胃癌微环境对大鼠骨髓间充质干细胞形态、生长及CD34、CD44表达的影响及细菌吸附重金属外文翻译

胃癌微环境对大鼠骨髓间充质干细胞形态、生长及CD34、CD44表达的影响文章快速阅读：骨髓间充质干细胞形态、生长及CD34骨髓间充质干细胞形态、生长及CD34、CD44表达与胃癌微环境的影响实验组对照组实验组对照组骨髓间充质干细胞单独常规培养建立胃癌微环境人胃癌BCG-823细胞与大鼠骨髓间充质干细胞非接触共培养倒置相差显微镜观察骨髓间充质干细胞形态的变化MTT法观察骨髓间充质干细胞的增殖率流式细胞术检测骨髓间充质干细胞周期及表面抗原CD34、CD44的表达 文题释义：肿瘤微环境：是一个复杂的综合系统，是肿瘤在其发生发展过程中所处的内环境，主要由肿瘤细胞本身、众多基质细胞及肿瘤细胞产生的细胞因子、蛋白水解酶、微血管、微淋巴管、组织液等共同构成。CD34分子：是高度糖基化的i型跨膜糖蛋白，选择性地表达于人类及其他哺乳动物造血干/祖细胞表面，并随细胞的成熟逐渐减弱至消失。目前已有愈来愈多的研究结果表明CD34分子在介导细胞间黏附作用中发挥着重要作用，可以参与造血干细胞的运输、定植，参与炎症反应以及淋巴细胞的归巢。摘要背景：临床胃癌组织及裸鼠体内致瘤组织分离得到的胃癌间质干细胞(GC-MSCs)，其生物学特性与骨髓间充质干细胞相似，并证明是肿瘤微环境的重要组成部分，可促进肿瘤的生长，作者推测骨髓间充质干细胞在胃癌微环境中生物学特性可能发生变化。目的：观察胃癌微环境对骨髓间充质干细胞形态、生长及CD34、CD44表达的影响。方法：设立骨髓间充质干细胞单独常规培养为对照组，实验组采用transwell小室将人胃癌BCG-823细胞与大鼠骨髓间充质干细胞非接触共培养建立胃癌微环境，倒置相差显微镜观察骨髓间充质干细胞形态的变化、四甲基偶氮唑盐比色法(MTT法)观察骨髓间充质干细胞的增殖率、流式细胞术(FCM)检测骨髓间充质干细胞周期及表面抗原CD34、CD44的表达。结果与结论：①实验组骨髓间充质干细胞排列紊乱，不规则，细胞间连接疏松，细胞变细、变长，胞核变小，呈团生长，与人胃BGC-823癌细胞形态有相似之处；②G1期细胞比例明显降低，S期细胞比例明显增加(P<0.01)、G2/M期细胞比率显著增加(P<0.05)；③实验组骨髓间充质干细胞的CD44阳性表达率显著降低，CD34阳性表达率显著降升高(P<0.01)；④结果表明，将人胃癌BCG-823细胞与大鼠骨髓间充质干细胞非接触共培养模拟胃癌微环境对骨髓间充质干细胞形态、生长增殖及CD34、CD44表达有显著影响，有向胃癌细胞恶性转化的趋势。关键词：主题词：间质干细胞；细胞增殖；CD34；CD44；组织工程基金资助：国家自然科学基金项目(81260525)MorphologyandproliferationofbonemarrowmesenchymalstemcellsandexpressionsofCD34andCD44understomachcancermicroenvironmentWuGao-feng1,LiuXi-ping1,YangBai-lin1,LiPei-qing1,MingHai-xia1,ZhangWei2(1BasicMedicineSchoolofGansuUniversityofChineseMedicine,Lanzhou73000,GansuProvince,China;2theFirstAffiliatedHospitalofLanzhouUniversity,Lanzhou73000,GansuProvince,China)AbstractBACKGROUND:Gastriccancermesenchyalstemcellsfromclinicalstomachcancerspecimensandtumorigenictissuesinnudemicearesimilartothebonemarrowmesenchymalstemcellsinbiologicalcharacteristics,whichhavebeenprovedtobeanimportantcomponentoftumormicroenvironmenttopromotetumorgrowth.Itisspeculatedthatbiologicalcharacteristicofbonemarrowmesenchymalstemcellsmaychangeinstomachcancermicroenvironment.OBJECTIVE:ToobservetheeffectofstomachcancermicroenvironmentonmorphologyandproliferationofbonemarrowmesenchymalstemcellsandexpressionsofCD34andCD44.METHODS:Ratbonemarrowmesenchymalstemcellswereculturedaloneascontrolgroup.Inthetestgroup,ratbonemarrowmesenchymalstemcellswereco-culturedwithhumanstomachcancerBGC-823cellsusingTranswellchamberassaytoestablishthestomachcancermicroenvironment.Then,cellmorphology,proliferation,cellcycleandCD34,CD44expressionswereobservedanddetectedusinginvertedphasecontrastmicroscope,MTTassay,andflowcytometry,respectively.RESULTSANDCONCLUSION:Inthetestgroup,bonemarrowmesenchymalstemcellsweresimilartohumanstomachcancercellsBGC-823thatarrangeddisorderlyandirregularly,wereinterconnectedloosely,becamethinnerandlonger,andgrewinclusterswithsmallernuclei.ThecellproportioninG1phasesignificantlydecreased,butthatinSandG2/Mphasessignificantlyincreased(P<0.01,P<0.05).ThepositiverateofCD44significantlydeclined,andtheCD34expressionsignificantlyraised(P<0.01).Inconclusion,stomachcancermicroenvironmentbynon-contactco-culturewithBCG-823cellshasanobviouseffectonthemorphology,proliferationandsurfaceantigensexpressionsofbonemarrowmesenchymalstemcellsthatwilltendtobemalignantgastriccancercells.Subjectheadings:MesenchymalStemCells;CellProliferation;CD34;CD44;TissueEngineeringFunding:theNationalNaturalScienceFoundationofChina,No.81260525Citethisarticle:WuGF,LiuXP,YangBL,LiPQ,MingHX,ZhangW.MorphologyandproliferationofbonemarrowmesenchymalstemcellsandexpressionsofCD34andCD44understomachcancermicroenvironment.ZhongguoZuzhiGongchengYanjiu.2016;20(14):2040-2045.WuGao-feng,Studyingformaster’sdegree,BasicMedicineSchoolofGansuUniversityofChineseMedicine,WuGao-feng,Studyingformaster’sdegree,BasicMedicineSchoolofGansuUniversityofChineseMedicine,Lanzhou730000,GansuProvince,ChinaCorrespondingauthor:LiuXi-ping,M.D.,Professor,BasicMedicineSchoolofGansuUniversityofChineseMedicine,Lanzhou730000,GansuProvince,China0引言Introduction骨髓间充质干细胞良好的迁移性、肿瘤趋向性及易于分离扩增和易于导入外源基因的特点[1]，使其成为肿瘤生物治疗的首选载体。利用骨髓间充质干细胞作为胃癌生物治疗的载体，追踪并运送肿瘤靶向药物到恶性肿瘤内部，或将基因修饰的骨髓间充质干细胞作为基因治疗的靶向载体以增强其抗肿瘤效应是胃癌治疗的全新思路与探索[2-3]，可能为胃癌的治疗带来希望。然而，随着对骨髓间充质干细胞生理特性和肿瘤研究的不断深入，发现骨髓间充质干细胞本身、尤其在肿瘤微环境中可能恶性转化，具有成瘤、促瘤风险[4-5]。据此，作者此次研究以“微环境诱导”理论为指导，采用transwell小室将人胃癌BCG-823细胞与大鼠骨髓间充质干细胞共培养方式建立胃癌微环境中骨髓间充质干细胞的生长转化模型，观察胃癌微环境对骨髓间充质干细胞的形态、生长及CD34、CD44表达的影响，以期进一步为胃癌安全应用骨髓间充质干细胞的生物治疗提供参考依据。1材料和方法Materialsandmethods1.1设计细胞学体外实验观察。1.2时间及地点于2014年1至7月在甘肃中医药大学科研实验中心完成。1.3材料实验动物：SPF级2周龄SD乳大鼠8只，体质量(45±5)g，由北京华阜康生物科技股份有限公司提供，合格证号SCXK(京)2014-0004。胃癌细胞：人胃癌BGC-823细胞，由上海中科院细胞库提供，用含有青链霉素各100u/mL的DMEM/F12培养基置于体积分数5%CO2、37℃条件下的培养箱中培养，定期换液传代，选择对数生长期的BGC-823细胞用于实验。胃癌微环境对大鼠骨髓间充质干细胞影响实验用主要试剂及仪器：试剂和仪器来源胎牛血清、PBS消化液、DMEM/F12培养基、FBS胃癌微环境对大鼠骨髓间充质干细胞影响实验用主要试剂及仪器：试剂和仪器来源胎牛血清、PBS消化液、DMEM/F12培养基、FBS消化液Hyclone公司0.05%胰酶-EDTA、1%青链霉素混合液Gibco公司MTT试剂盒、CD34/FITC抗体、CD44/PE抗体 英国abcam公司流式细胞仪 美国BectonDicknson公司Transwell小室 美国Costar公司1.4实验方法1.4.1大鼠骨髓间充质干细胞的分离及培养取SD乳大鼠，颈椎脱臼法处死后，分离出大鼠股骨和胫骨，从中剪断骨头，用2mL无菌注射器吸取DMEM/F12溶液冲出骨髓细胞多次，再用针头吹打，吸管吸入离心管内，100心管内，2000r/min离心15-25min，离心后溶液分4层，吸取中间骨髓基质细胞层(白色浑浊状)，PBS洗3次。用含体积分数15%胎牛血清及青链霉素的DMEM/F12以109L-1的细胞浓度接种于25cm2培养瓶中，置37℃1.4.2实验分组及细胞共培养体系的建立实验分为对照组(骨髓间充质干细胞单独常规培养)、实验组(骨髓间充质干细胞与BGC-823细胞共培养)。对照组以第3代骨髓间充质干细胞以2×105的量接种于Transwell小室内，下层为为含体积分数10%胎牛血清和1%青链霉素混合液的DMEM/F12基础培养液。实验组采用具有PET膜的Transwell悬挂式培养小室结合6孔板进行非接触式共培养，将第3代骨髓间充质干细胞以2×105的量种植于Transwell共培养系统的下室，再将Transwell的上室置于孔中，BGC-823细胞以2×105的量接种于上室内。分别在37℃、体积分数5%CO21.4.3各组细胞形态学观察各组细胞7d后终止培养，收集细胞移到培养瓶中继续培养，倒置相差显微镜下观察各组细胞的形态变化，并照相记录。1.4.4MTT法检测骨髓间充质干细胞的增殖收集各组细胞，用0.25%胰蛋白酶将骨髓间充质干细胞消化制成单细胞悬液，并调整细胞浓度为1×107L-1，分别接种于96孔培养板，每组细胞每个培养板接种3复孔，每孔200μL。同时设不加细胞只加培养液的空白对照孔，其操作与实验组和对照组一致。每组每天每孔加入20μL5g/LMTT溶液，将培养板在体积分数5%CO2、37℃、饱和湿度培养箱中培养内孵育4h。终止培养，小心吸去孔内培养液。沉淀内加入150μL二甲基亚砜，摇床上低速震荡10min，使结晶物充分溶解，用酶联免疫检测仪分别于第1，2，3，4，5，6，7天，选择490nm波长处测定吸光度(A值)，计算细胞增殖率：细胞增殖率(%)=实验组或对照组-空白孔/空白孔A1.4.5FCM检测骨髓间充质干细胞周期各组细胞培养7d后终止培养，用胰酶将骨髓间充质干细胞消化成单细胞悬液，离心3-5min后沉淀细胞，弃上清，以冰浴预冷的PBS重悬细胞；用4℃冰浴预冷的体积分数70%乙醇固定过夜，再以冰浴预冷的PBS重悬细胞，参考周期试剂盒说明，每管细胞样品中加入0.5mL碘化丙啶染色液，缓慢并充分重悬细胞沉淀，37℃避光温育30min。随后可以4℃或冰浴避光存放，染色完成后宜在24h内完成流式检测。