安岩20082895不同铜源对Caco-2细胞培养基质及细胞中酶活性的影响

青岛农业大学毕业论文题目：不同铜源对Caco-2细胞培养基质及细胞中酶活性的影响姓名：安岩学院：动物科技学院专业：动物医学专业班级：0801学号：20082895指导教师：朱连勤教授2012年06月08日青岛农业大学学士学位论文目录目录中文摘要........................................................................................................................1Abstract..........................................................................................................................1中英文对照表................................................................................................................1引言................................................................................................................................11材料与方法................................................................................................................41.1材料.........................................................................................................................41.1.1细胞株..................................................................................................................41.1.2主要试剂及耗材..................................................................................................41.1.3主要仪器..............................................................................................................41.1.4主要溶液配制......................................................................................................51.1.5培养用品准备......................................................................................................51.2试验方法.................................................................................................................61.2.1试验设计...............................................................................................................61.2.2检测方法...............................................................................................................61.3数据处理.................................................................................................................72结果............................................................................................................................72.1不同铜源对Caco-2细胞培养基质中的铜离子浓度的影响................................72.2不同铜源对Caco-2细胞培养基质及细胞中酶活性的影响................................73讨论............................................................................................................................93.1不同铜源对Caco-2细胞培养基质中的铜离子浓度的影响................................93.2不同铜源对Caco-2细胞培养基质及细胞中酶活性的影响................................94结论..........................................................................................................................10致谢..............................................................................................................................11参考文献......................................................................................................................12青岛农业大学学士学位论文中文摘要1不同铜源对Caco-2细胞培养基质及细胞中酶活性的影响动物医学专业安岩指导教师朱连勤摘要：本试验旨在利用Caco-2细胞模型，研究纳米氧化铜对Caco-2细胞中酶类的影响，为进一步探讨纳米氧化铜在肠道中的转运吸收机制奠定理论基础。取对数生长期Caco-2细胞，按2×105个/mL的细胞浓度接种于6孔板中，在37℃、5%CO2浓度及饱和湿度的条件下培养24h后，换成含有不同铜源的培养液。硫酸铜组（铜浓度分别为4mg/L、8mg/L、16mg/L、32mg/L）；微米氧化铜组（铜浓度分别为4mg/L、8mg/L、16mg/L、32mg/L）；纳米氧化铜组（铜浓度分别为4mg/L、8mg/L、16mg/L、32mg/L），每组设6个重复。作用24h后，收集培养基质检测铜离子浓度和LDH活性，收集细胞裂解液检测ALP、SOD活性。结果显示，铜水平16mg/L和32mg/L纳米氧化铜的溶解度分别显著高于同等铜浓度的微米氧化铜，低于硫酸铜（P0.05）；纳米氧化铜组（16mg/L、32mg/L）的LDH活性分别低于同等浓度下的硫酸铜组（P0.05）；在培养基中以硫酸铜形式添加铜32mg/L，以纳米氧化铜形式添加铜8mg/L、16mg/L和32mg/L，以氧化铜形式添加铜16mg/L时，Caco-2细胞中ALP活性显著低于对照组(P0.05)；添加纳米氧化铜的各组的SOD活性显著高于对照组和添加微米氧化铜的各组（P0.05）。由此表明，纳米氧化铜在Caco-2细胞培养基质中部分以铜离子形式存在，其对细胞的损伤要小于硫酸铜，同时能提高Caco-2细胞的抗氧化能力。关键词：Caco-2细胞；纳米氧化铜；乳酸脱氢酶；碱性磷酸酶；超氧化物歧化酶青岛农业大学学士学位论文Abstract1CopperfromDifferentSourcestotheEffectofEnzymesActivityintheCaco-2CellsCultureMatrixandintheCellsStudentmajoringinVeterinaryMedicineAnYanTutorZhuLian-qinAbstract:ThistestwasdesignedtotakeadvantageoftheCaco-2cellsinvitroexperimentalmodel,studynano-CuOtotheeffectoftheenzymesinCaco-2cellsandculturematrixandestablishthetheoreticalbasistofurtherinvestigateintotheintestinalabsorptionmechanismofnano-CuO.TheCaco-2cellsinthelogarithmicphaseweretakentovaccinatein6-wellplatesat2×105/mLcellconcentration.Afterincubatedat37℃,theconcentrationof5%CO2andsaturatedhumidityconditionsfor24h,wemadethemintodifferentcoppersourcessuspensions.CuSO4groups(copperlevelswere4mg/L,8mg/L,16mg/L,32mg/Lrespectively),micro-CuOgroups(copperlevelswere4mg/L,8mg/L,16mg/L,32mg/Lrespectively),nano-CuOgroups(copperlevelswere4mg/L,8mg/L,16mg/L,32mg/Lrespectively),andrepeatedsixtimesineachgroup.Thenafterincubatedfor24h,culturematrixeswerecollectedtodetecttheconcentrationofCu2+andtheactivityofLDH,andwecollectedthecelllysatestodetecttheactivityofALPandSOD.Theresultsshowedthat,inthecopperlevels16mg/Land32mg/L,thesolubilityofnano-CuOweresignificantlyhigherthanthesamecopperlevelsofmicro-CuO,lessthanCuSO4(P0.05);CuwasaddedintothemediumintheformofCuSO432mg/L,addedintheformofnano-CuO8mg/L,16mg/Land32mg/L,andaddedintheformofmicro-CuO16mg/L,theactivityofALPinCaco-2cellswassignificantlylowerthanthecontrolgroup(P0.05);theactivityofLDHinnano-