超顺磁氧化铁标记对大鼠脂肪干细胞内IRPs/IREs结合活力与铁含量的关联研究
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摘要
研究背景
随着基础实验医学的发展,干细胞移植技术也经历着日新月异的变化；其在人体多种疾病的治疗,比如堵塞血管的再通,受损组织的修复,以及糖尿病的移植治疗等等方面,前景十分的诱人。因此,近年来,干细胞研究愈发成为实验医学界的研究热点。
目前,在临床应用中,主要有以下两种干细胞的应用性较好：1、骨髓间充质干细胞(Bone Marrow Mesenchyme Stem Cells, BMSCs);2、胚胎干细胞(Embryonic Stem Cells, ESCs)。但是,其两者,在具备一定的临床应用价值的同时,都具有较显著的局限性：1、骨髓间充质干细胞具有来源比较不足、提取相对困难的缺点；2、胚胎干细胞由于是一种来源于异体的干细胞,其不可避免的具有免疫排斥反应；而且,胚胎干细胞在使用过程中,还不可避免地涉及到伦理学等种种问题。所以,急需找到一种来源充分、取材方便、且患者容易接受的干细胞来源,以使干细胞移植在临床应用方面,取得长足的进展与突破。
在2001年,Zuk等人研究宣布,从人体抽脂术所提取的脂肪组织悬液中,经过一定的不太繁杂的实验步骤,能够成功地分离提取出一种细胞：这种细胞具有干细胞的生物学特性,当时称之为脂肪间充质干细胞(Adipose Tissue-Derived Mesenchymal Stem Cells, ADMSCs).如今简称其为脂肪干细胞(Adipose Tissue-Derived Stem Cells, ADSCs)。脂肪干细胞的发现,打开了干细胞移植研究的一扇新窗户,为临床应用的干细胞来源提供了新的选择与希望。
干细胞移植入受试者体内后,及时和准确地了解细胞在受试者体内的迁徙、归巢、增殖以及分化等情况,非常有助于评估移植治疗的效果、对移植方法的优化以及对移植窗口期行更合适的选择。近年来,国内、外众多研究者使用超顺磁氧化铁(Supraparamagnetic Iron Oxide, SPIO)对移植的干细胞进行体外标记；超顺磁氧化铁实为一种磁共振阴性对比剂,经其标记后,能够借助磁共振成像(Magnetic Resonance Imaging, MRI)在体、无创、动态、实时地对移植细胞进行活体示踪成像,根据靶组织与器官内部的磁共振信号改变,可以判断移植干细胞在体内的迁徙、归巢以及增殖等情况。
诚然,SPIO标记干细胞,能够对标记细胞行体外示踪,但是,其对标记细胞最直接的影响就是引起细胞内的铁超载。研究证实,SPIO标记细胞后,细胞体内的铁浓度较未标记时的浓度增加数十倍甚至数百倍。铁,是细胞体内的一种非常重要的微量元素,它是维持细胞的增殖、生长以及机能活动的一种不可或缺的重要物质,它在细胞体内的电子传递、DNA合成以及氧气的运输等过程中,具有着举足轻重的作用。对干细胞行SPIO铁标记后,细胞内铁含量的时间-浓度关系,即铁标记后,细胞内的铁含量随时间的变化关系,未有研究明确涉及,有必要阐明此两者关系。
干细胞移植治疗的主要目的,是将干细胞移植到特定的靶器官或组织内,使用相应的技术手段,使其按照预想地程序分化成特定的组织细胞,执行特定器官或组织的相应功能。所以,要求SPIO在细胞内的停留应该能够持续足够的时间,让使用MRI对干细胞行示踪观察时,MRI能够准确、全面地判断移植干细胞在体内的各种情况,并对移植疗效进行精确评估。有研究证实,SPIO标记干细胞后,MRI对其行示踪观察的有效时间可长达21天。所以,有必要阐明SPIO标记干细胞后,细胞内铁含量,能否被MRI作定性、定量分析,这也是对移植细胞在体示踪的关键。
铁调节蛋白／铁反应元件系统(Iron Regulatory Proteins/Iron Responsive Elements System, IRPs/IREs系统)是细胞体内一种非常重要的调节系统,它能够维持细胞体内的铁稳态。IRPs/IREs系统包括：铁蛋白(Ferritin、转铁蛋白受体(Transferrin Receptor, TfR)以及铁调节蛋白(Iron Regulatory Proteins, IRPs),三者相互作用,共同维持细胞内的铁稳态；其中Fn和TfR的表达,均受到IRPs在转录后水平上的调控。IRPs则通过与TfR和Fn mRNA上的高度保守的非翻译区(Untranslated Region, UTR)作用,以实现调控细胞内铁稳态的功能。TfR和FnmRNA的UTR与IRPs相结合的位点被称为铁反应元件(Iron Response Elements, IREs)。IRPs/IREs对细胞内铁的基本调控机理如下：当细胞内铁超载时,(1)IRPs与TfR mRNA3'端UTR的IREs(TfR有5个IRE)结合活力下降或者不结合,导致TfR mRNA的不稳定,更易受到核酸酶的攻击从而发生降解,从而导致转铁蛋白受体翻译水平的下降,细胞摄取铁的减少。(2) IRPs与Fn mRNA5'端UTR的IRE(Fn有一个IRE)结合活力下降或者不结合,导致Fn mRNA的翻译增加,则铁蛋白的含量增多,从而螯合细胞质中的游离铁,细胞内过多的游离铁被转化为铁蛋白从而被封闭起来,得以维持了细胞内的铁稳态,降低了游离铁对细胞的毒性作用；相反的,如果细胞内的铁浓度过低时,则执行相反的调控程序。
那么,SPIO标记干细胞致细胞内铁超载数十倍甚至百倍,细胞内铁浓度的急剧升高,细胞内IRPs/IREs系统中的各个蛋白的蛋白表达情况呈现出怎么样的动态变化?铁超载后,对IRPs与IREs的结合活力存在何种影响,即细胞内铁浓度与IRPs与IREs的结合活力是否存在着“剂量--效应”关系?目前亦未见研究报道。
本研究使用转染剂左旋多聚赖氨酸(Poly-L-Lysine, PLL)介导SPIO标记大鼠ADSCs,运用电感耦合等离子体质谱仪Inductively Coupled Plasma Mass Spectrometry, ICP-MS)及MRI定量检测标记后干细胞不同时间点的铁含量,并初步探讨SPIO标记对干细胞IRPs与IREs的结合活力的影响。
研究目的
1.探讨PLL介导SPIO标记大鼠ADSCs后,对干细胞内铁浓度的影响。
2.初步探讨PLL介导SPIO标记大鼠ADSCs后,MRI扫描检测标记干细胞的可行性。
3.探讨PLL介导SPIO标记大鼠ADSCs后,对干细胞体内的转铁蛋白受体和铁蛋白的蛋白表达水平的影响,以及细胞内铁超载对IRPs与IREs的结合活力的影响。
材料与方法
1.大鼠ADSCs的分离、培养以及传代
取3周至4周龄、雄性SD大鼠,无菌条件下切开腹股沟区皮肤,切取皮下脂肪组织,冷PBS洗涤3至4遍后,剪成颗粒状,并用0.25%Ⅱ型胶原酶消化30-45分钟,离心(1500rpm,10min),弃上清液,用全培养液(含10%胎牛血清的低糖DMEM)重悬、接种,培养条件如下：饱和湿度、37℃、5%C02的标准环境。24小时后,原代培养细胞行第一次换液,以后每3天换液,贴壁细胞铺满瓶底的70%-80%时,进行传代培养。
2.大鼠ADSCs的鉴定(干细胞表面抗原的检测)
传至P4代的大鼠脂肪干细胞,吸去细胞培养液；加入5ml PBS洗去残留培养基,弃去PBS(重复二次)；加入1.5ml0.25%胰酶,在显微镜下观察细胞消化情况,在大部分细胞开始变小时,弃去胰酶,加入5ml PBS吹打,吸出PBS,置于离心管中,再加入1ml PBS吹打,吸出PBS,置于离心管中,1000rpm离心5min;弃去PBS,加入600ul PBS,吹打,使细胞均匀悬浮,平均分成6管,其中1管做为空白对照；每管分别加入5ul荧光素标记的抗体(CD29、CD31、 CD44、CD45),37℃下避光孵育30min离心(1000rpm,5min),弃上清；加入1ml PBS,将细胞吹打均匀,1000rpm离心5min,弃上清(重复二次)；每管加入200ul PBS;上机检测。
3. SPIO标记大鼠ADSCs,电感耦合等离子体质谱仪(ICP-MS)检测细胞内铁含量
本实验使用的SPIO试剂(原始浓度为28mg/ml)。SPIO和PLL加入无血清低糖培养基中,室温下,置于摇床摇晃30分钟混匀,待P2代有贴壁干细胞且铺满瓶底近80%-90%的培养瓶时,加入上述含SPIO及PLL培养基,再加入胎牛血清(终浓度10%)(SPIO终浓度为50μg/mL, SPIO/PLL为1：0.03),在标准环境下(饱和湿度、37-C、5%C02)培养。
未标记细胞及共培养12小时以内细胞(时间点分别为：0、2、4、8、12小时),在相应时间点,取标记干细胞,弃培养液,用PBS洗涤3次以去除残余SPIO标记液,用0.25%胰酶消化细胞,加入血清终止消化,离心(1200rpm,3分钟)。弃除上清液,用PBS重悬细胞,计数板行细胞计数,调整至1×106个/ml。取1mL细胞悬液于15mL离心管,2%硝酸溶液6mL充分消化溶解。电感耦合等离子体质谱仪(ICP-MS)定量分析铁含量。
测量时间点为超过12小时的细胞(16小时、1天、2天、4天、7天、14天、21天、28天),在共培养12小时后弃含SPIO培养液,冷PBS洗涤三次以去除残余SPIO及胎牛血清,加入10%全培养液继续培养,符合传代标准时可传代,在相应时间点按照上述步骤操作。ICP-MS测量标记细胞内铁含量。
4. SPIO标记大鼠ADSCs, MRI检测细胞内铁浓度
4.1磁共振扫描参数
磁共振扫描采用3.0T GE超导型磁共振扫描仪,头颅线圈。受检EP管装入自制容器内(灌满10%硫酸铜溶液)。行T2*WI扫描,并获取R2*mapping图。快速梯度回波T2加权像(FAST GRE T2*WI):行冠状位扫描,TR200ms, TE2.2ms,翻转角Flip angle60°,层厚2mm,层间距0mm,矩阵256×256,NEX10次信号采集。FOV15cm。
4.2标准浓度-磁共振扫描
配制Fe标准浓度的SPIO溶液：50ug/ml、40ug/ml、30ug/ml、25ug/ml、20ug/ml、15ug/ml、10ug/ml、5ug/ml、2.5ug/ml、1ug/ml.以上浓度溶液各取1ml,分装于2ml容量的EP管内。
将各标准浓度SPIO溶液的EP管放入自制容器内,灌满硫酸铜溶液(10mmol/l),行MRI扫描,获取T2*值及R2*值。R2*值与浓度拟合直线,获得直线回归方程。
4.3SPIO标记细胞-磁共振扫描
在有贴壁干细胞(P2代)且铺满瓶底约90%的培养瓶内,加入含SPIO的标记液10ml,在标准环境下培养(饱和湿度、37℃C、5%C02)。
共培养12小时以内细胞(时间点分别为：2、4、8、12小时),在相应时间点,取标记干细胞：弃除培养液,用冷PBS洗涤3次以去除残余SPIO标记液,用0.25%胰酶消化细胞,加入血清终止消化,离心(1200rpm,3分钟)。弃除上清液,用冷PBS重悬细胞,计数板行细胞计数,调整至2×106个细胞/nl。取0.5ml细胞悬液,加入0.5m1的8%的明胶溶液,充分混匀,稍待片刻,等气泡升起,然后放入冰块中让其冷却、凝固。最终浓度为1×106个细胞/ml。
测量时间点为超过12小时的细胞(16小时、1天、2天、4天、7天、14天、21天、28天),在共培养12小时后弃SPIO标记液,冷PBS洗涤三次以去除残余SPIO,加入10%全陪养液继续培养,符合传代标准时予以传代,在相应时间点按照上述步骤操作。
将SPIO标记后不同时间点提取细胞的EP管放入特制容器内,灌满硫酸铜溶液(10mmol/l)。行MRI扫描。获取T2*值及R2*值,R2*值代入上述直线回归方程,得出相应浓度值。
5Western-Blot检测PLL介导SPIO标记后干细胞内转铁蛋白受体、铁蛋白轻链及铁蛋白重链的蛋白表达的影响。