FCM在激发波长488nm波长处检测红色荧光，同时检测光散射情况，分析细胞DNA含量和光散射分析，计算G11.4.6FCM检测骨髓间充质干细胞的CD34和CD44表达取经胰酶消化的骨髓间充质干细胞单细胞悬液，调整细胞浓度为1×108L-1，PBS清洗后弃上清用于标记。每组细胞均按每管约106个细胞的比例，收集至Eppendorf管中。加入用StainingBuffer稀释后的一抗，4℃孵育30min。洗涤2次后，实验组与对照组加入异硫氢酸荧光素(FITC)标记的CD34和藻红蛋白(PE)标记的CD441.5主要观察指标1.6统计学分析实验结果采用SPSS17.0软件进行统计学分析，计量资料用±s表示，组间比较用配对样本的t检验，P<0.05为差异有显著性意义。2结果Results2.1胃癌微环境对骨髓间充质干细胞形态的影响结果见图1。对照组骨髓间充质干细胞形态呈短平状，长梭形，有序、均匀排列，似成纤维细胞样生长，有明显的折光性。与人胃BGC-823癌细胞共培养7d后，细胞排列紊乱，呈不规则型，细胞间连接疏松，细胞变细变长，胞核变小，呈团生长，与人胃BGC-823癌细胞形态有相似之处。2.2胃癌微环境对骨髓间充质干细胞增殖的影响结果见表1、图2。将骨髓间充质干细胞与人胃BGC-823癌细胞共培养后，相同时间点的骨髓间充质干细胞的增殖率高于单独培养组，差异有显著性意义(P<0.05或P<0.01)。表注：与对照组同时间比较，aP<0.01。时间表注：与对照组同时间比较，aP<0.01。时间对照组实验组1d1.51±1.201.72±1.362d32.23±2.1334.13±2.083d33.51±1.3750.24±2.464d38.51±1.4876.39±2.405d64.16±2.86165.92±0.046d78.15±2.06190.24±1.877d127.40±0.45244.43±0.09表1胃癌微环境对骨髓间充质干细胞增殖率的影响(±s，n=6，%)Table1Effectofstomachcancermicroenvironmentonthegrowthrateofbonemarrowmesenchymalstemcells2.3胃癌微环境对骨髓间充质干细胞周期的影响见图3、表2。FCM检测骨髓间充质干细胞周期显示，将骨髓间充质干细胞与人胃BGC-823癌细胞共培养后，G1期细胞比例明显降低，S期细胞比例明显增加(P<0.01)、G2/M期细胞比率显著增加(P<0.05)。表2胃癌微环境对骨髓间充质干细胞周期的影响(表2胃癌微环境对骨髓间充质干细胞周期的影响(±s，n=6)Table2Effectofstomachcancermicroenvironmentonthecellcycleofbonemarrowmesenchymalstemcells表注：与对照组比较，aP<0.05，bP<0.01。组别G1期S期G2/M期对照组87.64±0.2811.35±0.261.01±0.21实验组59.08±0.73b35.89±2.82b5.04±2.112.4胃癌微环境对骨髓间充质干细胞CD44、CD34表达的影响结果见图4、表3。经流式细胞术检测骨髓间充质干细胞表面标记物CD44、CD34，对照组CD44阳性表达率为(97.79±7.10)%，呈现高表达状态，CD34阳性表达率为(1.77±2.13)%，呈现低表达状态。实验组CD44阳性表达率为(70.67±4.37)%，与对照组比较显著降低(P<0.01)，CD34的阳性表达率为(27.93±5.32)%，与对照组比较显著升高(P<0.01)。表注：与对照组比较，aP<0.01。组别表注：与对照组比较，aP<0.01。组别CD44CD34对照组97.79±7.101.77±2.13实验组70.67±4.3727.93±5.32表3胃癌微环境对骨髓间充质干细胞表面标志抗原CD44、CD34表达的影响(±s，n=6，%)Table3EffectofstomachcancermicroenvironmentontheexpressionsofCD44andCD34ofbonemarrowmesenchymalstemcells3讨论Discussion干细胞生存的微环境亦称干细胞巢，它是干细胞维持稳态(homeostasis)，即保持细胞正常增殖、分化、代谢和功能活动的关键，巢的成分改变和巢中信号通路的改变可能导致正常的干细胞发生病变，导致干细胞分化受阻并过度增殖，向肿瘤细胞转化[6]。肿瘤微环境是一个复杂的综合系统，是肿瘤在其发生发展过程中所处的内环境，主要由肿瘤细胞本身、众多基质细胞及肿瘤细胞产生的细胞因子、蛋白水解酶、微血管、微淋巴管、组织液等共同构成[7]。肿瘤微环境能调控肿瘤细胞和骨髓间充质干细胞的增殖、分化等多种生物学行为[8]。已有研究表明，胶质瘤微环境[9]、肺癌微环境[10]、结肠癌微环境可诱导骨髓间充质干细胞引起细胞形态[11]、增殖能力等生物学特性的改变。研究认为胃癌发病与骨髓间充质干细胞有关，幽门螺杆菌等长期慢性感染，骨髓来源的干细胞会聚集到胃粘膜处，参与损伤修复并增生，久而久之发生肠化、不典型增生及癌肿[12]。临床胃癌组织及裸鼠体内致瘤组织分离得到的胃癌间质干细胞(GC-MSCs)，其生物学特性与骨髓间充质干细胞相似[13]，并证明是肿瘤微环境的重要组成部分，可促进肿瘤的生长，研究进一步显示在胃癌微环境中，骨髓间充质干细胞可向肿瘤相关成纤维母细胞(TAFs)转化，表达TAF的特异标志，体内可促进肿瘤细胞生长[14]。也有研究认为短时间的培养虽影响不到图2胃癌微环境对骨髓间充质干细胞增殖率的影响Figure2Effectofstomachcancermicroenvironmentonthegrowthrateofbonemarrowmesenchymalstemcells图注：将骨髓间充质干细胞与人胃BGC-823癌细胞共培养后的细胞增殖率高于单独培养组，图1各组细胞形态学的观察(倒置相差显微镜，×100)Figure1Morphologicalobservationofcellsinthecontrolandtestgroups(invertedphasecontrastmicroscope,×100)图注：图A为对照组；B为实验组；C为人胃BGC-823癌细胞。B1234567对照组实验组时间(d)AG1：87.75%G2：1.17%S：11.08%G1图2胃癌微环境对骨髓间充质干细胞增殖率的影响Figure2Effectofstomachcancermicroenvironmentonthegrowthrateofbonemarrowmesenchymalstemcells图注：将骨髓间充质干细胞与人胃BGC-823癌细胞共培养后的细胞增殖率高于单独培养组，图1各组细胞形态学的观察(倒置相差显微镜，×100)Figure1Morphologicalobservationofcellsinthecontrolandtestgroups(invertedphasecontrastmicroscope,×100)图注：图A为对照组；B为实验组；C为人胃BGC-823癌细胞。