CN111521815A - Lrg1作为诊断血栓闭塞性脉管炎的血清学标志物的应用 - Google Patents
Lrg1作为诊断血栓闭塞性脉管炎的血清学标志物的应用 Download PDFInfo
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Abstract
本发明提供了LRG1作为诊断血栓闭塞性脉管炎的血清学标志物的应用，以及LRG1作为诊断血栓闭塞性脉管炎炎症活动程度的血清学标志物的应用。本发明的应用为血栓闭塞性脉管炎的早期诊断和炎症预警提供新的辅助判断手段，与其他生物学标志物联合应用可以提高血栓闭塞性脉管炎的非创伤诊断效率。
Description
技术领域
本发明设计医药诊断领域，具体的说涉及LRG1作为诊断血栓闭塞性脉管炎的血清学标志物的应用。
技术背景
血栓闭塞性脉管炎(thromboangiitis obliterans,TAO)所介导的动脉闭塞及重度肢体缺血与动脉硬化闭塞症存在较大区别，特点是：1.好发于青壮年，2.累及中远端血管，破坏了流出道，使得支架/旁路等主流治疗难以奏效，3.自身免疫性炎症而非动脉粥样硬化是在疾病发生发展中最重要的因素。TAO患者的中小血管中存在大量炎性血栓，传统支架/旁路治疗下踝上截肢率高达31％。而且，该症好发于东南亚、中东及远东地区，我国发病率明显高于西方国家。目前众多国内外学者均在寻找行之有效的全新手段对TAO介导的重度肢体缺血进行治疗，这也是近年来外周动脉疾病方面研究的热点。
复旦大学附属中山医院在2009年于国内率先开展了自体外周血纯化CD34+细胞移植治疗TAO所致重度肢体缺血，有效的降低了致残率，术后6个月及5年保肢率分别达到91.2％和88.9％，尽管干细胞移植较传统治疗手段已大大改善了TAO患者预后，复旦大学附属中山医院还是发现存在着影响干细胞移植疗效的重要因素：TAO所致的肢体缺血根本病因在于血管内自身免疫性炎症，干细胞移植并非是根治TAO中自身免疫性的血管炎症手段，能否控制血管炎活动极大地影响干细胞移植疗效。临床上在干细胞治疗后症状缓解、血管炎转为静止期的患者中有约15％的患者术后一年内出现重度肢体缺血症状的复发。患者体内炎症的再活动被认为是引起血管闭塞进而产生缺血症状的根本原因。
因此，为了进一步改善TAO患者干细胞移植预后，也应当从以上方面着手：准确地监测TAO中血管炎的活动状态及早明确TAO炎症处于活动状态从而在重度肢体缺血症状复发前进行相关干预，从病因学角度治疗TAO。然而，临床中最常用的血清炎性标志物血沉和C反应蛋白(C-reactive protein,CRP)尽管可以较好地反应绝大部分炎症性疾病的炎症活动程度，但研究发现这两者在TAO诊断及急性活动期显示为阴性，因此目前仍缺乏准确TAO诊断及反应TAO炎症活动的血清学指标，亟需寻找到新的能跟踪TAO炎症活动的血清学标志物。
富含亮氨酸α-2糖蛋白1(leucine-rich-alpha-2-glycoprotein 1,LRG1)是富亮氨酸重复蛋白家族高度保守的成员之一，其不仅参与蛋白与蛋白之间的相互作用，且在信号转导、细胞粘附及发育过程中发挥重要作用。LRG1广泛存在于人体血液、体液和各器官组织中。
目前关于LRG1作为TAO诊断及反应TAO炎症活动的血清学标志物，还未见报道。
发明内容
本发明首先提供了LRG1作为诊断血栓闭塞性脉管炎(TAO)的血清学标志物的应用；通过检测血液中的LRG1，可以用于诊断或预示血栓闭塞性脉管炎。
本发明还提供了LRG1作为诊断血栓闭塞性脉管炎炎症活动程度的血清学标志物的应用；
通过检测血液中的LRG1，可以作为判断TAO是处于静止期还是活动期。
本发明还提供了一种用于诊断血栓闭塞性脉管炎的试剂盒，该试剂盒包括：
酶标板、LRG1标准品、样品稀释液、酶标试剂、显色剂A、显色剂B、终止液；
其中样品稀释液为：PBS；
酶标试剂为:LRG1抗体；
显色剂A为：醋酸钠13.6g,柠檬酸1.6g,30％双氧水0.3ml,蒸馏水加至500ml；
显色剂B为：乙二胺四乙酸二钠0.2g,柠檬酸0.95g,甘油50ml,0.15gTMB,蒸馏水加至500ml；
终止液为：2nmol/L H2SO4；
具体的说，本发明的一种用于诊断血栓闭塞性脉管炎的试剂盒，还包括说明书；
本发明还提供了应用上述试剂盒诊断血栓闭塞性脉管炎的方法，该方法包括如下步骤：
(9)加样：酶标板的孔内分别加入待测样品或标准品各50μL，同时设立空白对照；