本实验采用经转染剂PLL介导SPIO标记的大鼠ADSCs细胞,分别在标记前(0小时)、标记后1天、2天、4天、7天、14天、21天、28天用裂解液和蛋白酶抑制剂提取细胞总蛋白,并用BCA法测定不同时间点的蛋白浓度,进行Western Blot实验,其步骤大致如下：
5.1电泳
5.2电转
5.3对PVDF膜的免疫球蛋白结合位点行封闭
5.4洗膜,孵育一抗,洗膜,孵育二抗,洗膜三次
5.5ECL显示,并用胶片曝光。将所得结果用扫描仪扫描,应用FluorChem8900软件进行扫描灰度,定量分析上述不同时间点转铁蛋白受体、铁蛋白重链以及铁蛋白轻链的蛋白表达结果。
6SPIO标记大鼠ADSCs, RNA结合蛋白免疫共沉淀技术(RNA Binding Protein Immunoprecipitation, RIP)检测IREs/IRPs结合活力SPIO标记大鼠ADSCs,在相应时间点(标记前、标记后1天、2天、4天、
7天、14天、21天和28天),按照下述RIP实验,其步骤大致如下：
6.1使用抗体来捕获细胞质中的内源性RNA结合蛋白
6.2阻止非特异性RNA的结合
6.3通过免疫沉淀一并把RNA结合蛋白及其结合的RNA分离出来
6.4结合的RNA序列的鉴定：通过Microarray (RIP-Chip),定量RT-PCR方法
7统计学方法
所有统计计算用SPSS20.0统计分析系统进行,假设检验统一用双侧检验,给出检验统计量及其对应的P值。检验水准为0.05,即P<0.05认为差异有统计学意义。本实验所有数据均为计量资料,采用均数士标准差描述。
7.1ICP-MS测量不同时间点的干细胞内铁浓度的比较采用单组的重复测量方差分析,不同时间点间两两的比较采用基于单组重复测量方差分析的LSD检验。
7.2MRI测量：铁离子浓度与R2*值采用直线回归进行分析。MRI测量的细胞内铁浓度的预测值与ICP-MS测量的真实值之间的比较,采用配对t检验。
7.3比较大鼠ADSCs标记前与标记后不同时间点的Fn-H、Fn-L及TfR的蛋白表达水平,以及IRPs与IREs的结合活力水平,不同时间的比较采用单组的重复测量方差分析,不同时间点间两两的比较采用基于单组重复测量方差分析的LSD检验。
结果
1.大鼠腹股沟区的脂肪组织在使用0.25%Ⅱ型胶原酶消化后,经过多次的换液、传代,能够在体外成功地分离、培养出大鼠ADSCs。原代细胞培养24小时后即可看到细胞贴壁,细胞呈多种形态,之后经过多次的换液、传代,细胞数量明显增加,形态呈典型的长梭形(成纤维细胞样)外观,此时,细胞形态均一、排列呈漩涡状(有一定方向性)。在原代细胞培养到6-7天时,需进行第一次传代,之后约3-4天可传代一次。
2.从SD大鼠腹股沟区脂肪垫中分离提取的细胞,经流式细胞仪检测对其P4代细胞行检测,发现96.5%的细胞表达CD44,98.4%的细胞表达CD29,而只有8.4%的细胞表达CD31, CD45的细胞表达则仅有2.5%。结果表明：分离的细胞表型较均一。
3. ICP-MS检测SPIO标记大鼠ADSCs细胞内铁含量：未标记组细胞,铁浓度0.393±0.027pg/cell。SPIO标记后2小时测量,细胞内铁含量即可见增高,铁浓度2.627±0.207pg/cell,之后继续逐渐升高,在标记后第2天,铁离子浓度的均值达到最大值,35.315±2.041pg/cell,之后逐渐降低,第28天为1.350±0.260pg/cell,28天细胞内铁浓度仍未恢复标记前水平(P<0.05)。
经单组重复测量方差分析,不同时间的铁浓度有显著性差异(F=761.129,P<0.001)。基于单组重复测量方差分析的多重比较(LSD)方法进行分析,发现2h与3w,8h与12h,4d与1w差异无统计学意义(P>0.05),其他各组间的比较差异均有统计学意义(P<0.05)。
4MRI定量分析干细胞SPIO标记后细胞内铁含量
4.1MRI扫描标准浓度铁溶液
标准浓度铁溶液进行MRI扫描,测量得出T2*值,并转换成R2*值。铁浓度作为应变量,R2*值作为自变量,对铁浓度(Y)与R2*值(X)进行直线回归分析,最后的拟合的直线回归方程为：y=-5.672+0.296x,模型检验结果为,模型有统计学意义(F=4579.44, P<0.001), R=0.992, R square=0.983,铁浓度和R2*值存在很强的线性关系。经检验,回归方程的R2*值的系数有统计学意义(t=67.672,P<0.001)。
4.2SPIO标记细胞的磁共振扫描,计算细胞内铁浓度
磁共振扫描SPIO标记不同时间点细胞(与ICP-MS测量的为同一批次细胞,不同的处理方法),测量其T2*值及R2-值。根据SPIO标记细胞R2*值代入直线方程,得到铁离子浓度的预测值。ICP-MS测量的铁浓度与MRI预测的铁浓度两组间没有统计学差异(F=0.291,P=0.598),不同时间点之间存在统计学差异(F=1489.97,P<0.001)。对每个时间点进行配对t检验发现,2天,4天,7天,14天的两组间的比较均无统计学意义(P>0.05),其他时间点间均有统计学差异(P<0.05)。
5. SPIO标记对ADSCs Fn-L、Fn-H及TfR蛋白表达的影响
我们用Western Blot实验的方法,定量分析了SPIO标记大鼠脂肪干细胞后不同时间段,细胞内IRPs/IREs系统的铁蛋白重链Fn-H、铁蛋白轻链Fn-L及转铁蛋白受体TfR的蛋白表达水平。见表1。
5.1Fn-H蛋白表达水平
SPIO标记1天后,与标记前比较,Fn-H蛋白水平显著升高(P<0.001),PLL介导SPIO标记大鼠脂肪干细胞可促进Fn-H蛋白的表达,随着时间的推移Fn-H蛋白水平有恢复未标铁的趋势,在标记后28天,并未恢复到未标铁时的水平。
5.2Fn-L蛋白表达水平
SPIO标记1天后,与标记前比较,Fn-L的蛋白水平显著升高(P<0.05),之后逐渐降低,至SPIO标记后21天,Fn-L蛋白表达恢复至未标铁时的水平(P>0.05)。
5.3TfR蛋白表达水平
SPIO标记1天后,与标记前比较,TfR的蛋白水平显著降低(P<0.001),之后继续降低,至第4天,达到最低,之后逐渐升高,TfR蛋白表达在第21天,恢复至未标铁时的水平(P>0.05)。
6RIP检测SPIO标记干细胞对IRPs与IREs的结合活力
我们使用PLL转染剂介导SPIO对大鼠脂肪干细胞进行标记,分别在不同时间点采用RIP方法对细胞内的Fn-L mRNA与IRP1,以及TfR mRNA与IRP1的结合情况进行定量检测。
6.1Fn-L mRNA与IRP1结合活力
SPIO标记后第2天,Fn-L mRNA上IRP1蛋白的结合水平显著降低(P<0.05),表明IRP1结合Fn-L mRNA的活力显著降低；而随着SPIO标记后时间的推移,Fn-L mRNA上IRP1蛋白的结合水平逐渐回升,到14天时Fn-L mRNA上IRP1蛋白的结合水平与未标铁时的结合水平没有显著差异(P>0.05)。
6.2TfR mRNA与IRP1结合活力
SPIO标记后第4天、第7天的TfR mRNA上IRP1蛋白的结合水平显著降低(P<0.05),表明IRP1结合TfR mRNA的活力显著降低；而随着SPIO标记后时间的推移,TfR mRNA上IRP1蛋白的结合水平逐渐回升,到14天时TfRmRNA上IRP1蛋白的结合水平与未标铁时的结合水平没有显著差异(P>0.05)。
结论
大鼠腹股沟区皮下脂肪组织中能够成功提取脂肪间充质干细胞。提取的干细胞具有间充质干细胞的形态学特点,还具有提取、分离、培养简便的优点,且培养的细胞在体外增殖能力强、生物学特征较稳定。运用转染剂PLL介导SPIO共培养方式标记大鼠ADSCs,简便、高效。SPIO标记28天内的干细胞,ICP-MS能够精确检测干细胞内铁含量。MRI能够精确预测细胞内铁含量。MRI预测值与ICP-MS测量值有很高的一致性。SPIO标记后,干细胞内的Fn-L以及TfR的蛋白水平的改变只是短暂的,4周内会逐渐恢复至为标记前水平；而Fn-H则表现出了有恢复标记前水平的趋势。SPIO标记后,干细胞内Fn-L mRNA与IRPl以及TfRmRNA与IRP1的结合活力的改变也是短暂的,也会在2周内逐渐恢复至标记前水平。SPIO标记干细胞后,对其进行示踪,对于干细胞来说,安全、可靠。
Background
With the development of the experimental medicine, stem cell transplantation technology is also undergoing rapid changes. In recent years, stem cell transplantation is the hot spot in medical science research, because of the attractive prospect in repairing injuryed tissues such as myocardial infarct, myocardial infarct and diabetes.
Firstly, we should gain sufficient sources of stem cells in order to do stem cell transplantation. Candidates for such strategies include bone marrow mesenchyme stem cells(BMSCs) and embryonic stem cells(ESCs) in clinical application. There are still some limitations to their practical use, including lack of source(only10-20ml bone marrow per person) and a invasive way for BMSCs, immunologicrejection and ethical issues for ESCs. So the type of stem cells that is abundant and can be easily accepted by patient is badly in need. So, we should gain sufficient sources of stem cells in order to do stem cell transplantation.
Since Zuk et al reported that the stem cells in the fat was discovered at first time in2001, the Adipose tissue-Derived Mesenchymal Stem Cells (ADMSCs) became an new important source of stem cells, which has brought to people's attention.