B1234567对照组实验组时间(d)AG1：87.75%G2：1.17%S：11.08%G1：58.55%G2：4.04%S：37.41%B图3各组骨髓间充质干细胞周期的变化Figure3Changesofcellcycleofbonemarrowmesenchymalstemcellsinthetwogroups图注：图中A为对照组；B为实验组。将骨髓间充质干细胞与人胃BGC-823癌细胞共培养后，G1期细胞比例明显降低，S期细胞比例明显增加、G2/M期细胞比率显著增加。CA300250200150100500增殖率(%)图4各组骨髓间充质干细胞表面标志抗原CD44、CD34的表达图4各组骨髓间充质干细胞表面标志抗原CD44、CD34的表达Figure4ExpressionsofCD44andCD34ofbonemarrowmesenchymalstemcellsinthetwogroups图注：BCAPAGE其增殖，但处于胃癌微环境中的骨髓间充质干细胞生物学特性已发生一定的改变来适应肿瘤微环境[15]。研究采用transwell小室将人胃癌BCG-823细胞与大鼠骨髓间充质干细胞共培养方式建立胃癌微环境中骨髓间充质干细胞的生长转化模型。实验结果表明，人胃癌BCG-823细胞与大鼠骨髓间充质干细胞共培养后，细胞排列紊乱，不规则，细胞间连接疏松，细胞变细变长，胞核变小，呈团生长，与人胃BGC-823癌细胞形态有相似之处；G1期细胞比例明显降低，S期细胞比例明显增加、G2/M期细胞比率显著增加，骨髓间充质干细胞的增殖率明显增高，表明胃癌微环境对骨髓间充质干细胞的增殖产生显著影响，这与相关研究并不一致[14]，可能与培养时间差异有关。骨髓间充质干细胞的表面抗原并不是独特的，目前为止并未找到非常特异性的骨髓间充质干细胞标记分子。一般认为，整合素家族成员黏附分子CD44，是骨髓间充质干细胞的重要标志物[16]，而骨髓间充质干细胞不表达造血细胞表面抗原，如造血前体细胞标志抗原CD34[17]。此次研究流式细胞仪检测结果显示，单独培养的骨髓间充质干细胞CD44阳性率呈现高表达状态，CD34阳性表达率则呈现低表达状态，实验组经人胃癌BCG-823细胞与大鼠骨髓间充质干细胞共培养后骨髓间充质干细胞的CD44阳性表达率明显下降，CD34阳性表达率明显升高，提示经胃癌微环境诱导后骨髓间充质干细胞的表面标记物发生明显改变。作者此次研究初步证实，胃癌微环境对骨髓间充质干细胞形态、生长增殖及其表面标记物CD44、CD34产生显著影响，说明肿瘤微环境可以使骨髓间充质干细胞增殖分化调控失调，干细胞微环境的改变可引起细胞与细胞间质信号交流的异常，从而造成干细胞的失控性增殖，为利用骨髓间充质干细胞作为胃癌生物治疗的载体在胃癌治疗方面的安全应用提供了基础实验依据。但在胃癌微环境中骨髓间充质干细胞是否获得了胃癌细胞的相关生物学特性，值得进一步研究。作者贡献：实验设计为刘喜平教授，实验实施为吴高峰、杨柏林研究生完成；明海霞副教授，张炜主治医师为资料的搜集整理和统计学分析；实验评估为刘喜平、李沛清教授。利益冲突：所有作者共同认可文章内容不涉及相关利益冲突。伦理问题：实验动物在戊巴妥纳麻醉下进行所有的手术，并尽一切努力最大限度地减少其疼痛、痛苦和死亡。文章查重：文章出版前已经过CNKI反剽窃文献检测系统进行3次查重。文章外审：本刊实行双盲外审制度，文章经国内小同行外审专家审核，符合本刊发稿宗旨。作者声明：文章第一作者对研究和撰写的论文中出现的不端行为承担责任。论文中涉及的原始图片、数据(包括计算机数据库)记录及样本已按照有关规定保存、分享和销毁，可接受核查。文章版权：文章出版前杂志已与全体作者授权人签署了版权相关协议。4参考文献ReferencesNakamizoA,MariniF,AmanoT,etal.Humanboneofgliomas.CancerRes.2005;65(8):3307-3318.NakamuraK,ItoY,KawanoY,etal.Anti-tumoreffectofgeneticallyengineeredMesenchymalstemcellsinaratgliomas.GeneTherapy.2004;11(14):1155-1164.StudenyM,MariniFC,DembinskiJL,etal.MesenchymalStemCells:potentialprecursorsfortumorstromaandtargeted-deliveryvehiclesforanticanceragents.JNatlCancerInst.2004;96(21):1593-1603.MiuraaM,MiuraY,Padilla-NashHM,etal.Accumulatedchromo-somalinstabilityinmurinebonemarrowmesenchymalstemcellstomalignanttransformation.Stemcell.2006;24(4):1095-1103.RoslandGV,SvendsenA,TorsvikA,etal.Long-termculturesofbonemarrowderivedhumanmesenchymalstemcellsfrequentlyun-dergospontaneousmalignanttransformation.CancerRes.2009;69(13):5331-5339.LaconiE.Theevolvingconceptoftumormicroenvironments.Bioessays.2007;29(8):738-744.BexellD,GunnarssonS,TorminA,eta1.Bonemarrowmu1potentmesenchymalstormacellsactaspericyte-1ikemigratoryvehiclesinexperimentalgliomas.MolecularTherapy.2009;17(6):183-190BergfeldSA,DeClerckYA.Bonemarrow-derivedmesenchymalstemcellsandthetumormicroenvironment.CancerMetastasisRev.2010;29(2):249-261.张亚兰,朱静,田杰,等.肿瘤微环境中间充质干细胞的相关生物学特性分析.解放军医学杂志,2010,35(1):67-70.刘永琦,王倩,秦洁,等.肿瘤微环境对骨髓间充质干细胞形态、生长及增殖的影响[J].中国细胞生物学学报,2013,35(2):180-187.景明,陈正君,王雅莉,等.