CN107957498A - 一种含有载脂蛋白j的试剂盒及其在临床诊断中的应用 - Google Patents
一种含有载脂蛋白j的试剂盒及其在临床诊断中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，涉及一种载脂蛋白J的应用，特别是在制备心血管疾病和神经退行性疾病诊断试剂中的应用，本发明还包括心血管疾病和神经退行性疾病诊断的载脂蛋白J含量的检测试剂盒。本发明的方法是通过检测血浆中载脂蛋白J的含量变化情况，预警心血管疾病和神经退行性疾病。并且检测方法具有高度灵敏性，稳定性，准确性，同时线性范围广。
Description
技术领域
本发明属于医学免疫体外诊断领域，具体地，涉及一种含有载脂蛋白J的试 剂盒及其临床诊断中的应用。
背景技术
载脂蛋白J(apolipoprotein J，Apo J，又称Clusterin)是一种多功能的糖蛋 白，其结构与Lipocalin家族相关联。几乎所有类型的细胞都能分泌这种蛋白， 人类Apo J基因在人体组织中广泛存在，尤其在大脑、肾上腺髓质、垂体前叶和 后叶、卵巢、睾丸及肝脏中的mRNA水平很高。Apo J参与了许多生理功能， 包括凋亡、补体调控、保护细胞膜、细胞与细胞以及细胞与基质间相互作用的稳 定性，DNA的修复和致癌作用等。
Apo J主要存在于血浆中，并与高密度脂蛋白(HDL)相联系，尤其是与含有 Apo AI和胆固醇转移蛋活性的H DL亚型联系最密切。Apo J与载脂蛋白AI一 起形成高密度脂蛋白复合物，参与末端补体反应，调节补体功能，并参与脂质运 转、组织再生、生物活性肽转运等，在许多病理过程中也有所表达，因此，在生 理与病理过程中起着重要作用。
心脑血管疾病是心脏血管和脑血管疾病的统称，泛指由于高脂血症、血液黏 稠、动脉粥样硬化、高血压等所导致的心脏、大脑及全身组织发生的缺血性或出 血性疾病。心脑血管疾病是一种严重威胁人类，特别是50岁以上中老年人健康 的常见病，具有高患病率、高致残率和高死亡率的特点。
冠心病作为心脑血管疾病的一种，目前的最佳诊断方法是冠脉造影，但是诊 断过程相当麻烦。除此之外，冠心病等疾病较为简易的诊断方法为血液检验诊断， 目前为测定心肌酶谱(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、 天冬氨酸转氨酶(AST)的含量；前述四种酶含量应该综合考量来判断病情；上 述四种酶的血液检验来源于同一个原理，即测定心肌细胞死亡后释放进血液的酶 类，因此得病之后的患者血液中四种酶类含量急剧上升，但适用于较为严重的或 已经发生心肌梗死的病患。对于较轻的患者一般较难发现，不能及时，尽早地为 患者提供诊断和治疗方案。
此外，Apo J在多种神经系统疾病中均有高表达，如脑血管病、阿尔茨海默 病、帕金森病、神经肿瘤及多发性硬化等，且Apo J在这些病理过程中主要发挥 神经元保护性作用。
目前，世界上尚未有将载脂蛋白J在血浆中的含量检测应用于心脑血管疾病 或者神经系统疾病诊断的报道。
有鉴于此，特提出本发明。
发明内容
本发明的目的在于提供了载脂蛋白J在制备临床疾病诊断试剂盒中的应用。
本发明的另一个目的在于提供一种含有载脂蛋白J的试剂盒。
为实现上述技术目的，采用以下技术方案：
载脂蛋白J在制备心脑血管疾病诊断试剂中的应用。
进一步，所述的心脑血管疾病包括冠心病、缺血性心肌梗死、动脉粥样硬化、 缺血性慢性心衰、高脂血症、高血压。
进一步，载脂蛋白J在制备神经退行性疾病诊断试剂中的应用。
优选的，所述的神经退行性疾病包括阿尔茨海默病、帕金森病。
进一步，所述的诊断试剂中以载脂蛋白J作为诊断指标。
进一步，一种含有载脂蛋白J的试剂盒，所述的试剂盒包括：羊抗ApoJ、 载脂蛋白J标准品、叠氮化钠、聚乙二醇6000、缓冲液、显色液以及终止液。 优选的，所述的缓冲液为磷酸盐缓冲液、Tris-HCL缓冲液中的一种或几种。
一种如上述的含有载脂蛋白J的试剂盒在制备心脑血管疾病诊断试剂中的 应用。
具体的，所述的心脑血管疾病包括冠心病、心肌梗死、动脉粥样硬化、慢性 心衰、高脂血症、高血压。
一种如上述的含有载脂蛋白J的试剂盒在制备神经退行性疾病诊断试剂中 的应用。
具体的，所述的神经退行性疾病包括阿尔茨海默病、帕金森病。
本发明的有益效果：