The evaluation of stem cell therapy needs to reflect the situation of migrate, homing, proliferation and differentiation of transplanted stem cells in vivo, so that we can evaluate transplant curative effect, optimize transplant method and select appropriate transplant window period. Recently, many researches are focused on tracking of cells following transplantation, and magnetic resonance imaging (MRI) is considered to be one of the best techniques to determine the bio-distribution and migration of transplanted stem cells. Many researches found that cells labeled with superparemagnetic iron oxide(SPIO) could be intravital, dynamical, sensitive and non-invasive tracked by magnetic resonance imaging (MRI).
The direct effect of SPIO labeling on cells is iron overload. Initial studies showed that iron in labeled cells would be increased10to more than100times than that in unlabeled cells. In this case, the primary problem need to clarify is whether cellular iron homeostasis can be maintained. Iron, is a very important trace elements of the cells. When labeled with SPIO, the dynamic changes of iron concentration in the cells needs further study.
The main purpose of the stem cell transplant treatment, is transplanted stem cells into specific target organs or tissues, to make it differentiated into specific organization cells, perform specific organs or tissues of the corresponding function. Therefore, the SPIO should be stay enough time within cells, then, MRI could detect the labeling cells. MRI can accurately and comprehensively judge the transplanted stem cells in the body of all kinds of situation, and curative effect is accurate assessment for transplantation. Initial study reported that SPIO labeled cells could be visualized by MR more than21days. So, it is necessary to clarify the iron concentration after SPIO labeling of stem cells, whether the magnetic resonance imaging (MRI) could detect the labeling cell, it is also a key of stent cell transplantation.
Iron Regulatory Proteins/Iron Responsive Elements System(IRPs/IREs) is the most important system to maintain cellular iron homeostasis, which is consist of
Transferrin Receptor(TfR), Ferritin(Fn) and Iron Regulatory Proteins(IRPs). The expression of both TfR and Fn are regulated by IRPs in the post-transcriptional level. IRPs achieve regulatory function to combinewith highly conserved Untranslated Region(UTR) in TfR and Fn mRNA.The binding site between UTR in TfR, Fn mRNA and IRPs is called Iron Response Elements(IREs). As intracellular iron of SPIO labeled stem cells will be overload, whether cellular iron homeostasis can be maintained? When labeled with SPIO, the dynamic changes of protein in IRPs/IREs and the binding activity of IRPs/IREs needs further study.
Purpose
1. To investigate the effects of iron content on rat ADSCs labeling with SPIO.
2. To preliminarily investigate the feasibility of MRI to detect the rat ADSCs
labeling with SPIO.3. To investigate the effects of Fn-H, Fn-L and TfR protein expression, and the
binding activity of IRPs/IREs on rat ADSCs labeling with SPIO.
Materials and methods
1. Isolation and culture of rat ADSCs
Adipose tissue was obtained from the inguen of3-4weeks old male Sprague-Dawley(SD) rats. After washing with phosphate-buffered saline (PBS)3to4times, the adipose tissue was minced with eye scissors into less than lmm3per piece and dissociated in0.25%collagenase type II in PBS solution for35-45minutes at37℃with gentle shaking, followed by centrifugation at1500g for10minutes. Cells pellets were re-suspended in Dulbecco's modified Eagle's medium-low glucose (DMEM-LG), containing10%fetal bovine serum, and cultured at37℃in humid air with5%CO2atmosphere. After being seeded for24hours, unattached cells and debris were removed, and adherent cells were continue to culture. Fresh medium was changed every3days. The cells with70%-80%confluence were released with0.25%trypsin-EDTA and subsequently cultured for two passages prior to labeling.
2. Identification of rat ADSCs (Detection of the surface antigen on rat ADSCs)
The surface makers of stem cells such as CD29, CD31, CD44, and CD45were identified by Flow Cytometer.
3. ICP-MS detect the iron concentration on rat ADSCs labeling with SPIO.
The reagent of SPIO was Resovist (Schering, Berlin, Germany) with primitive concentration of28mg/ml. SPIO and PLL were put into a tube containing serum-free medium. Then, the solution containing SPIO and PLL was allowed to mix in a rotator for30minutes. After that, fetal bovine serum at final concentration of10%was added into the solution. Second passage rat ADSCs with80%-90%confluence of the surface area of culture plate were used to label. The SPIO was at final concentrations of50μg/ml (the ratio of SPIO and PLL was1:0.03). Incubating at37℃in humid air with5%CO2atmosphere.
After incubating for0hour(without labeled),2,4,8,12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and used for future research. Cells were resuspended in PBS, adjusted to1×106cells/ml. Take1ml cell suspension into the15ml centrifuge tube, and add6ml nitric acid solution(2%) to dissolve fully digest. Use inductively coupled plasma mass spectrometry (ICP-MS) to quantitative analysis of iron content.