(10)加酶：每孔加入酶标试剂100μL，空白对照除外；
(11)温育：用封板膜封板后置入37℃温育60分钟；
(12)洗涤：揭掉封板摸，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干；
(13)显色：每孔先加入显色剂A50μL,再加入显色剂B50μL，轻轻震荡混匀，37℃避光显色15分钟；
(14)终止：每孔加终止液50μL，终止反应(此时蓝色立转黄色)；
(15)测定：以空白孔凋零，450nm波长依序测量各孔的吸光度(OD值)；
(16)计算：以标准物的浓度为横坐标，OD值为纵坐标，在坐标纸上绘出标准曲线，根据样品的OD值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。
具体的说，是应用ELISA的方法，使用上述试剂盒中的试剂，检测待检样本血清中的LRG1含量；为TAO的临床诊断提供一种有效的血清学标志物并与影像学技术及其他生物学标志物联合应用从而提高TAO的非创伤诊断效率。
本发明的试剂盒使用酶联免疫吸附分析(ELISA)方法对TAO及健康人群血清LRG1含量进行了分析，并比较了炎症活动期TAO及炎症静止期TAO患者血清LRG1的含量。发现血清LRG1含量在TAO患者中较健康人群显著升高，且活动期TAO较静止期TAO患者血清LRG1含量显著升高。同时测定了这批入组人群中ESR、CRP及IL-6等临床中常用的炎症标志物，并绘制了ROC曲线进行分析，证实了血清LRG1在诊断TAO及判断TAO炎症活动程度上性能均优于ESR、CRP及IL-6为代表的传统炎症标志物。
因此，LRG1在TAO患者血清中含量显著增加，可以对TAO的诊断进行初步确定。而在活动期TAO患者血清中LRG1含量显著高于静止期TAO患者，因此，LRG1还可以作为判断TAO炎症活动程度的血清学标志物。
利用LRG1作为诊断TAO及判断TAO炎症活动程度的生物标志物，与目前常规应用的炎症标志物相比，可以更加准确地判断及预测TAO炎症活动程度；此外，该标志物可以用于制备筛查TAO或辅助TAO炎症活动程度判断的试剂盒。本发明为TAO的早期诊断和炎症预警提供新的辅助判断手段，其与其他生物学标志物联合应用可以提高TAO的非创伤诊断效率。
附图说明：
图1：活动期TAO、静止期TAO及对照组患者血清LRG1含量(pg/ml)
图2：LRG1、ESR、CRP、IL-6诊断TAO的ROC曲线
图3：LRG1、ESR、CRP、IL-6诊断TAO炎症活动的ROC曲线
具体实施方式
下面通过实施例对本发明做进一步详细说明，这些实施例仅用来说明本发明，并不吸纳之本发明的范围。
实施例1对TAO患者及正常对照的血清样本进行LRG1的ELISA检测
配制显色剂A和显色剂B：
显色剂A为：醋酸钠13.6g,柠檬酸1.6g,30％双氧水0.3ml,蒸馏水加至500ml；
显色剂B为：乙二胺四乙酸二钠0.2g,柠檬酸0.95g,甘油50ml,0.15gTMB,蒸馏水加至500ml；
终止液为：2nmol/L H2SO4水溶液
受试群体：
本发明所使用的研究方案得到了复旦大学附属中山医院医院伦理委员会的批准，并取得受试患者的知情同意，时间为2018年。
临床样本包括TAO组40例和对照组20例，TAO组分为两个亚组：炎症活动期TAO组及静止期TAO组各20例。样本来源于复旦大学附属中山医院血管外科。两组在年龄和性别上匹配。
其中TAO诊断标准如下：①长期吸烟史；②50岁以前发病；③膝下动脉闭塞；④常有游走性静脉炎或累计上肢；⑤无动脉粥样硬化的危险因素(吸烟除外)。
TAO患者存在以下三项中任意一项定义为活动期：①三个月内缺血症状加重，间跛缩短或者出现静息痛或者溃疡/坏疽；②三个月内反复发生的游走性静脉炎③血沉和/或CRP显著升高。
所有受试者都在采样前经历了一次标准的临床体检。包括常规病史采集，下肢动静脉血管体检及下肢动脉CTA。并均进行了ESR、CRP及IL-6等传统炎症标志物的检测。血清LRG1含量测定方法如下：
将采集得到的外周血血清进行ELISA检测，其中TAO组40例，对照组20例。所有血液样本均在患者体检后24h内采集，采集量为3-5ml,并于采集后24h内离心分离血清(作为待测样品)，分离条件为3000bpm、10min、4℃。然后按照ELISA方法进行检测LRG1含量，详细实验步骤如下：
