CN112083167B - S100a4作为评估甲氨蝶呤干预银屑病的生物标记物及其用途 - Google Patents
S100a4作为评估甲氨蝶呤干预银屑病的生物标记物及其用途 Download PDFInfo
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Abstract
本发明属生物技术领域，涉及S100A4作为评估甲氨蝶呤干预银屑病的生物标记物及其用途，本发明提供了S100A4在区分银屑病患者对甲氨蝶呤干预有效和无效的应用，具体涉及S100A4作为药物治疗筛选指标在评判银屑病患者是否给予甲氨蝶呤干预中的应用，还涉及测定S100A4蛋白表达水平的方法。有助于指导临床医生制定适合的甲氨蝶呤干预银屑病的方案，为提高甲氨蝶呤的临床疗效提供基础。
Description
技术领域
本发明属于生物技术和医学领域，涉及S100A4作为评估甲氨蝶呤干预银屑病的生物标记物及其用途，本发明提供了S100A4在区分银屑病患者对甲氨蝶呤干预有效和无效的应用，具体涉及S100A4(S100 Calcium Binding Protein A4)作为药物治疗筛选指标在评判银屑病患者是否给予甲氨蝶呤干预中的应用，还涉及测定S100A4蛋白表达水平的方法。
背景技术
现有技术公开了银屑病(psoriasis)是一种常见的慢性炎症性疾病，可累及皮肤、指甲和关节。据统计，该疾患影响全世界1-3％人口，在中国的发病率为0.47％；该疾患临床表现为红色鳞屑性斑块和丘疹，约20-30％的银屑病患者可出现关节损害，发展成银屑病关节炎(psoriatic arthritis，PsA)。临床实践显示，银屑病是一种系统性炎症反应，伴发心血管疾病、糖尿病、代谢综合症的概率远高于普通人群，给患者身心健康带来严重影响。
目前，临床实践中常采用的药物干预中，如，常使用的阿维A的有效率仅为20％左右，而且伴有引发血脂增高，增加患者心血管疾病的风险，且部分患者还会出现肝肾功能的损害，等；生物制剂的临床有效率虽然高在50％-80％左右，但由于价格昂贵，致使大多数患者无力承担；甲氨蝶呤(MTX)为目前较常采用的较经济、有效的治疗银屑病的药物，但由于疗效的不确定性和个体差异，及存在有骨髓抑制和肝纤维化影响的风险，严重限制了MTX在银屑病治疗中的广泛应用；因此，寻找一个生物标记预测甲氨蝶呤的干预效果，有利于提高临床疗效，该课题成为越来越多的本领域研究人员和医生关注的热点。
S100钙结合蛋白A4(S100 calcium-binding protein，S100A4)是S100蛋白家族成员之一。S100A4调控细胞运动、侵袭、增生，与细胞分化、凋亡和基质降解有关，其与肿瘤生物学的相关性已毋庸置疑。有研究表明，类风湿性关节炎患者滑膜成纤维细胞和血浆中S100A4水平显著升高，且与病情发展呈正相关。此外，抑制S100A4基因，能显著降低致炎细胞因子TNF-α、IL-β及促血管生成因子VEGF的表达，显著减轻关节滑膜的病理损伤。有研究表明银屑病患者真皮层S100A4蛋白的表达水平显著高于正常对照，拮抗S100A4蛋白的表达水平可显著抑制表皮的厚度，减少真皮内炎症细胞的浸润，减轻血管的扩张，研究结果提示S100A4有可能参与了银屑病的发生，但对S100A4表达水平是否与甲氨蝶呤干预银屑病的效果相关，目前尚未见相关报道。
基于现有技术的研究现状，本申请的发明人拟提供以S100A4作为分子标记物在用于评估甲氨蝶呤干预银屑病效果中的应用。更具体而言，本发明涉及S100A4作为药物治疗个体筛选指标用于评判银屑病患者是否给予甲氨蝶呤干预中的应用。
发明内容
本发明的目的是基于现有技术的现状，提供新的银屑病蛋白标志物，尤其是S100A4作为分子标记物在用于制备评估区分甲氨蝶呤干预效果的制品中的用途，更具体的，本发明涉及S100A4作为药物治疗个体筛选指标在用于制备评判银屑病患者是否给予甲氨蝶呤干预措施中的应用。