After incubating for12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and then, continue to culture the cells.
At16hours,1day,2,4,7,14,21,28days, the labeled cells were resuspended in PBS, adjusted to1×106cells/ml. Take1ml cell suspension into the15ml centrifuge tube, and add6ml nitric acid solution(2%) to dissolve fully digest. Use inductively coupled plasma mass spectrometry (ICP-MS) to quantitative analysis of iron content.
4. SPIO labeling rat ADSCs, MRI detect the iron concentration in the cell
4.1Magnetic resonance imaging (MRI) scan parameters
3.0T GE superconducting mri scanner, head coil, T2*WI scanning, and get the R2*mapping figure. FAST GRE T2*WI:line of coronary scanning, TR200ms, TE2.2ms, double Angle Flip Angle60°, thick layer2mm, layer spacing0mm, Matrix:256x256, NEX:10, FOV15cm.
4.2Standard concentration solution-MRI scan
The SPIO standard of Fe concentration in the solution:50ug/ml,40ug/ml,30ug/ml,25ug/ml,20ug/ml,15ug/ml,10ug/ml,5ug/ml,2.5ug/ml,1ug/ml. More than1ml solution concentration, partial shipments at EP tube (capacityof.2ml)
Put the standard concentration SPIO solution EP tube in a special container, filled with copper sulfate solution (10tendency/1), MRI scans, obtain T2*value and R2*value. R2*value and the concentration of fitting straight line, get linear equations.
4.3SPIO cell labeling solution-MRI scan
After incubating for2,4,8,12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and used for future research. Cells were resuspended in PBS, adjusted to2×106cells/ml. Take0.5ml cell suspension, add0.5ml gelatin solution(8%), thoroughly incorporated, then let it cool in the ice, frozen. Final concentration of1x106cells/ml.
After incubating for12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and then, continue to culture the cells. At16hours,1day,2,4,7,14,21,28days, the labeled cells were resuspended in PBS, adjusted to2×106cells/ml. Take0.5ml cell suspension, add0.5ml of gelatin solution(8%), thoroughly incorporated, then let it cool in the ice, frozen. Final concentration of1×106cells/ml.
Put the EP tube of different time after SPIO labeling in a special container that filled with copper sulfate solution (10mmol/1). MRI scans. Obtain T2*value and R2*value, put R2*value into linear equations, the corresponding density are obtained.
5Analyze the effect of the expression level of TfR and Fn's protein of the labeled ADSCs with SPIO and transfection agent PLL
The ADSCs were labeled with SPIO and transfection agent PLL as above. Lysis Buffer and PMSF weres used to extract total proteinat the time point of Oh,16h,24h,4d,lw,2w,3w,4w,and the protein concentration was measured by means of BCA. Western Blot:①the protein was degenerated and reducted,②SDS-PAGEwas preparated,③Sample was loaded,④Electrophoresis,⑤Transmembrane,⑥Ponceau staining,⑦Immunoglobulin binding sites at PVDF were closed,⑧Wash membrane, primary antibody incubation, wash membrane, secondary antibody inucubation, wash membrane,⑨Displayed by ECL,and exposured with film.The results were scaned by scanners, and the expression level of TfR and Fn's protein at the above time point were quantitative analysised by Molecular Analysis image analysis software.
6SPIO labeling rat ADSCs, RNA Binding Protein Immunoprecipitation(RIP) detect IREs/IRPs binding activity
SPIO labeling rat ADSCs, at corresponding time points (labeled before,1day,2days,4days,7days,14days,21days and28days), according to the following RIP experiment, the steps are as follows:6.1Using antibody to capture the endogenous RNA binding protein in the cytoplasm
6.2To prevent nonspecific combination of RNA
6.3Through precipitation along to separate RNA binding protein and its combination of RNA
6.4The combination of RNA sequence identification:by microarray (RIP-Chip), quantitative RT-PCR method
7Statistical methods
All calculation using SPSS20.0statistical analysis system, unified hypothesis testing with double side inspection, test statistics and their corresponding P values are given. Inspection level is0.05, or P<0.05think the difference was statistically significant. This experiment all the data for the measurement data, using the mean±standard deviation.
7.1ICP-MS measurement of different points in time the comparison of stem cells within the iron concentration using a single set of repetitive measure analysis of variance, the comparison of two different time points based on a single set of LSD test of repetitive measure analysis of variance.
7.2MRI measurements:iron ion concentration and R2*value using linear regression analysis. MRI measurement of intracellular iron concentration of the true value of the predicted values and the icp-ms measurement comparison between using matching t test.
7.3Compare rat ADSCs before and marking of different time points after Fn-H, Fn-L and TfR protein expression level, and the combination of IRPs and IREs activity level, the comparison of different time using a single set of repetitive measure analysis of variance, comparison of two different time points based on a single set of LSD test of repetitive measure analysis of variance.
Results
1. Rat ADSCs could be successfully isolated and cultured in vitro by means of0.25%Ⅱ collagenase solution. After culturing for24hours, some pleomorphic and adherent cells were present. Following by changing of medium and transferring of culture, cells were become more uniform, showing a typical fibroblast-like appearance with the long spindle morphology. After primarily culturing for6-7days, cells were needed to transfer. And then, cells were transferred every3-4days.
2The P4cells were identified by Flow Cytometer, which found that96.5%of the cells expressed CD44,98.4%of the cells expressed CD29, while only8.4%of the cells expressed CD31, CD45cells express is only2.5%. The results show that the separation of cell phenotype is relatively uniform. These results were correspondto the characteristic of mesenchymal stem cells.
3. ICP-MS detection SPIO labeling rat ADSCs iron content in the cell:not tag group of cells, iron concentration0.393+/-0.027pg/cell. Measurement,2hours after SPIO labeling higher iron content that is visible in the cell, concentration of iron2.627+/-0.207pg/cell, continue to rise gradually, after the second day after mark, the mean of iron ion concentration reached a maximum,35.315+/-2.041pg/cell, reduce gradually, after28days was1.350+/-0.260pg/cell.
By a single set of repetitive measure analysis of variance, iron concentration in different time have significant difference (F=761.129, P<0.001). Based on a single set of repetitive measure analysis of variance of multiple comparison (LSD) method were analyzed, and found2h and3w,12h,8h and4d there was no statistically significant difference with1w (P>0.05), the comparison of differences between other groups have statistical significance (P<0.05).
4MRI quantitative analysis of stem cells after SPIO labeling
4.1MRI scan standard iron concentration solution
Standard concentration iron solution by MRI scan, measured T2*value, translates into R2*value. Iron concentration as a dependent variable, R2*value as independent variables, the iron concentration (Y) and R2*value (X) linear regression analysis, the final fitting the linear regression equation is:the model test results for, was statistically significant (F=4579.44, P<0.001), R=0.992, R=0.983, square iron concentration and R2*value exists strong linear relationship. Upon examination, the regression equation of the R2*value coefficient was statistically significant (t=67.672, P<0.001). MRI scan standard concentration SPIO solution, concentration of iron and the R2*value linear fitting results are as follows:,y=-5.672+0.296x
4.2MRI scan the solution of cells labeling by SPIO, calculate iron concentration in the cell
MRI scans SPIO labeling cells in different time (with icp-ms measurement for the same batch of cells, different processing methods), measuring the T2*value and R2*value. According to R2*value SPIO labeling cells into linear equations, the predictive value of iron ion concentration. ICP ir-MS on concentration and MRI measurement of iron concentration no statistical differences between the two groups (F=0.291, P=0.291). For each time point matching t test found that2days,4days,7days and14days of the comparison between the two groups had no statistical significance (P>0.05), other time points were statistically significant (P<0.05).
5SPIO labeling of ADSCs, Fn-L, Fn-H and TfR protein expression
Our study found that the expression of Fn-L's, Fn-H's and TfR's protein were shown in Table1. Table1Effects of labeling of rADSCs with SPIO on the expression of Fn-H, Fn-L and TfR protein
6.1Binding activity of Fn-L mRNA and IRP1
2days after SPIO labeled, the binding activity of IRP1and Fn-L mRNA significantly reduced; and then, gradually recovered,14days later, recovered to the level of unlabeled (P>0.05).
6.2Binding activity of TfR mRNA and IRP1
4days and7days after SPIO labeled, the binding activity of IRP1and TfR mRNA significantly reduced; and then, gradually recovered,14days later, recovered to the level of unlabeled (P>0.05)
Conclusions
It can be successfully isolated ADSCs from rat adipose tissue. Rat ADSCs are one type of mesenchymal stem cells. They are not only easy to isolate, but also have active proliferation capability and stable biological characteristics. It is easy and efficient to label cells with SPIO by using PLL.
SPIO labeling stem cells, ICP can accurately detect the iron concentration of stem cells. MRI can accurately predict iron concentration in the cells. MRI has a high consistency with ICP. The influenceof expression of Fn-L's, TfR's and Fn-H's protein were just temporary, after intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles. These indicated that it would not induce irreversible influence on the expression of transferrin receptor, ferritin light chain's and high chain's protein on stem cells labeling with SPIO, within a certain range of concentration. SPIO-labeled stem cells, is safe and reliable.
