1987;A 5(4):1375-1384
[10]Lu W,Tan YZ,Hu KL,et al.Cationic albumin conjugated pegylatednanoparticle with its transcytosis ability and little toxicity against blood-brain barrier.Int J Pharm 2005W295(1-2):247-6
[11]白春礼.科学通报,2001;46(1):90
[12]Andrievsk R.A.J.Mater.Sci 1994;29:614
[13]Beck D.D,Siege R.W.J.Mater.Res.,1992;7:284
[14]Abdulkhadar M.,Thomas B.Nanostructured Mater.,1995:5(3):289
[15]杜仕国,施冬梅,邓辉.自然杂志,2000;22(2):101
[16]Hoet PH,Bruske-Hohlfeld I,Salata OV.Nanoparticles-known and unknown health risks.J Nanobiotechnology 2004 2:12
[17]Andre Nel,Tian Xia,Lutz Mdler,et al.Toxic potential of materials at thenanolevel.Science 2006 311:622-727
[18]Li N,Sioutas C,Cho A,et al.Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage[J].Environ Health Perspect,2003,111:455
[19]Derfus AM,Chan WCW,Bhatia SN.Probing the cytotoxicity of semiconductor quantum dots J.Nano Lett,2004,4(1):11
[20]Joo SH,Feitz AJ,Waite TD.Oxidative degradation of the carbothioate herbicide,molinate, using nanoscale zerovalentiron[J].Environ Sci Technol,2004,38:2242.
[21]Zhou YM,Zhong CY,Kennedy IM,et al.Oxidative stressand NFB activation in the lungs of rots:a synergistic interaction between soot and iron particles[J].Toxicol Appl Pharmacol,2003,190:157
[22]LiN,Sioutas C,ChoA,Schmitz D,Misra C,Sempf J,et al.Ultrafme particulate pollutants induce oxidative stress andmitochondrial damage[J].EnvironHealth Perspect,2003,111(4):455-456
[23]YamakoshiY,Umezawa N,Ryu A,Arakane K,Miyata N,GodaY,etal.Active oxygen species generated from photoexcitedfullerene(C60)as potentialmedicines:O2-versusl O2[J].J Am Chem Soc,2003,125(42):12803-12809
[24]Are diamond nanoparticles cytotoxic?J Phys Chem B.2007 Jan 11;111(1):2-7.
[25]Hughes LS,Cass GR,Gone J,et al.Physical and chemical characterization of atmospheric ultrafine particles in the Los Angeles area[J].Environ Sci Technol 1998,32:1153.
[26]Kulmala M.Formation and growth rates of ultrafine atmospheric particles:a review of observations[J].J Aerosol Sci,2004,35:143
[27]Compton M.M.,Cancer Metastasis Rev.,11,105-109(1992).
[28]Chen X,Deng C,Tang S,Zhang Mitochondria-dependent apoptosis induced by nanoscale hydroxyapatite in human gastric cancer SGC-7901 cells.Biol Pharm Bull.2007 Jan;30(1):128-32.
[29]Wang XB,Gao HY,Hou BL,Huang J,Xi RG,Wu LJ Nanoparticle realgar powders induce apoptosis in U937 cells through caspase MAPK and mitochondrial pathways.Arch Pharm Res.2007 May;30(5):653-8.