本发明发现冠心病患者、缺血性心梗患者、缺血性慢性心衰患者血浆中载脂 蛋白J显著低于正常人血液中的载脂蛋白J的含量，而老年痴呆患者、全身性炎 症反应肥胖病患者以及2型糖尿病患者的血浆中载脂蛋白J显著高于正常人血液 中的载脂蛋白J的含量。尤其对于早期心脑血管疾病、神经退行性疾病的人群进 行常规体检，在出现症状前及时发现问题，用于提示临床医生早期对患者采用相 关措施或者决策具有重要的意义。并且检测方法具有高度灵敏性，稳定性，准确 性，同时线性范围广。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例对本发明做进一步的说明。 本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不 应以此限制本发明的保护范围。
一、实验方法：
1、检测试剂盒的组成如下：
显色液A(过氧化氢脲)
显色液B(辣根过氧化氢酶)
终止液(盐酸)
具体测定方法如下：
(1)准备：将试剂盒和待测血浆从冰箱取出，于室温下复温平衡30min；
(2)配液：将Tween20用磷酸盐缓冲液或Tris缓冲液稀释成洗涤液；将叠氮化 钠和聚乙二醇6000按1:1比例配成稀释液；将羊抗载脂蛋白J抗体按1:10与磷 酸盐缓冲液配成工作液；
(3)加入标准品和待测的样本：在酶标包被板上分别设置空白对照孔、标准品 孔和待测样品孔，分别记录各孔的位置，空白对照孔不加液体；标准品孔加50ul 标准品；待测样品孔中先加入10ul待测样品，在加样品稀释液40ul(即样品稀 释5倍)；
(4)温育：37℃水浴锅温育30min；
(5)洗板：将液体弃去，拍干，用洗涤液加满每孔后静置1min，再甩去洗涤液， 拍干，如此反复洗涤4次；
(6)加液：每孔加入50ul酶标的工作液，空白对照孔不加液体；
(7)温育：37℃水浴锅温育30min；
(8)洗板：同步骤5；
(9)显色：每孔先加入50ul显色剂A液，再加入50ul显色剂B液，轻轻震荡 混匀30s，37℃电热恒温干烤箱避光显色15min；
(10)终止：将酶标板取出，每孔加50ul终止液，颜色由蓝色立即转为黄色；
(11)测定：用空白孔来调零，终止反应后15min内，用450nm的波长来测定 各孔的OD值；
(12)计算:根据标准样品的浓度及其相应的OD值，得出标准曲线的直线回归 方程，然后根据样品的OD值，在回归方程上计算出相应的样品浓度，最终的浓 度为实际测定浓度与稀释倍数的乘积。
实施例1
正常志愿者285人，基本信息见表1：
| 患者基本信息 | |
| 人数 | 300 |
| 年龄 | 50±15 |
| 男性 | 155 |
| 女性 | 130 |
采集上述正常志愿者的血浆；采用上述试剂盒以及测定方法测定正常志愿者 血浆中载脂蛋白J的含量。
实施例2
选取冠心病患者241位，其中，心电图以及冠状动脉造影均显示上述患者具 有冠心病，基本信息见表2：
采集上述冠心病患者的血浆，采用上述试剂盒以及测定方法测定冠心病患者 血浆中载脂蛋白J的含量。
实施例3
缺血性心梗患者235位，基本信息见表3：
| 患者基本信息 | |
| 人数 | 235 |
| 年龄 | 59±20 |
| 男性 | 118 |
| 女性 | 117 |
采集上述缺血性慢性心衰患者的血浆，采用上述试剂盒以及测定方法测定缺 血性心梗患者血浆中载脂蛋白J的含量。
实施例4
缺血性慢性心衰患者340位，基本信息见表4：
| 患者基本信息 | |
| 人数 | 340 |
| 年龄 | 50±20 |
| 男性 | 187 |
| 女性 | 153 |
采集上述缺血性慢性心衰患者的血浆，采用上述试剂盒以及测定方法测定缺 血性慢性心衰患者血浆中载脂蛋白J的含量。
实施例5
老年痴呆(阿尔茨海默病Alzheimer disease，AD)患者156位，基本信息见 表5：
采集上述老年痴呆患者的血浆，采用上述试剂盒以及测定方法测定老年痴呆 患者血浆中载脂蛋白J的含量。
实施例6
全身性炎症反应肥胖病患者189位，基本信息见表6：
| 患者基本信息 | |
| 人数 | 189 |
| 年龄 | 50+10 |
| 男性 | 92 |
| 女，性 | 97 |
采集上述全身性炎症反应肥胖病患者的血浆，采用上述试剂盒以及测定方法 测定全身性炎症反应肥胖病患者血浆中载脂蛋白J的含量。
实施例7
2型糖尿病患者212位，基本信息见表7：
| 患者基本信息 | |
| 人数 | 212 |
| 年龄 | 50±20 |
| 男性 | 98 |
| 女性 | 114 |
采集上述2型糖尿病患者的血浆，采用上述试剂盒以及测定方法测定2型糖 尿病患者血浆中载脂蛋白J的含量。