随着基础实验医学的发展,干细胞移植技术也经历着日新月异的变化；其在人体多种疾病的治疗,比如堵塞血管的再通,受损组织的修复,以及糖尿病的移植治疗等等方面,前景十分的诱人。因此,近年来,干细胞研究愈发成为实验医学界的研究热点。
目前,在临床应用中,主要有以下两种干细胞的应用性较好：1、骨髓间充质干细胞(Bone Marrow Mesenchyme Stem Cells, BMSCs);2、胚胎干细胞(Embryonic Stem Cells, ESCs)。但是,其两者,在具备一定的临床应用价值的同时,都具有较显著的局限性：1、骨髓间充质干细胞具有来源比较不足、提取相对困难的缺点；2、胚胎干细胞由于是一种来源于异体的干细胞,其不可避免的具有免疫排斥反应；而且,胚胎干细胞在使用过程中,还不可避免地涉及到伦理学等种种问题。所以,急需找到一种来源充分、取材方便、且患者容易接受的干细胞来源,以使干细胞移植在临床应用方面,取得长足的进展与突破。
在2001年,Zuk等人研究宣布,从人体抽脂术所提取的脂肪组织悬液中,经过一定的不太繁杂的实验步骤,能够成功地分离提取出一种细胞：这种细胞具有干细胞的生物学特性,当时称之为脂肪间充质干细胞(Adipose Tissue-Derived Mesenchymal Stem Cells, ADMSCs).如今简称其为脂肪干细胞(Adipose Tissue-Derived Stem Cells, ADSCs)。脂肪干细胞的发现,打开了干细胞移植研究的一扇新窗户,为临床应用的干细胞来源提供了新的选择与希望。
干细胞移植入受试者体内后,及时和准确地了解细胞在受试者体内的迁徙、归巢、增殖以及分化等情况,非常有助于评估移植治疗的效果、对移植方法的优化以及对移植窗口期行更合适的选择。近年来,国内、外众多研究者使用超顺磁氧化铁(Supraparamagnetic Iron Oxide, SPIO)对移植的干细胞进行体外标记；超顺磁氧化铁实为一种磁共振阴性对比剂,经其标记后,能够借助磁共振成像(Magnetic Resonance Imaging, MRI)在体、无创、动态、实时地对移植细胞进行活体示踪成像,根据靶组织与器官内部的磁共振信号改变,可以判断移植干细胞在体内的迁徙、归巢以及增殖等情况。
诚然,SPIO标记干细胞,能够对标记细胞行体外示踪,但是,其对标记细胞最直接的影响就是引起细胞内的铁超载。研究证实,SPIO标记细胞后,细胞体内的铁浓度较未标记时的浓度增加数十倍甚至数百倍。铁,是细胞体内的一种非常重要的微量元素,它是维持细胞的增殖、生长以及机能活动的一种不可或缺的重要物质,它在细胞体内的电子传递、DNA合成以及氧气的运输等过程中,具有着举足轻重的作用。对干细胞行SPIO铁标记后,细胞内铁含量的时间-浓度关系,即铁标记后,细胞内的铁含量随时间的变化关系,未有研究明确涉及,有必要阐明此两者关系。
干细胞移植治疗的主要目的,是将干细胞移植到特定的靶器官或组织内,使用相应的技术手段,使其按照预想地程序分化成特定的组织细胞,执行特定器官或组织的相应功能。所以,要求SPIO在细胞内的停留应该能够持续足够的时间,让使用MRI对干细胞行示踪观察时,MRI能够准确、全面地判断移植干细胞在体内的各种情况,并对移植疗效进行精确评估。有研究证实,SPIO标记干细胞后,MRI对其行示踪观察的有效时间可长达21天。所以,有必要阐明SPIO标记干细胞后,细胞内铁含量,能否被MRI作定性、定量分析,这也是对移植细胞在体示踪的关键。
铁调节蛋白／铁反应元件系统(Iron Regulatory Proteins/Iron Responsive Elements System, IRPs/IREs系统)是细胞体内一种非常重要的调节系统,它能够维持细胞体内的铁稳态。IRPs/IREs系统包括：铁蛋白(Ferritin、转铁蛋白受体(Transferrin Receptor, TfR)以及铁调节蛋白(Iron Regulatory Proteins, IRPs),三者相互作用,共同维持细胞内的铁稳态；其中Fn和TfR的表达,均受到IRPs在转录后水平上的调控。IRPs则通过与TfR和Fn mRNA上的高度保守的非翻译区(Untranslated Region, UTR)作用,以实现调控细胞内铁稳态的功能。TfR和FnmRNA的UTR与IRPs相结合的位点被称为铁反应元件(Iron Response Elements, IREs)。IRPs/IREs对细胞内铁的基本调控机理如下：当细胞内铁超载时,(1)IRPs与TfR mRNA3'端UTR的IREs(TfR有5个IRE)结合活力下降或者不结合,导致TfR mRNA的不稳定,更易受到核酸酶的攻击从而发生降解,从而导致转铁蛋白受体翻译水平的下降,细胞摄取铁的减少。(2) IRPs与Fn mRNA5'端UTR的IRE(Fn有一个IRE)结合活力下降或者不结合,导致Fn mRNA的翻译增加,则铁蛋白的含量增多,从而螯合细胞质中的游离铁,细胞内过多的游离铁被转化为铁蛋白从而被封闭起来,得以维持了细胞内的铁稳态,降低了游离铁对细胞的毒性作用；相反的,如果细胞内的铁浓度过低时,则执行相反的调控程序。
那么,SPIO标记干细胞致细胞内铁超载数十倍甚至百倍,细胞内铁浓度的急剧升高,细胞内IRPs/IREs系统中的各个蛋白的蛋白表达情况呈现出怎么样的动态变化?铁超载后,对IRPs与IREs的结合活力存在何种影响,即细胞内铁浓度与IRPs与IREs的结合活力是否存在着“剂量--效应”关系?目前亦未见研究报道。
本研究使用转染剂左旋多聚赖氨酸(Poly-L-Lysine, PLL)介导SPIO标记大鼠ADSCs,运用电感耦合等离子体质谱仪Inductively Coupled Plasma Mass Spectrometry, ICP-MS)及MRI定量检测标记后干细胞不同时间点的铁含量,并初步探讨SPIO标记对干细胞IRPs与IREs的结合活力的影响。
研究目的
1.探讨PLL介导SPIO标记大鼠ADSCs后,对干细胞内铁浓度的影响。
2.初步探讨PLL介导SPIO标记大鼠ADSCs后,MRI扫描检测标记干细胞的可行性。
3.探讨PLL介导SPIO标记大鼠ADSCs后,对干细胞体内的转铁蛋白受体和铁蛋白的蛋白表达水平的影响,以及细胞内铁超载对IRPs与IREs的结合活力的影响。
材料与方法
1.大鼠ADSCs的分离、培养以及传代
取3周至4周龄、雄性SD大鼠,无菌条件下切开腹股沟区皮肤,切取皮下脂肪组织,冷PBS洗涤3至4遍后,剪成颗粒状,并用0.25%Ⅱ型胶原酶消化30-45分钟,离心(1500rpm,10min),弃上清液,用全培养液(含10%胎牛血清的低糖DMEM)重悬、接种,培养条件如下：饱和湿度、37℃、5%C02的标准环境。24小时后,原代培养细胞行第一次换液,以后每3天换液,贴壁细胞铺满瓶底的70%-80%时,进行传代培养。
2.大鼠ADSCs的鉴定(干细胞表面抗原的检测)
传至P4代的大鼠脂肪干细胞,吸去细胞培养液；加入5ml PBS洗去残留培养基,弃去PBS(重复二次)；加入1.5ml0.25%胰酶,在显微镜下观察细胞消化情况,在大部分细胞开始变小时,弃去胰酶,加入5ml PBS吹打,吸出PBS,置于离心管中,再加入1ml PBS吹打,吸出PBS,置于离心管中,1000rpm离心5min;弃去PBS,加入600ul PBS,吹打,使细胞均匀悬浮,平均分成6管,其中1管做为空白对照；每管分别加入5ul荧光素标记的抗体(CD29、CD31、 CD44、CD45),37℃下避光孵育30min离心(1000rpm,5min),弃上清；加入1ml PBS,将细胞吹打均匀,1000rpm离心5min,弃上清(重复二次)；每管加入200ul PBS;上机检测。
3. SPIO标记大鼠ADSCs,电感耦合等离子体质谱仪(ICP-MS)检测细胞内铁含量
本实验使用的SPIO试剂(原始浓度为28mg/ml)。SPIO和PLL加入无血清低糖培养基中,室温下,置于摇床摇晃30分钟混匀,待P2代有贴壁干细胞且铺满瓶底近80%-90%的培养瓶时,加入上述含SPIO及PLL培养基,再加入胎牛血清(终浓度10%)(SPIO终浓度为50μg/mL, SPIO/PLL为1：0.03),在标准环境下(饱和湿度、37-C、5%C02)培养。
未标记细胞及共培养12小时以内细胞(时间点分别为：0、2、4、8、12小时),在相应时间点,取标记干细胞,弃培养液,用PBS洗涤3次以去除残余SPIO标记液,用0.25%胰酶消化细胞,加入血清终止消化,离心(1200rpm,3分钟)。弃除上清液,用PBS重悬细胞,计数板行细胞计数,调整至1×106个/ml。取1mL细胞悬液于15mL离心管,2%硝酸溶液6mL充分消化溶解。电感耦合等离子体质谱仪(ICP-MS)定量分析铁含量。
测量时间点为超过12小时的细胞(16小时、1天、2天、4天、7天、14天、21天、28天),在共培养12小时后弃含SPIO培养液,冷PBS洗涤三次以去除残余SPIO及胎牛血清,加入10%全培养液继续培养,符合传代标准时可传代,在相应时间点按照上述步骤操作。ICP-MS测量标记细胞内铁含量。
4. SPIO标记大鼠ADSCs, MRI检测细胞内铁浓度
4.1磁共振扫描参数
磁共振扫描采用3.0T GE超导型磁共振扫描仪,头颅线圈。受检EP管装入自制容器内(灌满10%硫酸铜溶液)。行T2*WI扫描,并获取R2*mapping图。快速梯度回波T2加权像(FAST GRE T2*WI):行冠状位扫描,TR200ms, TE2.2ms,翻转角Flip angle60°,层厚2mm,层间距0mm,矩阵256×256,NEX10次信号采集。FOV15cm。
4.2标准浓度-磁共振扫描
配制Fe标准浓度的SPIO溶液：50ug/ml、40ug/ml、30ug/ml、25ug/ml、20ug/ml、15ug/ml、10ug/ml、5ug/ml、2.5ug/ml、1ug/ml.以上浓度溶液各取1ml,分装于2ml容量的EP管内。
将各标准浓度SPIO溶液的EP管放入自制容器内,灌满硫酸铜溶液(10mmol/l),行MRI扫描,获取T2*值及R2*值。R2*值与浓度拟合直线,获得直线回归方程。
4.3SPIO标记细胞-磁共振扫描
在有贴壁干细胞(P2代)且铺满瓶底约90%的培养瓶内,加入含SPIO的标记液10ml,在标准环境下培养(饱和湿度、37℃C、5%C02)。
共培养12小时以内细胞(时间点分别为：2、4、8、12小时),在相应时间点,取标记干细胞：弃除培养液,用冷PBS洗涤3次以去除残余SPIO标记液,用0.25%胰酶消化细胞,加入血清终止消化,离心(1200rpm,3分钟)。弃除上清液,用冷PBS重悬细胞,计数板行细胞计数,调整至2×106个细胞/nl。取0.5ml细胞悬液,加入0.5m1的8%的明胶溶液,充分混匀,稍待片刻,等气泡升起,然后放入冰块中让其冷却、凝固。最终浓度为1×106个细胞/ml。
测量时间点为超过12小时的细胞(16小时、1天、2天、4天、7天、14天、21天、28天),在共培养12小时后弃SPIO标记液,冷PBS洗涤三次以去除残余SPIO,加入10%全陪养液继续培养,符合传代标准时予以传代,在相应时间点按照上述步骤操作。
将SPIO标记后不同时间点提取细胞的EP管放入特制容器内,灌满硫酸铜溶液(10mmol/l)。行MRI扫描。获取T2*值及R2*值,R2*值代入上述直线回归方程,得出相应浓度值。
5Western-Blot检测PLL介导SPIO标记后干细胞内转铁蛋白受体、铁蛋白轻链及铁蛋白重链的蛋白表达的影响。
本实验采用经转染剂PLL介导SPIO标记的大鼠ADSCs细胞,分别在标记前(0小时)、标记后1天、2天、4天、7天、14天、21天、28天用裂解液和蛋白酶抑制剂提取细胞总蛋白,并用BCA法测定不同时间点的蛋白浓度,进行Western Blot实验,其步骤大致如下：
