CN117054652A - 一种用于辅助检测心肌肥厚的生物标志物及其应用 - Google Patents
一种用于辅助检测心肌肥厚的生物标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于辅助检测心肌肥厚的生物标志物及其应用。本发明筛选出了一种在心肌肥厚患者的血清中特异性高表达的生物标志物PGI。该生物标志物PGI可作为生物标志物用于血清学检查，判断机体是否发生心肌肥厚，有助于检测和诊断心肌肥厚，从而尽早采取相应治疗措施。本发明提供的PGI蛋白对于鉴别心肌肥厚患者的灵敏度和特异度高，在一些实施例中，其灵敏度和特异度分别可达到82.78％和91.45％；本发明中提供的PGI蛋白，能够进一步提高心肌肥厚的诊断准确性，作为心肌肥厚的辅助诊断方法。
Description
技术领域
本发明属于医疗技术领域，具体涉及一种用于辅助检测心肌肥厚的生物标志物及其应用。
背景技术
心肌肥厚(cardiac hypertrophy)是多种心血管疾病终末期共同的病理表现。目前认为，心肌肥厚是心肌对心功能不全的适应性改变，初期代偿具有一定有益作用，但后期进入失代偿后会产生不利影响，导致心肌细胞肥大、间质细胞增殖、胶原纤维异常堆积、心肌内冠脉系统形成异常，最终引起心律失常、心肌梗死、充血性心力衰竭等疾病，甚至导致猝死。因此，明确心肌肥厚发生的分子机制，开发诊断和治疗心肌肥厚的靶点和手段，是临床上亟待解决的重要问题。
早期、准确的心肌肥厚临床诊断是提高患者生存率的关键。目前，对心肌肥厚的诊断主要依靠影像学手段，但其缺乏及时性、方便性、快速性。而血清学检查具有灵敏、简单、经济的特点，被广泛应用于多种疾病的诊断。近年来报道的心肌肥厚的生物标志物主要有：多种微小RNA(Micro-RNA)，NAD依赖性脱乙酰酶sirtuin-3(SIRT3)，钙调素依赖性蛋白激酶II(calmodulin-dependent protein kinase II，CaMKII)等。然而由于敏感性和特异性均不高，这些生物标志物未能在临床上广泛推广。因此寻找高效、灵敏的心肌肥厚生物标志物迫在眉睫。
磷酸葡萄糖异构酶(Phosphoglucose Isomerase，PGI)是糖代谢途径中的关键酶，主要参与催化糖酵解途径中葡萄糖-6-磷酸(glucose 6-phosphate，G6P)变构生成果糖-6-磷酸(fructose 6-phosphate，F6P)。PGI又称自分泌运动因子(Autocrine MotilityFactor，AMF)，通过与AMF受体结合发挥促进肿瘤迁移、侵袭的作用。此外，PGI在类风湿性关节炎、缺血性心脏病、二型糖尿病心肌病等病理条件下表达异常。但其在心肌肥厚中的诊断和预测作用尚未阐明，仍需进一步的探究。
发明内容
本发明的目的在于提供一种用于辅助检测心肌肥厚的生物标志物及其应用，该生物标志物为心肌肥厚的临床早期诊断及预后评估提供科学依据。
本发明的目的可以通过以下技术方案实现：
PGI蛋白作为生物标志物在制备用于辅助诊断心肌肥厚患者的诊断试剂或诊断试剂盒中的应用。
用于检测血清中PGI蛋白含量的物质在制备用于辅助诊断心肌肥厚患者的诊断试剂或诊断试剂盒中的应用。优选的，用于检测血清中PGI蛋白含量的物质可以为ELISA试剂。
一种用于辅助诊断心肌肥厚患者的诊断试剂或诊断试剂盒，该诊断试剂或诊断试剂盒中包含用于检测血清中PGI蛋白含量的物质。
本发明筛选出了一种用于检测心肌肥厚的生物标志物，该生物标志物为PGI。本发明研究结果表明，PGI蛋白在心肌肥厚病人血清中表达水平升高。
检测血清中该生物标志物的含量可以区分正常人和心肌肥厚患者，所以该生物标志物可以用来制备用于区分正常人和心肌肥厚患者的诊断(检测)试剂或诊断(检测)试剂盒。
PGI的蛋白序列在网址https://www.ncbi.nlm.nih.gov/protein/P06744.4上公开。
采用PGI作为生物标志物诊断心肌肥厚患者的方法，包括如下步骤：
(1)收集待测患者的血清样品，并以正常人血清样品作为对照；
(2)采用ELISA方法检测正常人与待测患者的血清样品中生物标志物的含量，并将所得结果进行比对；
(3)根据比对结果指示待测患者是否为心肌肥厚患者；若待测对象血清样品中所用的生物标志物的含量高于对照，则指示所述待测对象为心肌肥厚患者。
本发明所述的室温为25±10℃。
本发明的有益效果：
本发明提供的PGI作为生物标志物可用于血清学检查，判断机体是否发生心肌肥厚，有助于检测和诊断心肌肥厚，从而尽早采取相应治疗措施。
本发明提供的PGI蛋白对于鉴别心肌肥厚患者的灵敏度和特异度高，在一些实施例中，其灵敏度和特异度分别可达到82.78％和91.45％；
本发明中提供的PGI蛋白在心肌肥厚患者的血清中特异性高表达，能够进一步提高心肌肥厚的诊断准确性，作为心肌肥厚的辅助诊断方法。
附图说明
图1为正常人和心肌肥厚患者的血清中的PGI含量比较；
其中，横坐标是正常人组(n＝117)和心肌肥厚患者组(n＝151)，纵坐标是PGI含量(nmol/mL)，***p<0.001。