(1)分组设置：设置标准品孔、样本孔(采集的60例血液样本)和空白对照孔(空白对照孔不加样品及酶标试剂，其余各步操作相同)。
其中，标准品孔各加不同浓度的标准品溶液(PBS稀释)50μL(浓度分别为0，50，100，200，400，800pg/ml)；
待测样品孔中先加样品稀释液(PBS)40μL，然后再加待测样品10uL。加样将样品加于酶标板孔地步，尽量不触及孔壁，轻轻晃动混匀。
(2)加酶：每孔加入酶标试剂LRG1抗体(上海将来实业股份有限公司)100μL，空白板除外；
(3)温育：用封板膜封板后置入37℃温育60分钟；
(4)配液：将20倍浓缩洗涤液(PBST)用蒸馏水20倍稀释后备用；
(5)洗涤：小心揭掉封板摸，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干；
(6)显色：每孔先加入显色剂A50μL,再加入显色剂B50μL，轻轻震荡混匀，37℃避光显色15分钟；
(7)终止：每孔加终止液50μL，终止反应(此时蓝色立转黄色)；
(8)测定：以空白孔凋零，450nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液15分钟以内进行；
(9)计算：以标准物的浓度为横坐标，OD值为纵坐标，在坐标纸上汇出标准曲线，根据样品的OD值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。
所有浓度值进行TAO组与对照组、TAO组两个亚组与对照组进行统计学分析，由GraphPad Prism 7进行统计分析与制图，两组之间定量资料用非配对t检验，并通过ROC曲线等统计学方法与目前常见临床炎症标志物进行比较和评估LRG1对TAO的诊断价值。结果显示，LRG1在TAO患者(965.40±146.24pg/ml)中显著高于对照组(568.34±192.97pg/ml)，且在TAO患者两个亚组中，活动期TAO患者血清LRG1水平(1048.14±121.11pg/ml)显著高于静止期TAO患者(882.66±121.72pg/ml)(图1)。因此，经分析显示本发明所涉及的LRG1作为TAO患者的辅助诊断及炎症活动程度判断的血清标志物是具有可行性的。
基于ROC曲线的血清LRG1对TAO的诊断及炎症活动程度判断的分析表明，相比于ESR、CRP及IL-6等一系列传统炎症标志物，LRG1的诊断效能更强，可靠性更高(图2，图3)，进一步确证了其可以作为TAO诊断及TAO炎症活动程度判断的更有力的新型血清学标志物。
Claims (6)
1.LRG1作为诊断血栓闭塞性脉管炎的血清学标志物的应用。
2.LRG1作为诊断血栓闭塞性脉管炎炎症活动程度的血清学标志物的应用。
3.一种用于诊断血栓闭塞性脉管炎的试剂盒，该试剂盒包括：
酶标板、LRG1标准品、样品稀释液、酶标试剂、显色剂A、显色剂B、终止液。
4.根据权利要求3所述的试剂盒，其特征在于
其中样品稀释液为：PBS；
酶标试剂为:LRG1抗体；
显色剂A为：醋酸钠13.6g,柠檬酸1.6g,30％双氧水0.3ml,蒸馏水加至500ml；
显色剂B为：乙二胺四乙酸二钠0.2g,柠檬酸0.95g,甘油50ml,0.15gTMB,蒸馏水加至500ml；
终止液为：2nmol/L H2SO4溶液。
5.根据权利要求3或4任一项所述的试剂盒，其特征在于还包括说明书。
6.一种应用权利要求3所述的试剂盒诊断血栓闭塞性脉管炎的方法，该方法包括如下步骤：
(1)加样：酶标板的孔内分别加入待测样品或标准品各50μL，同时设立空白对照；
(2)加酶：每孔加入酶标试剂100μL，空白对照除外；
(3)温育：用封板膜封板后置入37℃温育60分钟；
(4)洗涤：揭掉封板摸，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干；
(5)显色：每孔先加入显色剂A50μL,再加入显色剂B50μL，轻轻震荡混匀，37℃避光显色15分钟；
(6)终止：每孔加终止液50μL，终止反应；
(7)测定：以空白孔凋零，450nm波长依序测量各孔的吸光度；
(8)计算：以标准物的浓度为横坐标，OD值为纵坐标，在坐标纸上绘出标准曲线，根据样品的OD值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。
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