CN112415199B - Application of CETP detection reagent in preparation of lung cancer screening kit - Google Patents

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a CETP detection reagent in preparation of a lung cancer screening kit. The invention discovers for the first time that CETP protein level in plasma exosomes of lung cancer patients is significantly higher than that of healthy people. The reagent for detecting CETP protein is used for preparing the lung cancer screening kit, so that the lung cancer can be effectively screened.

Description

Translated fromChinese

CETP检测试剂在制备肺癌筛查试剂盒中的用途Use of CETP detection reagents in preparing lung cancer screening kits

技术领域Technical field

本发明涉及体外诊断试剂领域，具体涉及CETP检测试剂在制备肺癌筛查试剂盒中的用途。The present invention relates to the field of in vitro diagnostic reagents, and specifically relates to the use of CETP detection reagents in preparing lung cancer screening kits.

背景技术Background technique

肺癌是世界上最常见的恶性肿瘤之一，其发病率和死亡率呈逐年上升趋势，目前发病率居世界首位，严重威胁着人类健康和生命。Lung cancer is one of the most common malignant tumors in the world, and its incidence and mortality rates are increasing year by year. The current incidence rate ranks first in the world, seriously threatening human health and life.

肺癌是一种善于隐匿的疾病，经常在疾病发展到晚期才表现出临床症状，70～80％的肺癌患者在诊断出患有肺癌症状时已是中、晚期，癌细胞已经扩散，错过了最佳治愈时机，五年生存率低。对于早期的肺癌患者，经过及时治疗可大大提高患者的5年及以上生存率和生存质量。因此肺癌的早期诊断和进行有效的筛查至关重要。Lung cancer is a disease that is good at hiding. It often does not show clinical symptoms until the disease has developed to an advanced stage. 70 to 80% of lung cancer patients are already in the middle or late stage when they are diagnosed with lung cancer symptoms. The cancer cells have spread and they have missed the earliest stage of the disease. The best opportunity for cure is that the five-year survival rate is low. For early-stage lung cancer patients, timely treatment can greatly improve the patient's survival rate and quality of life for 5 years or more. Therefore, early diagnosis and effective screening of lung cancer are crucial.

肺癌的筛查，是指对那些没有肺癌相关症状的人群进行常规体检，在出现症状前及时发现肺癌。如果可以找到血浆里面的肺癌分子标志物，用于提示临床医生早期对患者采取相关的治疗措施或者决策具有重要的意义。Lung cancer screening refers to routine physical examinations for people who do not have symptoms related to lung cancer to detect lung cancer in time before symptoms appear. If the molecular markers of lung cancer in plasma can be found, it will be of great significance to prompt clinicians to take relevant treatment measures or decisions for patients at an early stage.

CETP蛋白(Uniprot编号:P11597)全称胆固醇酯转移蛋白(Cholesteryl estertransfer protein)。该蛋白是脂代谢中重要的转运蛋白之一，主要介导胆固醇的逆向转运过程，即在高密度脂蛋白(HDL)颗粒与其他脂蛋白或细胞膜之间不断地进行脂质成分交换，同时增加胆固醇从组织细胞中的流出，使其转运至肝脏。血浆CETP蛋白水平与脂质代谢平衡密切相关。The full name of CETP protein (Uniprot number: P11597) is Cholesteryl estertransfer protein. This protein is one of the important transport proteins in lipid metabolism. It mainly mediates the reverse transport process of cholesterol, that is, the continuous exchange of lipid components between high-density lipoprotein (HDL) particles and other lipoproteins or cell membranes, while increasing The efflux of cholesterol from tissue cells transports it to the liver. Plasma CETP protein levels are closely related to lipid metabolism balance.

目前尚未见CETP蛋白与肺癌相关的现有技术。There is no existing technology related to CETP protein and lung cancer.

发明内容Contents of the invention

本发明的目的在于提供一种新的类的肺癌标志物，以及该标志物的检测试剂在制备肺癌筛查试剂盒中的用途。The object of the present invention is to provide a new type of lung cancer marker and the use of a detection reagent for the marker in preparing a lung cancer screening kit.

本发明的技术方案包括：The technical solutions of the present invention include:

检测CETP蛋白的试剂在制备肺癌筛查试剂盒中的用途。Use of reagents for detecting CETP protein in preparing lung cancer screening kits.

如前述的用途，所述检测CETP蛋白的试剂为酶联免疫吸附试验用试剂。As mentioned above, the reagent for detecting CETP protein is an enzyme-linked immunosorbent assay reagent.