其中，测定结果显示：正常人平均血液ApoJ含量大约在50-60微克/毫升； 其中，实施例1中241位冠心病患者血液ApoJ含量大约低于正常人的20-50％， 至25-45微克/毫升；进一步将上述241位冠心病患者测定心肌酶谱(CK)、肌 酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、天冬氨酸转氨酶(AST)的含 量，其中有163位患者四种酶含量均升高，48位患者仅心肌酶谱(CK)、肌酸 激酶同工酶(CK-MB)升高，30位患者仅肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、天冬氨酸转氨酶(AST)升高。
同样，实施例2中缺血性心梗患者，血液ApoJ含量大约低于正常人的 20-35％，至38-48微克/毫升；
实施例3中缺血性慢性心衰患者，血液ApoJ含量大约低于正常人的23-42％， 至36-45微克/毫升；
由此可见，冠心病、缺血性心梗患者、缺血性慢性心衰患者血浆中载脂蛋白 J显著低于正常人血液中的载脂蛋白J的含量。
采用Apo J含量测定进行心血脑管疾病诊断的原理与现有技术中采用测定 心肌酶谱(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、天冬氨酸 转氨酶(AST)的含量上述四种酶类完全不同，对于早期心脑血管疾病患者，并 未出现上述四种酶均升高，而由于ApoJ对心血管组织的保护作用，Apo J在血 液中含量的下降可能预示着潜在的心血管意外；患者在心血管疾病早中晚全程都 可以出现Apo J下降的现象，因此对Apo J的测定可以对心血管疾病进行预测、 可以诊断早中期心脑血管疾病。并且检测方法具有高度灵敏性，稳定性，准确性， 同时线性范围广。
进一步，实施例4中老年痴呆患者，血液ApoJ含量大约高于正常人的 25-40％；至66-85微克/毫升；
实施例5中全身性炎症反应肥胖病患者，血液ApoJ含量大约高于正常人的 20-30％，至63-75微克/毫升；
实施例6中2型糖尿病患者，血液ApoJ含量大约高于正常人的60％，至80-90 微克/毫升。
由此可见，老年痴呆患者、全身性炎症反应肥胖病患者以及2型糖尿病患者 的血浆中载脂蛋白J显著高于正常人血液中的载脂蛋白J的含量。由于Apo J在 多种神经系统疾病中均有高表达，如阿尔茨海默病、帕金森病等，且Apo J在这 些病理过程中主要发挥神经元保护性作用。Apo J在血液中含量的升高可能预示 着潜在的神经系统疾病。因此对Apo J的测定可以对神经退行性疾病进行预测、 可以诊断早中期疾病。
由此可见，对那些没有心脑血管疾病、神经退行性疾病的人群进行常规体检， 在出现症状前及时发现问题，用于提示临床医生早期对患者采用相关措施或者决 策具有重要的意义，临床应用前景良好。
Claims (11)
1.载脂蛋白J在制备心脑血管疾病诊断试剂中的应用。
2.根据权利要求1所述的载脂蛋白J在制备心脑血管疾病诊断试剂中的应用，其特征在于：所述的心脑血管疾病包括冠心病、缺血性心肌梗死、动脉粥样硬化、缺血性慢性心衰、高脂血症、高血压。
3.载脂蛋白J在制备神经退行性疾病诊断试剂中的应用。
4.根据权利要求3所述的载脂蛋白J在制备神经退行性疾病诊断试剂中的应用，其特征在于：所述的神经退行性疾病包括阿尔茨海默病、帕金森病。
5.根据权利要求1-4中任一种所述的应用，其特征在于：所述的诊断试剂中以载脂蛋白J作为诊断指标。
6.一种含有载脂蛋白J的试剂盒，其特征在于：所述的试剂盒包括：羊抗ApoJ、载脂蛋白J标准品、叠氮化钠、聚乙二醇6000、缓冲液、显色液以及终止液。
7.根据权利要求6所述的一种含有载脂蛋白J的试剂盒，其特征在于：所述的缓冲液为磷酸盐缓冲液、Tris-HCL缓冲液中的一种或几种。
8.一种如权利要求6或7中所述的含有载脂蛋白J的试剂盒在制备心脑血管疾病诊断试剂中的应用。
9.根据权利要求8所述的含有载脂蛋白J的试剂盒在制备心脑血管疾病诊断试剂中的应用，其特征在于：所述的心脑血管疾病包括冠心病、心肌梗死、动脉粥样硬化、慢性心衰、高脂血症、高血压。
10.一种如权利要求6或7中所述的含有载脂蛋白J的试剂盒在制备神经退行性疾病诊断试剂中的应用。
11.根据权利要求10所述的含有载脂蛋白J的试剂盒在制备心脑血管疾病诊断试剂中的应用，其特征在于：所述的神经退行性疾病包括阿尔茨海默病、帕金森病。
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