结肠癌微环境对骨髓间充质干细胞形态、增殖及CD13、CD133表达的影响,2014,33(9):497-500.OgasawaraN,TsukamotoT,InadaK,etal.Frequentc-Kitgenemutationsnotonlyingastrointestinalstromaltumorsbutalsoinintersti-tialcellsofCajalinsurroundingnormalmucosa.CancerLett.2005;230(2):199-210HoughtonJ,StoicovC,NomuraS,etal.Gastriccanceroriginatingfrombonemarrow-derivedcells.Science.2004;306(3):1568-1571李争艳,刘杨,翟丽丽,等.胃癌细胞SGC-7901对骨髓来源的间充质干细胞作用的研究[J].临床与实验病理学杂志,2011,27(8):856-859JonesDL,WagersAJ.Noplacelikehome:anatomyandfunctionofthestemcellniche.NatureReviewsMolecularCellBiology.2008;9(1):11-21.DeUgarteDA,AlfonsoZ,ZukPA,etal.Differentialexpressionofstemcellmobilization-associatedmoleculesonmulti-lineagecellsfromadiposetissueandbonemarrow.ImmunolLett.2003;89(3):267.ShahdadfarA,FronsdalK,HaugT,etal.Invitroexpansionofhumanmesenchymalstemcellschoiceofserumisadeterminantofcellproliferation,differentiation,geneexpression,andtranscriptomestability.StemCells.2005;23(9):1357-1366.BacterialSorptionofHeavyMetalsFourbacteria,Bacilluscereus,B.subtilis,Escherichiacoli,andPseudomonasaeruginosa,wereexaminedfortheabilitytoremoveAg+,Cd2+,Cu2+,andLa3+fromsolutionbybatchequilibrationmethods.CdandCusorptionovertheconcentrationrange0.001to1mMwasdescribedbyFreundlichisotherms.At1mMconcentrationsofbothCd2+andCu2+,P.aeruginosaandB.cereuswerethemostandleastefficientatmetalremoval,respectively.FreundlichKconstantsindicatedthatE.coliwasmostefficientatCd2+removalandB.subtilisremovedthemostCu2+.RemovalofAg+fromsolutionbybacteriawasveryefficient;anaverageof89%ofthetotalAg+wasremovedfromthe1mMsolution,whileonly12,29,and27%ofthetotalCd2+,Cu2+,andLa3+,respectively,weresorbedfrom1mMsolutions.ElectronmicroscopyindicatedthatLa3+accumulatedatthecellsurfaceasneedlelike,crystallineprecipitates.Silverprecipitatedasdiscretecolloidalaggregatesatthecellsurfaceandoccasionallyinthecytoplasm.NeitherCd2+norCu2+providedenoughelectronscatteringtoidentifythelocationofsorption.TheaffinityseriesforbacterialremovalofthesemetalsdecreasedintheorderAg>La>Cu>Cd.Theresultsindicatethatbacterialcellsarecapableofbindinglargequantitiesofdifferentmetals.Adsorptionequationsmaybeusefulfordescribingbacterium-metalinteractionswithmetalssuchasCdandCu;however,thisapproachmaynotbeadequatewhenprecipitationofmetalsoccurs.Thefateoftoxicmetalliccationsinthesoilenvironmentdependslargelyontheinteractionsofthesemetalswithinorganicandorganicsurfaces.Theextenttowhichametalliccationinteractswiththesesurfacesdeterminestheconcentrationofmetalinsolutionand,consequently,thepotentialformovementintogroundwateroruptakebyplants.Aconsiderableamountofworkhasbeendonetoevaluatetheadsorptionorcomplexationofvariousheavymetalsbysoilsandsoilconstituents,suchasclaysandorganicmatterfractions.Onepotentiallyimportantorganicsurfacewhichhasreceivedlittleattentionisthatofthesoilmicrobialpopulation.Soilmicroorganismsaretypicallyassociatedwiththeclayandorganicfractionsofthesoilmicroenvironmentsandwouldbeexpectedtoparticipateinthemetaldynamicstypicallyascribedtothesefractions.Bacteriahaveahighsurfacearea-to-volumeratioand,asastrictlyphysicalcellularinterface,shouldhaveahighcapacityforsorbingmetalsfromsolution