5.1电泳
5.2电转
5.3对PVDF膜的免疫球蛋白结合位点行封闭
5.4洗膜,孵育一抗,洗膜,孵育二抗,洗膜三次
5.5ECL显示,并用胶片曝光。将所得结果用扫描仪扫描,应用FluorChem8900软件进行扫描灰度,定量分析上述不同时间点转铁蛋白受体、铁蛋白重链以及铁蛋白轻链的蛋白表达结果。
6SPIO标记大鼠ADSCs, RNA结合蛋白免疫共沉淀技术(RNA Binding Protein Immunoprecipitation, RIP)检测IREs/IRPs结合活力SPIO标记大鼠ADSCs,在相应时间点(标记前、标记后1天、2天、4天、
7天、14天、21天和28天),按照下述RIP实验,其步骤大致如下：
6.1使用抗体来捕获细胞质中的内源性RNA结合蛋白
6.2阻止非特异性RNA的结合
6.3通过免疫沉淀一并把RNA结合蛋白及其结合的RNA分离出来
6.4结合的RNA序列的鉴定：通过Microarray (RIP-Chip),定量RT-PCR方法
7统计学方法
所有统计计算用SPSS20.0统计分析系统进行,假设检验统一用双侧检验,给出检验统计量及其对应的P值。检验水准为0.05,即P<0.05认为差异有统计学意义。本实验所有数据均为计量资料,采用均数士标准差描述。
7.1ICP-MS测量不同时间点的干细胞内铁浓度的比较采用单组的重复测量方差分析,不同时间点间两两的比较采用基于单组重复测量方差分析的LSD检验。
7.2MRI测量：铁离子浓度与R2*值采用直线回归进行分析。MRI测量的细胞内铁浓度的预测值与ICP-MS测量的真实值之间的比较,采用配对t检验。
7.3比较大鼠ADSCs标记前与标记后不同时间点的Fn-H、Fn-L及TfR的蛋白表达水平,以及IRPs与IREs的结合活力水平,不同时间的比较采用单组的重复测量方差分析,不同时间点间两两的比较采用基于单组重复测量方差分析的LSD检验。
结果
1.大鼠腹股沟区的脂肪组织在使用0.25%Ⅱ型胶原酶消化后,经过多次的换液、传代,能够在体外成功地分离、培养出大鼠ADSCs。原代细胞培养24小时后即可看到细胞贴壁,细胞呈多种形态,之后经过多次的换液、传代,细胞数量明显增加,形态呈典型的长梭形(成纤维细胞样)外观,此时,细胞形态均一、排列呈漩涡状(有一定方向性)。在原代细胞培养到6-7天时,需进行第一次传代,之后约3-4天可传代一次。
2.从SD大鼠腹股沟区脂肪垫中分离提取的细胞,经流式细胞仪检测对其P4代细胞行检测,发现96.5%的细胞表达CD44,98.4%的细胞表达CD29,而只有8.4%的细胞表达CD31, CD45的细胞表达则仅有2.5%。结果表明：分离的细胞表型较均一。
3. ICP-MS检测SPIO标记大鼠ADSCs细胞内铁含量：未标记组细胞,铁浓度0.393±0.027pg/cell。SPIO标记后2小时测量,细胞内铁含量即可见增高,铁浓度2.627±0.207pg/cell,之后继续逐渐升高,在标记后第2天,铁离子浓度的均值达到最大值,35.315±2.041pg/cell,之后逐渐降低,第28天为1.350±0.260pg/cell,28天细胞内铁浓度仍未恢复标记前水平(P<0.05)。
经单组重复测量方差分析,不同时间的铁浓度有显著性差异(F=761.129,P<0.001)。基于单组重复测量方差分析的多重比较(LSD)方法进行分析,发现2h与3w,8h与12h,4d与1w差异无统计学意义(P>0.05),其他各组间的比较差异均有统计学意义(P<0.05)。
4MRI定量分析干细胞SPIO标记后细胞内铁含量
4.1MRI扫描标准浓度铁溶液
标准浓度铁溶液进行MRI扫描,测量得出T2*值,并转换成R2*值。铁浓度作为应变量,R2*值作为自变量,对铁浓度(Y)与R2*值(X)进行直线回归分析,最后的拟合的直线回归方程为：y=-5.672+0.296x,模型检验结果为,模型有统计学意义(F=4579.44, P<0.001), R=0.992, R square=0.983,铁浓度和R2*值存在很强的线性关系。经检验,回归方程的R2*值的系数有统计学意义(t=67.672,P<0.001)。
4.2SPIO标记细胞的磁共振扫描,计算细胞内铁浓度
磁共振扫描SPIO标记不同时间点细胞(与ICP-MS测量的为同一批次细胞,不同的处理方法),测量其T2*值及R2-值。根据SPIO标记细胞R2*值代入直线方程,得到铁离子浓度的预测值。ICP-MS测量的铁浓度与MRI预测的铁浓度两组间没有统计学差异(F=0.291,P=0.598),不同时间点之间存在统计学差异(F=1489.97,P<0.001)。对每个时间点进行配对t检验发现,2天,4天,7天,14天的两组间的比较均无统计学意义(P>0.05),其他时间点间均有统计学差异(P<0.05)。
5. SPIO标记对ADSCs Fn-L、Fn-H及TfR蛋白表达的影响
我们用Western Blot实验的方法,定量分析了SPIO标记大鼠脂肪干细胞后不同时间段,细胞内IRPs/IREs系统的铁蛋白重链Fn-H、铁蛋白轻链Fn-L及转铁蛋白受体TfR的蛋白表达水平。见表1。
5.1Fn-H蛋白表达水平
SPIO标记1天后,与标记前比较,Fn-H蛋白水平显著升高(P<0.001),PLL介导SPIO标记大鼠脂肪干细胞可促进Fn-H蛋白的表达,随着时间的推移Fn-H蛋白水平有恢复未标铁的趋势,在标记后28天,并未恢复到未标铁时的水平。
5.2Fn-L蛋白表达水平
SPIO标记1天后,与标记前比较,Fn-L的蛋白水平显著升高(P<0.05),之后逐渐降低,至SPIO标记后21天,Fn-L蛋白表达恢复至未标铁时的水平(P>0.05)。
5.3TfR蛋白表达水平
SPIO标记1天后,与标记前比较,TfR的蛋白水平显著降低(P<0.001),之后继续降低,至第4天,达到最低,之后逐渐升高,TfR蛋白表达在第21天,恢复至未标铁时的水平(P>0.05)。
6RIP检测SPIO标记干细胞对IRPs与IREs的结合活力
我们使用PLL转染剂介导SPIO对大鼠脂肪干细胞进行标记,分别在不同时间点采用RIP方法对细胞内的Fn-L mRNA与IRP1,以及TfR mRNA与IRP1的结合情况进行定量检测。
6.1Fn-L mRNA与IRP1结合活力
SPIO标记后第2天,Fn-L mRNA上IRP1蛋白的结合水平显著降低(P<0.05),表明IRP1结合Fn-L mRNA的活力显著降低；而随着SPIO标记后时间的推移,Fn-L mRNA上IRP1蛋白的结合水平逐渐回升,到14天时Fn-L mRNA上IRP1蛋白的结合水平与未标铁时的结合水平没有显著差异(P>0.05)。
6.2TfR mRNA与IRP1结合活力
SPIO标记后第4天、第7天的TfR mRNA上IRP1蛋白的结合水平显著降低(P<0.05),表明IRP1结合TfR mRNA的活力显著降低；而随着SPIO标记后时间的推移,TfR mRNA上IRP1蛋白的结合水平逐渐回升,到14天时TfRmRNA上IRP1蛋白的结合水平与未标铁时的结合水平没有显著差异(P>0.05)。
结论
大鼠腹股沟区皮下脂肪组织中能够成功提取脂肪间充质干细胞。提取的干细胞具有间充质干细胞的形态学特点,还具有提取、分离、培养简便的优点,且培养的细胞在体外增殖能力强、生物学特征较稳定。运用转染剂PLL介导SPIO共培养方式标记大鼠ADSCs,简便、高效。SPIO标记28天内的干细胞,ICP-MS能够精确检测干细胞内铁含量。MRI能够精确预测细胞内铁含量。MRI预测值与ICP-MS测量值有很高的一致性。SPIO标记后,干细胞内的Fn-L以及TfR的蛋白水平的改变只是短暂的,4周内会逐渐恢复至为标记前水平；而Fn-H则表现出了有恢复标记前水平的趋势。SPIO标记后,干细胞内Fn-L mRNA与IRPl以及TfRmRNA与IRP1的结合活力的改变也是短暂的,也会在2周内逐渐恢复至标记前水平。SPIO标记干细胞后,对其进行示踪,对于干细胞来说,安全、可靠。
Background
With the development of the experimental medicine, stem cell transplantation technology is also undergoing rapid changes. In recent years, stem cell transplantation is the hot spot in medical science research, because of the attractive prospect in repairing injuryed tissues such as myocardial infarct, myocardial infarct and diabetes.
Firstly, we should gain sufficient sources of stem cells in order to do stem cell transplantation. Candidates for such strategies include bone marrow mesenchyme stem cells(BMSCs) and embryonic stem cells(ESCs) in clinical application. There are still some limitations to their practical use, including lack of source(only10-20ml bone marrow per person) and a invasive way for BMSCs, immunologicrejection and ethical issues for ESCs. So the type of stem cells that is abundant and can be easily accepted by patient is badly in need. So, we should gain sufficient sources of stem cells in order to do stem cell transplantation.
Since Zuk et al reported that the stem cells in the fat was discovered at first time in2001, the Adipose tissue-Derived Mesenchymal Stem Cells (ADMSCs) became an new important source of stem cells, which has brought to people's attention.