本发明的进一步提供了用于预测甲氨蝶呤治疗银屑病疗效的检测试剂盒。本发明中，通过检测S100A4蛋白表达水平与甲氨蝶呤治疗银屑病临床疗效的关系，如银屑病患者外周血PBMC、血清及皮损中S100A4表达水平高低可预测甲氨蝶呤治疗银屑病的临床疗效，进一步用于指导临床医生制定个体化给药方案。
本发明中，通过iTRAQ技术分析了4名对甲氨蝶呤治疗有效(疾病严重程度评分好转75％以上)和4名对甲氨蝶呤治疗无效(疾病严重程度好转50％一下)的银屑病患者，及4名正常对照外周血单个核细胞(PBMC)上蛋白表达水平，结果显示：(1)银屑病患者外周血PBMC上S100A4蛋白表达水平显著高于正常对照(p＜0.01)；(2)对甲氨蝶呤干预有效的银屑病患者外周血PBMC上S100A4蛋白表达水平显著高于无效组(p＜0.05)和正常对照(p＜0.0001)，对甲氨蝶呤干预无效的银屑病患者外周血PBMC上S100A4蛋白表达水平高于正常对照(p＜0.05)；(3)对甲氨蝶呤干预有效的银屑病患者外周血PBMC上S100A4蛋白表达水平在干预第8周显著下调(p＜0.05)；(4)对甲氨蝶呤干预无效的银屑病患者外周血PBMC上S100A4蛋白表达水平在干预第8周显著下调(p＜0.05)。
本发明通过ELISA方法检测了10名正常对照和29名银屑病患者(15名寻常型银屑病患者和14名关节型银屑病患者)在甲氨蝶呤干预第0周和第12周外周血清中S100A4的水平，结果显示：(1)银屑病患者血清S100A4水平显著高于正常对照(p＜0.0001)；(2)对甲氨蝶呤干预有效的银屑病患者血清S100A4水平显著高于正常对照(p＜0.01)；(3)对甲氨蝶呤干预有效的银屑病患者血清S100A4水平在干预第12周显著下调(p＜0.05)；(4)对甲氨蝶呤干预无效的银屑病患者血清S100A4水平在干预第12周无显著变化；(5)寻常型银屑病(PsO)患者血清S100A4水平显著高于关节型银屑病(PsA，p＜0.05)和正常对照(p＜0.01)；关节型银屑病患者血清S100A4水平与正常对照无显著差异；(6)寻常型银屑病患者血清中S100A4水平在干预第12周显著下调(p＜0.01)；(7)关节型银屑病患者血清中S100A4水平在干预第12周无显著变化。
本发明中，进行了采用S100A4区分对甲氨蝶呤干预有效和对甲氨蝶呤干预无效的临床研究，通过检测银屑病患者血清中S100A4的水平预测甲氨蝶呤干预银屑病的疗效，实验结果有助于临床医生制定个体化干预方案，有利于提高患者生活质量。
本发明提供了新的预测甲氨蝶呤干预银屑病的临床疗效的生物标志物。本发明通过实验检测结果表明，对患者血清和外周血PBMC上S100A4表达量的检测，在预测MTX临床疗效方面具有很大价值。
本发明进一步提供了银屑病生物标志物S100A4的检测试剂盒，针对患者血清和外周血中S100A4的不同含量，达到预测甲氨蝶呤临床疗效的目的。
本发明所述S100A4基因和蛋白的具体信息，可通过下述渠道查询：
https：//www.uniprot.org/uniprot/P26447
附图说明
图1显示了对甲氨蝶呤干预有效和无效组外周血PBMCs上S100A4表达水平的检测结果，其中，
(a)银屑病患者(n＝8)外周血中PBMC上S100A4水平与正常对照(n＝4)相比显著升高；(b)MTX有效干预组(n＝4)和无效干预组(n＝4)患者的外周血PBMC上S100A4水平与正常对照(n＝4)相比均显著升高，无效组银屑病患者外周血PBMC上S100A4水平显著高于正常对照；(c)有效干预组(n＝4)接受MTX干预后8周的外周血PBMC上S100A4水平与基线水平相比显著下降；(d)无效干预组(n＝4)接受MTX干预后8周的外周血PBMC上S100A4水平与基线水平相比显著下降。
图2显示了银屑病患者血清S100A4的检测的结果，其中，