[30]陈国明,刘岚.柠檬酸包埋的四氧化三铁纳米颗粒安全性研究[J].江苏药学与临床研究,2003,11(6):4-6
[31]Hansen T,Boutrand JP,Eloy R,et al.Biological tolerance of different nanoparticles in a rat model-clinical and histological findings.7th World Biomaterials Congress 2004:752
[32]Thomas K,Sayre P.Research Strategies for Safety Evaluation of Nanomaterials,Part Ⅰ:Evaluating the Human Health Implications of Exposureto Nanoscale Materials.Toxicol Sci 2005W87(2):316-321
[33]尹其华,刘璐,顾宁,等,59Fe示踪谷氨酸包被三氧化二铁纳米颗粒的制备及其在小鼠体内的生物学分布[J].医学研究生学报,2005,18(4):312-6
[34]Revell PA,Gatti AM,Gambarel li A,et al.Detection of cocr particles in the spleen of guinea pigs six weeks after their intra-osseous implanta-tion.7th World Biomaterials Congress 2004:753
[35]Hussain SM,Hess KL,Gearhart JM,et al.in vitro toxicity of nanoparti-cles in BRL 3A rat liver cells.Toxicol In Vitro 2005W19(7):975-83
[36]孟纯阳,安洪.纳米羟基磷灰石/聚酰胺66与人胚胎成纤维细胞的生物相容性研究[J].重庆医科大学学报,2005,30(2):169-72
[37]戴伯军,李家顺,贾连顺,等.纳米氧化锆强韧化高孔隙率磷酸钙人工古细胞支架生物学评价[J].中国矫形外科杂志,2004,12(13):998-1001
[38]许艳慧,李四群,李志安.纳米羟基磷灰石复合材料的研究-纳米羟基磷灰石的细胞毒性[J].实用口腔医学杂志,2004,20(2):147-50
[39]Chen CN,Kuo CC,Chen LT.Evaluation of cellular compatibility andblood compatibility of electrospum bioabsorbable Nanofiber.Tth WoddBiomaterials Congress 2004:1741
[40]Andre Nel,Tian Xia,Lutz M^dler,et al.Toxic potential of materials at thenanolevel.Science 2006W311:622-727
[41]李兴,邱宗荫,潘卫等 肝癌细胞线粒体的纯化及双向凝胶电泳分析[J].重庆医科大学学报,2005:30(2):163-167
[42]Wallase KB,Eells J T,Madeira V M,et al.Fundam Appl Toxicol.1997 38(1):23-27.
[43][美]DL.斯佩克特,RD.戈德曼,LA.莱因万德等著,黄培堂等译.细胞实验指南.科学出版社.
[44]李鸣真,等中西医结台杂志 1989;9(7):412
[45]季静娟,等酶检测法在线粒体功能及混杂程度评价中的应用.临床输血与检验,2004;6(3):167-168
[46]王小平,等脱辅基细胞色素c定向于线粒体的转运特异性研究.中国生物化学与分子生物学报,1999:15(5):813-819
[47]李兴,等肝癌细胞线粒体的纯化及双向凝胶电泳分析.重庆医科大学学报,2005;30(2):163-177
[48]冯庆玲,崔福斋,张伟.纳米羟基磷灰石/胶原骨修复材料[J].中国医学科学院学报,2002,24(2):124-128.
[49]Shinomiya K,Itoh S,Kawauchi T.Development of a novel hydroxyapatite/collagen composite biomaterial[C].International Congress Series,1 222(Tissue Engineering for Therapeutic Use 5),2001,165-177.
[50]#12
[51]Hideki Aoki,et al.Effects of HAP-sol on cell growth[J].Report of the institute forMedical and Dental Engineering,1992,26(1):15-21.
[52]张士成,李世普等.磷灰石超微粉对癌细胞作用的初步研究[J].武汉工业大学学报,1996,18(1):5-8.
[53]DentonRMoccmrackJG.on the role of the calcium transport cycle in heart and other mammalian mitochondrial.EFBS(Fed Eur Biochem Soc)Lett.1980,119:1-8.
[54]DnentonRM,Moccmrxack JG.Ca~(2+)transport by mammalian mitochondria and its role in hormone action.AorleinhmornoeaCtion.AmJphysiol.1985,249:E 543-E554.
[55]Dnenton RM MmoccrackJG.Ca~(2+)as a second messenger within the mitochondria of the heart and other tissues Annu Rev Physiol 1990,52:451-466.