如前述的用途，所述检测CETP蛋白的试剂为western blot试剂。As mentioned above, the reagent for detecting CETP protein is a western blot reagent.

如前述的用途，所述检测CETP蛋白的试剂为蛋白芯片检测方法用试剂。As mentioned above, the reagent for detecting CETP protein is a reagent for protein chip detection method.

如前述的用途，所述检测CETP蛋白的试剂是检测人血浆外泌体中CETP蛋白的试剂。As mentioned above, the reagent for detecting CETP protein is a reagent for detecting CETP protein in human plasma exosomes.

一种肺癌筛查试剂盒，它包括用于检测CETP蛋白的试剂。A lung cancer screening kit, which includes a reagent for detecting CETP protein.

如前述的试剂盒，所述检测CETP蛋白的试剂为酶联免疫吸附试验用试剂。As in the aforementioned kit, the reagent for detecting CETP protein is an enzyme-linked immunosorbent assay reagent.

如前述的试剂盒，所述检测CETP蛋白的试剂为western blot试剂。As in the aforementioned kit, the reagent for detecting CETP protein is a western blot reagent.

如前述的试剂盒，所述检测CETP蛋白的试剂为蛋白芯片检测方法用试剂。As in the aforementioned kit, the reagent for detecting CETP protein is a reagent for protein chip detection method.

如前述的试剂盒，所述检测CETP蛋白的试剂是检测人血浆外泌体中CETP蛋白的试剂。As in the aforementioned kit, the reagent for detecting CETP protein is a reagent for detecting CETP protein in human plasma exosomes.

本发明的关键在于，确定了人血浆外泌体中CETP的含量与患肺癌的风险显著相关，因此可以通过检测人血浆外泌体中CETP的含量来判断患肺癌的风险，至于具体检测人血浆外泌体中CETP手段，可以采用现有技术公开的各种手段，本发明实施例具体采用酶联免疫分析方法(蛋白芯片)进行检测，但不仅仅限于该手段，任何能够检测CETP含量的方法均可用于肺癌筛查。The key to the present invention is that it is determined that the content of CETP in human plasma exosomes is significantly related to the risk of lung cancer. Therefore, the risk of lung cancer can be judged by detecting the content of CETP in human plasma exosomes. As for the specific detection of human plasma CETP in exosomes can be detected by various methods disclosed in the prior art. In the embodiment of the present invention, an enzyme-linked immunoassay method (protein chip) is specifically used for detection, but it is not limited to this method. Any method that can detect CETP content is used. Both can be used for lung cancer screening.

本发明提供了一种新的肺癌筛查标记物和一种新的肺癌筛查试剂盒，能够实现肺癌的有效筛查；且能以血浆外泌体作为检测样品，对患者伤害很低。本发明具备良好的应用前景。The present invention provides a new lung cancer screening marker and a new lung cancer screening kit, which can achieve effective screening of lung cancer; and can use plasma exosomes as detection samples, causing very little harm to patients. The invention has good application prospects.

显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。Obviously, according to the above content of the present invention, according to the common technical knowledge and common means in the field, without departing from the above basic technical idea of the present invention, various other forms of modifications, replacements or changes can also be made.

以下通过具体实施方式对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above contents of the present invention will be further described in detail below through specific embodiments. However, this should not be understood to mean that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

附图说明Description of the drawings

图1：肺癌患者(LC)、健康对照(NC)血浆外泌体中CETP水平对比。Figure 1: Comparison of CETP levels in plasma exosomes of lung cancer patients (LC) and healthy controls (NC).

图2：肺癌患者(LC)与健康对照(NC)ROC分析。Figure 2: ROC analysis of lung cancer patients (LC) and healthy controls (NC).

具体实施方式Detailed ways

实施例1血浆中CETP与肺癌的关系Example 1 The relationship between CETP in plasma and lung cancer

一、临床资料1. Clinical data

选取肺癌患者40例，健康对照40例，基本信息如下：40 patients with lung cancer and 40 healthy controls were selected. The basic information is as follows:

基本信息Basic Information肺癌患者lung cancer patients健康对照healthy control人数number of people40404040年龄age58.8±11.558.8±11.550.1±14.350.1±14.3男性比例Male proportion33(82.5％)33(82.5%)17(44.7％)17(44.7%)

二、检测原理2. Detection principle

同基本的酶联免疫吸附试验(ELISA)检测方法。Same as the basic enzyme-linked immunosorbent assay (ELISA) detection method.