.Thereisevidencethatbacterialcellsaremoreefficientatmetalremovalthanclaymineralsonadry-weightbasis.Kurekandco-workersobservedthatsorptionofCd2+bydeadcellsofaParacoccussp.andSerratiamarcescenswasgreaterthanthatofmontmorillonitewhenthesolid-to-solutionratiowasthesameforbothbacteriaandclay.LivecellsaccumulatedaboutthesamequantityofCd2+asdidclay.Severalinvestigationshaveshownthatrelativelylargequantitiesofmetalliccationsarecomplexedbyalgae,bacteria,andfungi.Metalbindingbyisolatedgram-positiveandgram-negativebacterialcellwallshasalsobeenevaluated.Cellwallsofthegram-positivebacteriaBacillussubtilisandB.licheniformiswereobservedtobindlargerquantitiesofseveralmetalsthancellenvelopesofthegram-negativebacteriumEscherichiacoli.Weareinterestedintheroleofmicroorganismsinthebehaviorofvariousheavymetalsinthesoilenvironment.Theobjectivesofthisworkweretodeterminethemetal-bindingcapacitiesofwholecellsoftwogram-positiveandtwogram-negativebacteriaandtodeterminewhetheranequilibriummodel,theFreundlichadsorptionisotherm,wouldadequatelydescribebacterialmetalsorption.B.cereus,B.subtilis,andPseudomonasaeruginosawereexaminedasrepresentativesofcommonspeciesfrequentlyisolatedfromsoils.E.coliwasalsousedasasecondgram-negativebacteriumbecauseitisawell-characterizedmicroorganismanditscellenvelopehasbeenshowntobindlessmetalthandoB.subtiliscellwalls.ThefourmetallicionsusedinthisinvestigationwereAg+,Cd2+,Cu2+,andLa3+.Cadmiumandcopperarebothtoxiccationsofenvironmentalimportance.Silverandlanthanum,representativeofmonovalentandtrivalentheavymetals,respectively,arealsotoxicbutarelessfrequentlyfoundintheenvironment.MATERIALSANDMETHODSBacteriaandgrowthconditions.ThebacteriausedintheseexperimentswereB.cereusATCC11778;P.aeruginosaATCC14886,bothobtainedfromtheAmericanTypeCultureCollection;B.subtilis168;andE.coliK-12strainAB264,bothfromtheUniversityofGuelph.Thebacteriawereroutinelyculturedin0.5xbrainheartinfusionbroth(BBLMicrobiology.Systems)am.ended.with.2.4g.of.HEPES(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid)liter-1and2.0gofMES(2-N-morpholinoethanesulfonicacid)liter-1tobufferthemediumacidity.ThemediumaciditywasadjustedtopH6.8with0.5MKOH.Two2-millilitersamplesoflate-exponential-phasestartercultureswereusedtoinoculate800mlofbrothin3-literErlenmeyerflasks.Cellsweregrowntothelate-exponentialphaseatroomtemperature(approximately23°C)onanorbitalshakerat150rpm.CellsofB.cereusgrowninthismannerremainedinthevegetativestate.Thecellswereharvestedbycentrifugationandwashedtwicewithcold10mMCa(NO3)2whichhadbeenadjustedtopH4.0with0.5MHNO3.ThewashedcellswereresuspendedintheCa(NO3)2solutionataconcentrationofapproximately12mg(dryweight)ml-1andstoredfor2to4hat5°Cbeforeuse.TheCa(NO3)2Metalsorptionstudies.ThefourmetalsaltsusedinthisstudywereAgNO3，Cd(NO3)2·4H2O，Cu(NO3)2·2.5H2OandLa(NO3)2·6H2O.ThemetalsolutionswereadjustedtopH4.0with0.5MHNO3toavoidprecipitationofCuasCuCO3.IdentificationofthesemetalsinsolutionatpH4.0wasdonewiththeGEOCHEMcomputerprogram.AtpH4.0inaCa(NO3)2matrix,