(a)银屑病患者(n＝29)血清S100A4水平与正常对照(n＝10)相比显著升高(288.0±33.9vs 129.9±6.2，p＝0.0007)；(b)MTX有效干预组(n＝21)和无效干预组(n＝8)患者的血清S100A4水平与正常对照(n＝10)相比均显著升高(302.9±45.19vs 248.9±32.32vs129.9±6.2，p＜0.01，p＜0.05)；(c)MTX有效干预组的血清S100A4水平在干预第12周时与基线水平相比显著下降(n＝21，302.9±45.19vs 228.9±25.6，p＜0.05)；(d)MTX无效干预组的血清S100A4水平在干预第12周时与基线水平相比无明显差异(n＝8，248.9±32.32vs179.8±31.5，p＞0.05)(e)无关节炎的银屑病患者(PsO，n＝15)的血清S100A4水平显著高于伴关节炎的银屑病患者(PsA，n＝14)和正常对照(n＝10)(349.1±57.8vs 222.5±25.3vs 129.9±6.2，p＜0.01and p＜0.05)(f)无关节炎的银屑病患者(Ps0，n＝15)接受MTX干预后12周的血清S100A4水平与基线水平相比显著下降(n＝15，349.1±57.8vs 219.9±35.7，p＜0.01).(g)伴关节炎的银屑病患者(PsA，n＝14)接受MTX干预后12周的血清S100A4水平与基线水平相比无明显差异(n＝12，222.5±25.3vs 210.5±23.1，p＞0.05)。
具体实施方式
实施例1：对甲氨蝶呤干预有效和无效组外周血PBMCs上S100A4表达水平的检测
材料与方法：
1)银屑病患者：选定到华山医院皮肤科银屑病专病门诊就诊的8名银屑病患者何4名正常对照为观察对象。其中4名对甲氨蝶呤治疗有效的患者(治疗第8周疾病严重程度评分改善75％以上)和4名对甲氨蝶呤治疗无效的银屑病患者(治疗第8周疾病严重程度评分改善低于50％)，8名患者和4名正常对照签署知情同意书。
2)实验方法：给予8名银屑病患者甲氨蝶呤(MTX)干预，收集用药第0周和第8周的外周血PBMC样本。
制备方法
1)从临床抽取血液样本(加入肝素抗凝剂)，用等体积的PBS稀释；
2)在离心管中加入等体积的人外周血淋巴细胞分离液(总体积不要超过离心管的三分之二)，将稀释后的血液缓慢平铺于稀释液上方，此时，二者形成明显的分层界面；
3)室温，2000r离心20min。离心后将出现明显的分，从上往下依次为血浆层、淋巴细胞层、分离层和红细胞层；
4)取出淋巴细胞层(白膜层)，放于新的离心管中，加入10ml PBS清洗细胞。500r离心10min；
5)弃上清，加入5ml PBS重悬细胞。500r离心10min。重复此步骤一次；
6)弃上清，用细胞冻存液重悬细胞，并存放于-80℃备用；
7)将实验所需的样本统一时间解冻，用2ml PBS清洗，1500r离心3min。弃上清，重复一次清洗；
8)弃上清，加入SDT细胞裂解液，获得PBMC全细胞裂解液，震荡混匀，95℃加热10min促进蛋白变性。
(1).色氨酸荧光定量方法
1)用SDT缓冲液将色氨酸(Tryptophan)配制成1ug/μL母液并稀释为300ng/μL，200ng/μL，150ng/μL，100ng/uL 75ng/μL，50ng/μL，25ng/μL，0ng/μL，的八个浓度梯度点的标准品；
2)将标准品和样品各取10μL加入1mL Dilution buffer中，常温摇床5min震荡混匀，离心；
3)取用新的Nunc96孔黑板，将混匀的样品按照既定的顺序依次加入到对应的空中，每个样品三个重复，每孔200μL(保证加样无气泡)；
4)将荧光分光光度计设定为激发光波长295nm、发射光波长350nm、光栅10nm的程序，检测色氨酸的荧光强度；
5)拷贝数据，用EXCEL处理，用标准液做出标准曲线，然后通过标准曲线公式计算样品中色氨酸的含量，最后通过色氨酸在蛋白中所占的比例(13‰)进行换算成蛋白的含量。