图2为PGI在正常人组与心肌肥厚患者组之间进行对比分析的ROC曲线。
具体实施方式
下面结合附图，对本发明的具体实施方式进行详细描述，但本发明不受具体实施方式的限制。
实施例1：心肌肥厚患者血清标志物样本测试
依据性别和年龄随机分组，根据超声心动图结果，选取正常人117名作为对照，心肌肥厚患者151名，进行ELISA实验验证PGI能否判断患者患有心肌肥厚。ELISA试剂盒购于南京森贝伽生物科技有限公司。实验步骤如下：
(1)实验所需试剂及耗材：
①ELISA试剂盒，如表1：
表1 ELISA试剂盒组成
| 试剂盒组成 | 规格(96T) |
| 说明书 | 1份 |
| 封板膜 | 2片 |
| 密封袋 | 1个 |
| 酶标包被板 | 1×96 |
| 标准品：13.5nmol/mL | 0.5mL×1瓶 |
| 标准品稀释液 | 1.5mL×1瓶 |
| 酶标试剂 | 6mL×1瓶 |
| 样品稀释液 | 6mL×1瓶 |
| 显色剂A液 | 6mL×1瓶 |
| 显色剂B液 | 6mL×1瓶 |
| 终止液 | 6mL×1瓶 |
| 浓缩洗涤液 | (20mL×30倍)×1瓶 |
②所需自备实验器材：酶标仪(450nm)；37℃恒温箱。
(2)检测前准备工作：
①提前30min从冰箱中取出试剂盒、样本，平衡至室温。
②将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。
(3)检测：
①标准品的稀释与加样：在酶标包被板上设标准品孔10孔，在第一、第二孔中分别加标准品100μL，然后在第一、弟二孔中加标准品稀释液50μL，混匀；然后从第一孔、第二孔中各取100μL分别加到第三孔和第四孔，再在第三、第四孔分别加标准品稀释液50μL，混匀；然后在第三孔和第四孔中先各取50μL弃掉，再各取50μL分别加到第五、第六孔中，再在第五、第六孔中分别加标准品稀释液50μL，混匀；混匀后从第五、第六孔中各取50μL分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液50μL，混匀后从第七、第八孔中分别取50μL加到第九、第十孔中，再在第九、第十孔分别加标准品稀释液50μL，混匀后从第九第十孔中各取50μL弃掉。(稀释后各孔加样量都为50μL,浓度分别为9nmol/mL，6nmol/mL，3nmol/mL，1.5nmol/mL，0.75nmol/mL)。
②加样:分别设空白孔(空白对照孔不加样品及酶标试剂，其余各步操作相同)、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液40μL，然后再加待测样品l0μL(样品最终稀释度为5倍)。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。
③温育：用封板膜封板后置37℃温育30分钟。
④洗涤：小心揭掉封板膜，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加酶：每孔加入酶标试剂50μL，空白孔除外。
⑥温育：操作同3。
⑦洗涤：操作同5。
⑧显色：每孔先加入显色剂A 50μL，再加入显色剂B 50μL,轻轻震荡混匀，37℃避光显色15分钟。
⑨终止：每孔加终止液50μL，终止反应(此时蓝色立转黄色)。
⑩测定：以空白孔调零，450nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液后15分钟以内进行。
(4)统计学方法
实验中每个样本设置3个复孔，结果为3个复孔的平均值，所得数据采用Prism8.0统计软件进行统计分析，不同组之间采用非参数秩和检验，认为P<0.05时具有统计学意义；采用Prism8.0统计软件绘制受试者工作特征(ROC)曲线，明确PGI能否成为新的心肌肥厚诊断标志物应用于临床。
(5)结果分析
如图1所示，该图为正常人和心肌肥厚患者的血清中的PGI含量数值，结果显示：与正常组相比，心肌肥厚患者血清中PGI水平显著升高，差异具有统计学意义(P<0.001)，说明PGI能够特异地区分健康人与心肌肥厚患者。
如图2和表2所示，该图为PGI在正常人组与心肌肥厚患者组之间进行对比分析的ROC曲线，结果显示：曲线下面积为0.9496，最佳截断值为173.23，灵敏度为82.78％，特异度为91.45％，说明其对心肌肥厚具有很好的诊断价值。
表2PGI用于区分正常人和心肌肥厚患者时的ROC统计结果
Claims (3)
1.PGI蛋白作为生物标志物在制备用于辅助诊断心肌肥厚患者的诊断试剂或诊断试剂盒中的应用。
2.用于检测血清中PGI蛋白含量的物质在制备用于辅助诊断心肌肥厚患者的诊断试剂或诊断试剂盒中的应用。
3.一种用于辅助诊断心肌肥厚患者的诊断试剂或诊断试剂盒，其特征在于，该诊断试剂或诊断试剂盒中包含用于检测血清中PGI蛋白含量的物质。
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