[2]张立德,等 纳米材料科学[M]沈阳:辽宁出版社,1994
[3]张立德,等 开拓原子和物质的中间领域-纳米微粒与纳米固体物理,1992.21(3):167-172
[4]郑玉峰 李莉著 生物医用材料 哈尔滨工业[M]大学出版社2005
[5]Bermudez E,Mangum JB,Wong BA,et al.Pulmonary responses of rats,mice and hamsters to subchronic inhalation of ultrafine titaniumdi oxide particles.Toxicol Sci 2004 77(2):347-57
[6]Renwick LC,Donaldson K,Clouter A.Impairment of alveolarmacrophage phagocytosis by ultrafine particles.Toxicol Appl Pharmacol 2001 172:119-27
[7]金一和,孙鹏,张颖花.纳米材料对人体潜在性影响问题[J].自然,2002,23(5):306-7
[8]Birringer R,Gleiter H,Klein HP,et al.N anocr2ystalline meterials:A n app roach to a novel so 12id st ructurew ith gas like diso rder?Phy Let A,1984;102:365-369
[9]Ch ikara Hayash i.U lt rafine part icales.J V alcumScience and Techno logy,1987;A 5(4):1375-1384
[10]Lu W,Tan YZ,Hu KL,et al.Cationic albumin conjugated pegylatednanoparticle with its transcytosis ability and little toxicity against blood-brain barrier.Int J Pharm 2005W295(1-2):247-6
[11]白春礼.科学通报,2001;46(1):90
[12]Andrievsk R.A.J.Mater.Sci 1994;29:614
[13]Beck D.D,Siege R.W.J.Mater.Res.,1992;7:284
[14]Abdulkhadar M.,Thomas B.Nanostructured Mater.,1995:5(3):289
[15]杜仕国,施冬梅,邓辉.自然杂志,2000;22(2):101
[16]Hoet PH,Bruske-Hohlfeld I,Salata OV.Nanoparticles-known and unknown health risks.J Nanobiotechnology 2004 2:12
[17]Andre Nel,Tian Xia,Lutz Mdler,et al.Toxic potential of materials at thenanolevel.Science 2006 311:622-727
[18]Li N,Sioutas C,Cho A,et al.Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage[J].Environ Health Perspect,2003,111:455
[19]Derfus AM,Chan WCW,Bhatia SN.Probing the cytotoxicity of semiconductor quantum dots J.Nano Lett,2004,4(1):11
[20]Joo SH,Feitz AJ,Waite TD.Oxidative degradation of the carbothioate herbicide,molinate, using nanoscale zerovalentiron[J].Environ Sci Technol,2004,38:2242.
[21]Zhou YM,Zhong CY,Kennedy IM,et al.Oxidative stressand NFB activation in the lungs of rots:a synergistic interaction between soot and iron particles[J].Toxicol Appl Pharmacol,2003,190:157
[22]LiN,Sioutas C,ChoA,Schmitz D,Misra C,Sempf J,et al.Ultrafme particulate pollutants induce oxidative stress andmitochondrial damage[J].EnvironHealth Perspect,2003,111(4):455-456
[23]YamakoshiY,Umezawa N,Ryu A,Arakane K,Miyata N,GodaY,etal.Active oxygen species generated from photoexcitedfullerene(C60)as potentialmedicines:O2-versusl O2[J].J Am Chem Soc,2003,125(42):12803-12809
[24]Are diamond nanoparticles cytotoxic?J Phys Chem B.2007 Jan 11;111(1):2-7.
[25]Hughes LS,Cass GR,Gone J,et al.Physical and chemical characterization of atmospheric ultrafine particles in the Los Angeles area[J].Environ Sci Technol 1998,32:1153.
[26]Kulmala M.Formation and growth rates of ultrafine atmospheric particles:a review of observations[J].J Aerosol Sci,2004,35:143
[27]Compton M.M.,Cancer Metastasis Rev.,11,105-109(1992).
[28]Chen X,Deng C,Tang S,Zhang Mitochondria-dependent apoptosis induced by nanoscale hydroxyapatite in human gastric cancer SGC-7901 cells.Biol Pharm Bull.2007 Jan;30(1):128-32.
[29]Wang XB,Gao HY,Hou BL,Huang J,Xi RG,Wu LJ Nanoparticle realgar powders induce apoptosis in U937 cells through caspase MAPK and mitochondrial pathways.Arch Pharm Res.2007 May;30(5):653-8.