(2).超滤膜辅助酶解方法(FASP)酶解
1)取用30KD超滤膜，加入UA清洗活化滤膜，10000g离心约30min；
2)每个超滤管中加入100μg蛋白，并加入150uL UA缓冲液，震荡混匀，放入离心机，配平后旋紧盖子，将转速调至10000g离心30-40min，至液体全部离心进入收集管；
3)再次加入150μL UA缓冲液，震荡混匀，放入离心机，配平后旋紧盖子，将转速调至10000g离心30-40min，至液体全部离心进入收集管，重复2次；
4)加入100μL的50mM IAA，将样品放在暗处，避光静置20min，放入离心机，配平后旋紧盖子，将转速调至10000g离心30min，至液体全部离心进入收集管；
5)取150μL的0.1M TEAB缓冲液洗取盐离子，每次都在10000g下离心30-40min；至液体全部离心进入收集管，重复4遍；
6)更换新的收集管，然后按质量比1∶50的比例加入Trypsin酶，将样品放于37℃震荡酶解过夜，酶解时间不要超过16h；
7)第二次按质量比1∶50的比例加入Trypsin酶，将样品放于37℃震荡器酶解4h(总的酶解时间不要超过20h)，酶解完成后，12000g转速离心收集肽段，加入100ul 0.1M TEAB重复2次冻干，-80℃冻存备用。
(3).iTRAQ标记
根据BCA法定量结果，每组各取40ug肽段冻干后使用7.5ul 0.5M TEAB充分混匀溶解。从20°取出iTRAQ标记试剂后静置到室温后打开，每管标记试剂各加50ul异丙醇充分溶解；将溶解的Itraq试剂分别加入到对应的待标记肽段中混匀，在Thermomixer上20℃、300rpm震荡反应2h；每管加50ul MILIQ水震荡混匀15min后终止反应；将8管样品汇集到一个新的EP管中，充分混匀后等分成2分，冻干(一份分级一份备用)，
表1.样品标记方案
其中，，NP表示正常血糖、GR表示甲氨蝶呤治疗的有效患者、NR表示甲氨蝶呤治疗无效的患者；N表示正常人，PS表示银屑病患者，以上字母之后的数字表示不同样品的序号；mix是混合样本，用于作为内参；
(4).High pH RP分级
标记的样本用24μL bufferA溶解，高速离心两遍，取20μL上清液置于上样瓶中；进样体积：18μL吸样速度：200μL/min打样速度：200μL/min洗针方式：Flush Port；洗针时间：10s柱温：45℃紫外吸收波长：214nm，带宽16nm，参比波长360nm，带宽100nm-254nm，带宽4nm，参比波长360nm，带宽100nm流速：0.2mL/min；
洗脱梯度为：5min，8％B；40min，18％B，62min，32％B；64min，95％B；68min，95％B；69min，5％B；75min，5％B；
样品收集：先分成28级，每2min接一管，冻干浓缩后合并成14管，按照F1-14、F2-15……合并，冻干，-20℃保存；
(5).多维液相色谱串联质谱(LC-MS/MS)分析
液相色谱为Easy-nLC-1000系统(ThermoFisher Scientific)；反相柱为自制C18反相柱(75μm×150mm，3μm填料)；反相的洗脱液为0.1％甲酸的水溶液(A)和0.1％甲酸的乙腈溶液(B)，样品上样到反相柱上后经由流速为0.3μL/min的反相洗脱体系，有效洗脱梯度为：2min，5％B；66min，25％B，81min，35％B；84min，90％B；90min，90％B，质谱分析时间为90min。
(6).数据库搜索
质谱采集到的RAW文件用Maxquant1.5.1.0软件进行正反搜库，采用的数据库是Uniprot homo sapiens(2016年3月下载)数据库。搜库参数设置如下：报告离子搜库模式为iTRAQ八标，子离子质量偏差20ppm以内，蛋白酶设置为胰蛋白酶，最大允许漏切位点为2，可变修饰设置为蛋白N端乙酰化和甲硫氨酸氧化，蛋白质和肽段的假阳性率(falsediscovery rate，FDR)设置为0.01。