The evaluation of stem cell therapy needs to reflect the situation of migrate, homing, proliferation and differentiation of transplanted stem cells in vivo, so that we can evaluate transplant curative effect, optimize transplant method and select appropriate transplant window period. Recently, many researches are focused on tracking of cells following transplantation, and magnetic resonance imaging (MRI) is considered to be one of the best techniques to determine the bio-distribution and migration of transplanted stem cells. Many researches found that cells labeled with superparemagnetic iron oxide(SPIO) could be intravital, dynamical, sensitive and non-invasive tracked by magnetic resonance imaging (MRI).
The direct effect of SPIO labeling on cells is iron overload. Initial studies showed that iron in labeled cells would be increased10to more than100times than that in unlabeled cells. In this case, the primary problem need to clarify is whether cellular iron homeostasis can be maintained. Iron, is a very important trace elements of the cells. When labeled with SPIO, the dynamic changes of iron concentration in the cells needs further study.
The main purpose of the stem cell transplant treatment, is transplanted stem cells into specific target organs or tissues, to make it differentiated into specific organization cells, perform specific organs or tissues of the corresponding function. Therefore, the SPIO should be stay enough time within cells, then, MRI could detect the labeling cells. MRI can accurately and comprehensively judge the transplanted stem cells in the body of all kinds of situation, and curative effect is accurate assessment for transplantation. Initial study reported that SPIO labeled cells could be visualized by MR more than21days. So, it is necessary to clarify the iron concentration after SPIO labeling of stem cells, whether the magnetic resonance imaging (MRI) could detect the labeling cell, it is also a key of stent cell transplantation.
Iron Regulatory Proteins/Iron Responsive Elements System(IRPs/IREs) is the most important system to maintain cellular iron homeostasis, which is consist of
Transferrin Receptor(TfR), Ferritin(Fn) and Iron Regulatory Proteins(IRPs). The expression of both TfR and Fn are regulated by IRPs in the post-transcriptional level. IRPs achieve regulatory function to combinewith highly conserved Untranslated Region(UTR) in TfR and Fn mRNA.The binding site between UTR in TfR, Fn mRNA and IRPs is called Iron Response Elements(IREs). As intracellular iron of SPIO labeled stem cells will be overload, whether cellular iron homeostasis can be maintained? When labeled with SPIO, the dynamic changes of protein in IRPs/IREs and the binding activity of IRPs/IREs needs further study.
Purpose
1. To investigate the effects of iron content on rat ADSCs labeling with SPIO.
2. To preliminarily investigate the feasibility of MRI to detect the rat ADSCs
labeling with SPIO.3. To investigate the effects of Fn-H, Fn-L and TfR protein expression, and the
binding activity of IRPs/IREs on rat ADSCs labeling with SPIO.
Materials and methods
1. Isolation and culture of rat ADSCs
Adipose tissue was obtained from the inguen of3-4weeks old male Sprague-Dawley(SD) rats. After washing with phosphate-buffered saline (PBS)3to4times, the adipose tissue was minced with eye scissors into less than lmm3per piece and dissociated in0.25%collagenase type II in PBS solution for35-45minutes at37℃with gentle shaking, followed by centrifugation at1500g for10minutes. Cells pellets were re-suspended in Dulbecco's modified Eagle's medium-low glucose (DMEM-LG), containing10%fetal bovine serum, and cultured at37℃in humid air with5%CO2atmosphere. After being seeded for24hours, unattached cells and debris were removed, and adherent cells were continue to culture. Fresh medium was changed every3days. The cells with70%-80%confluence were released with0.25%trypsin-EDTA and subsequently cultured for two passages prior to labeling.
2. Identification of rat ADSCs (Detection of the surface antigen on rat ADSCs)
The surface makers of stem cells such as CD29, CD31, CD44, and CD45were identified by Flow Cytometer.
3. ICP-MS detect the iron concentration on rat ADSCs labeling with SPIO.
The reagent of SPIO was Resovist (Schering, Berlin, Germany) with primitive concentration of28mg/ml. SPIO and PLL were put into a tube containing serum-free medium. Then, the solution containing SPIO and PLL was allowed to mix in a rotator for30minutes. After that, fetal bovine serum at final concentration of10%was added into the solution. Second passage rat ADSCs with80%-90%confluence of the surface area of culture plate were used to label. The SPIO was at final concentrations of50μg/ml (the ratio of SPIO and PLL was1:0.03). Incubating at37℃in humid air with5%CO2atmosphere.
After incubating for0hour(without labeled),2,4,8,12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and used for future research. Cells were resuspended in PBS, adjusted to1×106cells/ml. Take1ml cell suspension into the15ml centrifuge tube, and add6ml nitric acid solution(2%) to dissolve fully digest. Use inductively coupled plasma mass spectrometry (ICP-MS) to quantitative analysis of iron content.
After incubating for12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and then, continue to culture the cells.