[30]陈国明,刘岚.柠檬酸包埋的四氧化三铁纳米颗粒安全性研究[J].江苏药学与临床研究,2003,11(6):4-6
[31]Hansen T,Boutrand JP,Eloy R,et al.Biological tolerance of different nanoparticles in a rat model-clinical and histological findings.7th World Biomaterials Congress 2004:752
[32]Thomas K,Sayre P.Research Strategies for Safety Evaluation of Nanomaterials,Part Ⅰ:Evaluating the Human Health Implications of Exposureto Nanoscale Materials.Toxicol Sci 2005W87(2):316-321
[33]尹其华,刘璐,顾宁,等,59Fe示踪谷氨酸包被三氧化二铁纳米颗粒的制备及其在小鼠体内的生物学分布[J].医学研究生学报,2005,18(4):312-6
[34]Revell PA,Gatti AM,Gambarel li A,et al.Detection of cocr particles in the spleen of guinea pigs six weeks after their intra-osseous implanta-tion.7th World Biomaterials Congress 2004:753
[35]Hussain SM,Hess KL,Gearhart JM,et al.in vitro toxicity of nanoparti-cles in BRL 3A rat liver cells.Toxicol In Vitro 2005W19(7):975-83
[36]孟纯阳,安洪.纳米羟基磷灰石/聚酰胺66与人胚胎成纤维细胞的生物相容性研究[J].重庆医科大学学报,2005,30(2):169-72
[37]戴伯军,李家顺,贾连顺,等.纳米氧化锆强韧化高孔隙率磷酸钙人工古细胞支架生物学评价[J].中国矫形外科杂志,2004,12(13):998-1001
[38]许艳慧,李四群,李志安.纳米羟基磷灰石复合材料的研究-纳米羟基磷灰石的细胞毒性[J].实用口腔医学杂志,2004,20(2):147-50
[39]Chen CN,Kuo CC,Chen LT.Evaluation of cellular compatibility andblood compatibility of electrospum bioabsorbable Nanofiber.Tth WoddBiomaterials Congress 2004:1741
[40]Andre Nel,Tian Xia,Lutz M^dler,et al.Toxic potential of materials at thenanolevel.Science 2006W311:622-727
[41]李兴,邱宗荫,潘卫等 肝癌细胞线粒体的纯化及双向凝胶电泳分析[J].重庆医科大学学报,2005:30(2):163-167
[42]Wallase KB,Eells J T,Madeira V M,et al.Fundam Appl Toxicol.1997 38(1):23-27.
[43][美]DL.斯佩克特,RD.戈德曼,LA.莱因万德等著,黄培堂等译.细胞实验指南.科学出版社.
[44]李鸣真,等中西医结台杂志 1989;9(7):412
[45]季静娟,等酶检测法在线粒体功能及混杂程度评价中的应用.临床输血与检验,2004;6(3):167-168
[46]王小平,等脱辅基细胞色素c定向于线粒体的转运特异性研究.中国生物化学与分子生物学报,1999:15(5):813-819
[47]李兴,等肝癌细胞线粒体的纯化及双向凝胶电泳分析.重庆医科大学学报,2005;30(2):163-177
[48]冯庆玲,崔福斋,张伟.纳米羟基磷灰石/胶原骨修复材料[J].中国医学科学院学报,2002,24(2):124-128.
[49]Shinomiya K,Itoh S,Kawauchi T.Development of a novel hydroxyapatite/collagen composite biomaterial[C].International Congress Series,1 222(Tissue Engineering for Therapeutic Use 5),2001,165-177.
[50]#12
[51]Hideki Aoki,et al.Effects of HAP-sol on cell growth[J].Report of the institute forMedical and Dental Engineering,1992,26(1):15-21.
[52]张士成,李世普等.磷灰石超微粉对癌细胞作用的初步研究[J].武汉工业大学学报,1996,18(1):5-8.
[53]DentonRMoccmrackJG.on the role of the calcium transport cycle in heart and other mammalian mitochondrial.EFBS(Fed Eur Biochem Soc)Lett.1980,119:1-8.
[54]DnentonRM,Moccmrxack JG.Ca~(2+)transport by mammalian mitochondria and its role in hormone action.AorleinhmornoeaCtion.AmJphysiol.1985,249:E 543-E554.
[55]Dnenton RM MmoccrackJG.Ca~(2+)as a second messenger within the mitochondria of the heart and other tissues Annu Rev Physiol 1990,52:451-466.