thesemetalliccationswerepredictedtobefoundprimarilyinthefreeionicform(>97%inallcases).Alloftheplasticwareusedinthesestudieswasleachedin3MHNO3andrinsedseveraltimeswithdouble-deionizedwaterbeforeusetoavoidmetalcontamination.Abatchequilibrationmethodwasusedtodeterminesorptionofmetalsbybacteria.Twomillilitersofwashedcellswasplacedina10-mlpolypropylenecentrifugetubecontaining6mlofcold10mMCa(NO3)2;and1mlofmetalstocksolutionwasadded.Thetubeswerecapped,placedonaninvertingshaker,andequilibratedfor2hat5°C.After2h,thecellswereremovedfromsolutionbycentrifugationandthesupernatantwascollectedandusedformetalanalysis.EquilibriummetalconcentrationsweredeterminedbyinductivelycoupledargonplasmaspectroscopyonaThermoJarrell-AshPlasma300spectrometer.Theamountofmetalremovedbythecellswasdeterminedonadry-weightbasis.Todeterminesorptionisotherms,finalmetalconcentrationsforCd2+were1,0.1,0.01,and0.001mM.InitialexperimentsindicatedthatsorptionofCu2+fromthe0.001Mmtreatmentresultedinequilibriumconcentrationsbelowthedetectionlimit.Subsequently,themostdiluteconcentrationofCu2+usedwas0.005mM.SorptionofAg+andLa3+from0.01and0.001mMsolutionsalsotypicallyresultedinconcentrationsbelowthedetectionlimit.TheconcentrationsofAgandLaevaluatedwere10,1,0.1,and0.01mM.Thesorptionexperimentwassetupasanunbalancedfour-by-fourlatticewiththreereplicationsovertime).Eachblockwithinreplicatescontainedfourdifferentbacterium-metalcombinationsatallfourmetalconcentrations.Whenappropriate,thesorptionisothermswereconstructedbythemethodsoutlinedbyDaoetal.withtheGLMprocedureoftheSASstatisticalprogram.Electronmicroscopy.Electronmicroscopywasdonetovisualizethelocationofmetalsonthebacterialcells.Cellswereequilibratedwith1mMmetalsolutionsasdescribedaboveandfixedfor30minatroomtemperaturein5%glutaraldehyde(EMgrade;PolysciencesInc.)containingthemetalofinterestataconcentrationapproximatelyequaltotheequilibrationconcentration.Thecellswerethenwashedfreeofglutaraldehydewiththemetalsolution,enrobedin2%Nobleagar(DifcoLaboratories),dehydratedthroughanethanol-propyleneoxideseries,andembeddedinSPURR(PolysciencesInc.).TheembeddedcellswerethinsectionedonaReichertUltracutEultramicrotome,andsectionswerecollectedonFormvarcarbon-coated200-meshcopperoraluminumgrids.Sectionsofmetal-treatedcellswerenotstained;theelectronscatteringprovidedbythesorbedmetalsactedasacontrastingagent.Somecontrolcellswerelightlystainedwith2%uranylacetatefor2mintoprovidebettervisualizationofthesecells.Electronmicros-copywasperformedat100kVonaPhilipsEM-400TequippedwithanEDAXenergy-dispersiveX-rayspectrometerinterfacedwithaTracorNorthernmultichannelanalyzer.Energy-dispersiveX-rayanalysiswasusedtoconfirmtheidentitiesofmetalsonthecells.Affinityseriesdetermination.Tofurtherelucidatetheaffinityofthebacterialcellsforthesemetals,thecellswereequilibratedfor2hat5°Cinsolutionscontainingeitherthesinglemetalataninitialconcentrationof1mMorallfourmetalsat1mMeach.Asdescribedabove,allmetalsolutionsweremadeinpH4.010mMCa(NO3)2.Afterthecellswereharvestedbycentrifugation,metalsinthesupernatantweredeterminedbyinductivelycoupledargonplasmaspectroscopy.Theexperimentwasreplicatedthreetimes.DataweresubjectedtoanalysisofvarianceproceduresoftheSASstatisticalprogram,andmeanswereseparatedbytheleast-significant-differencemethod.RESULTSBacterialsorptionofCd2+andCu2+fromsolutionwasdescribedwellbythelinearizedFreundlichadsorptionisothermequation,log10S=log1OK+nlog1OC,whereSistheamountofmetaladsorbedinmicromolespergram,Cistheequilibriumsolutionconcentrationinmicromolesperliter,andKandnareFreundlichconstants.TheFreundlichconstantsforCd2+andCu2+sorptionbythefourbacteriaaregiveninTable1.TheconstantKrepresentsthepredictedquantityofmetalremovedinmicromolesofmetalpergramofdrycellsatanequilibriumconcentrationof1uM,andnistheslopeoftheisotherm.ExaminationofKvaluesforCdsorptionshowedthatthegram-negativebacteriumE.coliwasmostefficientatCdsorptionandP.aeruginosaalsotendedtosorbmoreCd2+thandidthegram-positivebacteria.B.subtilisremovedthemostCu2+atanequilibriumconcentrationof1uM.However,onlya1.9-folddifferenceinCusorptionwasobservedbetweenthemostandleastefficientbacteria,B.subtilisandB.cereus,respectively.RepresentativeplotsofadsorptionisothermsforCd2+andCu2+areshowninFig.1.Analysisofcovarianceindicatedthattheslopesoftheisothermsweredifferentforthefourbacteria;athighequilibriumconcentrations,P.aeruginosaandB.cereusweremostandleastefficient,respectively,atremovingCd2+andCu2+fromsolution.FreundlichisothermswerenotusefulfordescribingtheremovalofAg+andLa3+fromsolutionbecausethereweretoofewdatumpointsoverawiderangeofconcentrations.EquilibriumconcentrationsofLa3+werebelowdetectionlimitswhentheinitialconcentrationwas10uM.SeveraloftheobservationsforAg+equilibriumconcentrationswerealsobelowdetectionlimitsatthe10uMconcentration.Whentheequilibriumconcentrationwasbelowdetectionlimits,thetotalmetalboundonadry-weightbasiswascalculatedwiththeassumptionthatessentiallyalloftheAg+orLa3+insolutionwasbound.A10mMtreatmentwasincludedforthesemetalstoextendtheconcentrationrangeexamined.AgoodrelationshipwasfoundforremovalofAg+fromsolutionasafunctionoftheinitialAg+concentrationfrom10to1,000p.M(Fig.2).TherewerenosignificantdifferencesinAg+removalamongbacteria.SaturationofthecellswithAg+apparentlyoccurredinthe10mMAg+treatment,asthetotalAg+boundincreasedonlyabout2-foldovera10-foldincreaseinAg+concentration.SignificantdifferencesamongthebacteriaforLa3+removalwerefoundinthe1mMtreatmentwhen33,70,114,and144p.molg-1wereremovedbyB.cereus,E.coli,B.subtilis,P.aeruginosa,respectively(Fig.3).TherewerenosignificantdifferencesamongbacteriaforLa3+removalfromthe10or100uMsolutions.ThebacterialcellswereevidentlysaturatedwithLaatthe1mMconcentration,asverylittleadditionalLa3+wasboundbythesecellsfromthe10mMLa3+treatment(Fig.3).Silverwasremovedfromsolutionmuchmoreefficientlythanweretheothermetalsatthe1and0.1mMconcentrations(Table2).Anaverageof99%ofthetotalAg+wasremovedfromsolutioninthe0.1mMtreatment.Cadmiumwasboundbythecellstoamuchlesserextent,withonly