At16hours,1day,2,4,7,14,21,28days, the labeled cells were resuspended in PBS, adjusted to1×106cells/ml. Take1ml cell suspension into the15ml centrifuge tube, and add6ml nitric acid solution(2%) to dissolve fully digest. Use inductively coupled plasma mass spectrometry (ICP-MS) to quantitative analysis of iron content.
4. SPIO labeling rat ADSCs, MRI detect the iron concentration in the cell
4.1Magnetic resonance imaging (MRI) scan parameters
3.0T GE superconducting mri scanner, head coil, T2*WI scanning, and get the R2*mapping figure. FAST GRE T2*WI:line of coronary scanning, TR200ms, TE2.2ms, double Angle Flip Angle60°, thick layer2mm, layer spacing0mm, Matrix:256x256, NEX:10, FOV15cm.
4.2Standard concentration solution-MRI scan
The SPIO standard of Fe concentration in the solution:50ug/ml,40ug/ml,30ug/ml,25ug/ml,20ug/ml,15ug/ml,10ug/ml,5ug/ml,2.5ug/ml,1ug/ml. More than1ml solution concentration, partial shipments at EP tube (capacityof.2ml)
Put the standard concentration SPIO solution EP tube in a special container, filled with copper sulfate solution (10tendency/1), MRI scans, obtain T2*value and R2*value. R2*value and the concentration of fitting straight line, get linear equations.
4.3SPIO cell labeling solution-MRI scan
After incubating for2,4,8,12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and used for future research. Cells were resuspended in PBS, adjusted to2×106cells/ml. Take0.5ml cell suspension, add0.5ml gelatin solution(8%), thoroughly incorporated, then let it cool in the ice, frozen. Final concentration of1x106cells/ml.
After incubating for12hours, the labeled cells were washing three times with PBS to remove excess SPIO-PLL, and then, continue to culture the cells. At16hours,1day,2,4,7,14,21,28days, the labeled cells were resuspended in PBS, adjusted to2×106cells/ml. Take0.5ml cell suspension, add0.5ml of gelatin solution(8%), thoroughly incorporated, then let it cool in the ice, frozen. Final concentration of1×106cells/ml.
Put the EP tube of different time after SPIO labeling in a special container that filled with copper sulfate solution (10mmol/1). MRI scans. Obtain T2*value and R2*value, put R2*value into linear equations, the corresponding density are obtained.
5Analyze the effect of the expression level of TfR and Fn's protein of the labeled ADSCs with SPIO and transfection agent PLL
The ADSCs were labeled with SPIO and transfection agent PLL as above. Lysis Buffer and PMSF weres used to extract total proteinat the time point of Oh,16h,24h,4d,lw,2w,3w,4w,and the protein concentration was measured by means of BCA. Western Blot:①the protein was degenerated and reducted,②SDS-PAGEwas preparated,③Sample was loaded,④Electrophoresis,⑤Transmembrane,⑥Ponceau staining,⑦Immunoglobulin binding sites at PVDF were closed,⑧Wash membrane, primary antibody incubation, wash membrane, secondary antibody inucubation, wash membrane,⑨Displayed by ECL,and exposured with film.The results were scaned by scanners, and the expression level of TfR and Fn's protein at the above time point were quantitative analysised by Molecular Analysis image analysis software.
6SPIO labeling rat ADSCs, RNA Binding Protein Immunoprecipitation(RIP) detect IREs/IRPs binding activity
SPIO labeling rat ADSCs, at corresponding time points (labeled before,1day,2days,4days,7days,14days,21days and28days), according to the following RIP experiment, the steps are as follows:6.1Using antibody to capture the endogenous RNA binding protein in the cytoplasm
6.2To prevent nonspecific combination of RNA
6.3Through precipitation along to separate RNA binding protein and its combination of RNA
6.4The combination of RNA sequence identification:by microarray (RIP-Chip), quantitative RT-PCR method
7Statistical methods
All calculation using SPSS20.0statistical analysis system, unified hypothesis testing with double side inspection, test statistics and their corresponding P values are given. Inspection level is0.05, or P<0.05think the difference was statistically significant. This experiment all the data for the measurement data, using the mean±standard deviation.
7.1ICP-MS measurement of different points in time the comparison of stem cells within the iron concentration using a single set of repetitive measure analysis of variance, the comparison of two different time points based on a single set of LSD test of repetitive measure analysis of variance.
7.2MRI measurements:iron ion concentration and R2*value using linear regression analysis. MRI measurement of intracellular iron concentration of the true value of the predicted values and the icp-ms measurement comparison between using matching t test.
7.3Compare rat ADSCs before and marking of different time points after Fn-H, Fn-L and TfR protein expression level, and the combination of IRPs and IREs activity level, the comparison of different time using a single set of repetitive measure analysis of variance, comparison of two different time points based on a single set of LSD test of repetitive measure analysis of variance.
Results
1. Rat ADSCs could be successfully isolated and cultured in vitro by means of0.25%Ⅱ collagenase solution. After culturing for24hours, some pleomorphic and adherent cells were present. Following by changing of medium and transferring of culture, cells were become more uniform, showing a typical fibroblast-like appearance with the long spindle morphology. After primarily culturing for6-7days, cells were needed to transfer. And then, cells were transferred every3-4days.
2The P4cells were identified by Flow Cytometer, which found that96.5%of the cells expressed CD44,98.4%of the cells expressed CD29, while only8.4%of the cells expressed CD31, CD45cells express is only2.5%. The results show that the separation of cell phenotype is relatively uniform. These results were correspondto the characteristic of mesenchymal stem cells.
3. ICP-MS detection SPIO labeling rat ADSCs iron content in the cell:not tag group of cells, iron concentration0.393+/-0.027pg/cell. Measurement,2hours after SPIO labeling higher iron content that is visible in the cell, concentration of iron2.627+/-0.207pg/cell, continue to rise gradually, after the second day after mark, the mean of iron ion concentration reached a maximum,35.315+/-2.041pg/cell, reduce gradually, after28days was1.350+/-0.260pg/cell.
By a single set of repetitive measure analysis of variance, iron concentration in different time have significant difference (F=761.129, P<0.001). Based on a single set of repetitive measure analysis of variance of multiple comparison (LSD) method were analyzed, and found2h and3w,12h,8h and4d there was no statistically significant difference with1w (P>0.05), the comparison of differences between other groups have statistical significance (P<0.05).
4MRI quantitative analysis of stem cells after SPIO labeling
4.1MRI scan standard iron concentration solution
Standard concentration iron solution by MRI scan, measured T2*value, translates into R2*value. Iron concentration as a dependent variable, R2*value as independent variables, the iron concentration (Y) and R2*value (X) linear regression analysis, the final fitting the linear regression equation is:the model test results for, was statistically significant (F=4579.44, P<0.001), R=0.992, R=0.983, square iron concentration and R2*value exists strong linear relationship. Upon examination, the regression equation of the R2*value coefficient was statistically significant (t=67.672, P<0.001). MRI scan standard concentration SPIO solution, concentration of iron and the R2*value linear fitting results are as follows:,y=-5.672+0.296x
4.2MRI scan the solution of cells labeling by SPIO, calculate iron concentration in the cell
MRI scans SPIO labeling cells in different time (with icp-ms measurement for the same batch of cells, different processing methods), measuring the T2*value and R2*value. According to R2*value SPIO labeling cells into linear equations, the predictive value of iron ion concentration. ICP ir-MS on concentration and MRI measurement of iron concentration no statistical differences between the two groups (F=0.291, P=0.291). For each time point matching t test found that2days,4days,7days and14days of the comparison between the two groups had no statistical significance (P>0.05), other time points were statistically significant (P<0.05).
5SPIO labeling of ADSCs, Fn-L, Fn-H and TfR protein expression
Our study found that the expression of Fn-L's, Fn-H's and TfR's protein were shown in Table1. Table1Effects of labeling of rADSCs with SPIO on the expression of Fn-H, Fn-L and TfR protein
6.1Binding activity of Fn-L mRNA and IRP1
2days after SPIO labeled, the binding activity of IRP1and Fn-L mRNA significantly reduced; and then, gradually recovered,14days later, recovered to the level of unlabeled (P>0.05).
6.2Binding activity of TfR mRNA and IRP1
4days and7days after SPIO labeled, the binding activity of IRP1and TfR mRNA significantly reduced; and then, gradually recovered,14days later, recovered to the level of unlabeled (P>0.05)
Conclusions
It can be successfully isolated ADSCs from rat adipose tissue. Rat ADSCs are one type of mesenchymal stem cells. They are not only easy to isolate, but also have active proliferation capability and stable biological characteristics. It is easy and efficient to label cells with SPIO by using PLL.
SPIO labeling stem cells, ICP can accurately detect the iron concentration of stem cells. MRI can accurately predict iron concentration in the cells. MRI has a high consistency with ICP. The influenceof expression of Fn-L's, TfR's and Fn-H's protein were just temporary, after intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles. These indicated that it would not induce irreversible influence on the expression of transferrin receptor, ferritin light chain's and high chain's protein on stem cells labeling with SPIO, within a certain range of concentration. SPIO-labeled stem cells, is safe and reliable.
引文
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