(7).统计学及生物信息学分析
所有样品数据经过中位数矫正，然后除以内参样品，做对数处理。组间两两比较用t-test检验，经FDR矫正。所有数据分析和统计学检验都使用R安装包或者Excel完成。
结果显示，1.银屑病患者外周血中PBMC上S100A4水平显著高于正常对照(p＜0.01，图1a)；2.对甲氨蝶呤治疗有效组银屑病患者外周血PBMCs上S100A4蛋白水平显著高于无效组(p＜0.05，图1b)和正常对照(p＜0.0001，图1b)，对甲氨蝶呤治疗无效银屑病患者外周血PBMCs上S100A4水平高于正常对照(p＜0.05，图1b)。3.对甲氨蝶呤治疗有效银屑病患者外周血PBMCs上S100A4水平在治疗第8周显著下调(p＜0.05，图1c)4.对甲氨蝶呤治疗无效银屑病患者外周血PBMCs上S100A4水平在治疗第8周显著下调(p＜0.05，图1d)。
实施例2：银屑病患者血清S100A4的检测
材料与方法：
1)银屑病患者：选定到华山医院皮肤科银屑病专病门诊就诊的29名银屑病患者为观察对象(患者的一般信息如表1所示)。其中无关节炎的银屑病患者(PsO)共15名，银屑病关节炎患者(PsA)共14名。银屑病关节炎的诊断符合CASPAR诊断标准。29名患者填写完整流行病学调查表，签署知情同意书。
2)实验方法：给予29名银屑病患者甲氨蝶呤(MTX)治疗，收集用药第0周和第12周的血清。有效组定义为治疗第12周疾病严重程度改善在75％以上，无效组定义为疾病严重程度改善在50％以下。测定不同组别的血清S100A4水平，结果如图2所示：
1.银屑病患者血清S100A4水平与正常对照相比显著升高，且与关节症状的有无和MTX疗效相关；2.无关节炎的银屑病患者(PsO)血清S100A4水平显著高于伴关节炎的银屑病患者(PsA)和正常对照；3.无关节炎的银屑病患者(PsO)接受MTX治疗后12周的血清S100A4水平与基线水平相比显著下降，而伴关节炎的银屑病患者(PsA)治疗前后则无明显差异；4.MTX有效治疗组的血清S100A4水平在治疗第12周时与基线水平相比显著下降，而无效治疗组治疗前后则无明显差异；5.免疫组化结果显示银屑病皮损区S100A4表达增加。
表2. 29名患者的一般情况
Claims (7)
1.S100A4 在制备作为评估甲氨蝶呤干预无关节炎的银屑病患者的临床疗效的生物标记物中 的用途，其特征在于，
所述的 S100A4 为 S100 钙结合蛋白 A4；
银屑病为无关节炎的银屑病患者；
检测无关节炎的银屑病患者的样品为血清。
2.按权利要求 1 所述的用途， 其特征在于， 所述的用途中， 通过检测患者血清样本中S100A4 表达量，预测甲氨蝶呤干预银屑病的效果。
3.S100A4 在制备作为评估甲氨蝶呤干预无关节炎的银屑病患者的临床疗效的制品中的应用，其特征在于，通过检测对象血清样品中的 S100A4 水平评判是否给予甲氨蝶呤干预。
4.按权利要求3 所述的应用，其特征在于，所述的制品是检测 S100A4 表达水平的试剂或试剂盒。
5.按权利要求3所述的应用，其特征在于，所述的制品还选自针对 S100A4 的抗体、针对S100A4 的特异性探针、针对 S100A4 的基因芯片或蛋白质芯片。
6. 如权利要求 3 或 4 所述的应用，其中，所述的应用是，通过如下方法中的一种或多种检测 S100A4，多维液相色谱串联质谱分析;免疫组化法;化学发光法;放射性同位素法;
荧光发光法;免疫荧光法;酶标法;胶体金法;实时定量反转录 PCR;生物芯片检测法;DNA 印迹法;RNA 印记法原位杂交法;蛋白质印迹法。
7.如权利要求4 所述的应用， 其中，所述的试剂或试剂盒用于检测 S100A4 DNA水平、mRNA 水平和/或蛋白质水平。
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