蛋白芯片上固定有CETP蛋白CETP抗体(特异性识别CETP蛋白的ASYPDITGEK氨基酸序列(SEQ ID NO.1))，加血浆外泌体蛋白孵育后，若外泌体蛋白中存在CETP蛋白，则CETP蛋白会结合上去；清洗去除未结合的抗体和其它蛋白质，再用荧光标记的CETP抗体结合到CETP蛋白上检测，通过荧光扫描仪读取信号，信号的强弱与抗体的亲和力和数量呈正相关。The CETP protein CETP antibody (which specifically recognizes the ASYPDITGEK amino acid sequence of CETP protein (SEQ ID NO.1)) is fixed on the protein chip. After incubation with plasma exosome protein, if CETP protein is present in the exosome protein, the CETP protein It will bind; wash to remove unbound antibodies and other proteins, then use fluorescently labeled CETP antibodies to bind to the CETP protein for detection, and read the signal through a fluorescence scanner. The strength of the signal is positively related to the affinity and quantity of the antibody.

三、方法3. Method

1.血浆外泌体蛋白的制备方法如下：1. The preparation method of plasma exosome protein is as follows:

样品从-80℃取出，4℃，12000g离心15分钟，上清液转移至新的离心管，0.22μM微孔滤膜过滤后，用PTM公司生产的PTM-EVs试剂盒参照说明书去分离外泌体。加入终浓度为8M的Urea以及蛋白酶抑制剂超声裂解，利用BCA试剂盒进行蛋白浓度测定。Take the sample out from -80℃, centrifuge it at 12000g for 15 minutes at 4℃, transfer the supernatant to a new centrifuge tube, filter it with a 0.22μM microporous filter, and use the PTM-EVs kit produced by PTM Company to isolate exocrine according to the instructions. body. Urea with a final concentration of 8M and protease inhibitors were added for ultrasonic lysis, and the protein concentration was determined using a BCA kit.

2.血浆外泌体CETP蛋白检测2. Detection of CETP protein in plasma exosomes

试剂如下：The reagents are as follows:

步骤如下：Proceed as follows:

1)复温：将芯片从-80℃冰箱取出，置于4℃冰箱复温半小时，然后置于室温复温15min；1) Rewarming: Take the chip out of the -80°C refrigerator, place it in a 4°C refrigerator for half an hour, and then place it at room temperature for 15 minutes;

2)封闭：复温后的芯片，固定14个blocks围栏，固定好之后，向每个block中加入封闭液，并放置侧摆摇床上，室温封闭3hr；2) Sealing: After rewarming the chip, fix 14 blocks in the fence. After fixing, add blocking solution to each block, place it on a side-swing shaker, and seal at room temperature for 3 hours;

3)外泌体蛋白样本孵育：封闭完成后，倒尽封闭液，然后迅速加入预先准备好的血浆外泌体蛋白孵育液，上样体积为200μL，侧摆摇床20rpm，4℃过夜孵育(血浆外泌体蛋白加孵育液以1：50比例稀释，得血浆外泌体蛋白孵育液)；( Add plasma exosome protein to the incubation solution and dilute it at a ratio of 1:50 to obtain plasma exosome protein incubation solution);

4)清洗：将芯片及芯片围栏一同取出，吸去样本，然后迅速加入等体积的PBST，如此循环数次，保证在拆除芯片围栏时血浆外泌体蛋白间无交叉污染。拆除芯片围栏后，将芯片置于加有清洗液的芯片清洗盒，水平摇床，室温80rpm，清洗3次，每次10min；4) Cleaning: Take out the chip and chip fence together, aspirate the sample, and then quickly add an equal volume of PBST. Repeat this cycle several times to ensure that there is no cross-contamination between plasma exosome proteins when the chip fence is removed. After removing the chip fence, place the chip in a chip cleaning box with cleaning solution, on a horizontal shaker, at room temperature 80 rpm, and clean 3 times, 10 minutes each time;

5)二抗孵育：将芯片转移到加入了3mL二抗孵育液的孵育盒中，侧摆摇床40rpm，避光，室温1hr；5) Secondary antibody incubation: Transfer the chip to an incubation box with 3 mL of secondary antibody incubation solution, with a side-swing shaker at 40 rpm, protected from light, and room temperature for 1 hr;

6)清洗：将芯片取出(注意不能触及或划破芯片的上表面)，置于加有清洗液的芯片清洗盒，水平摇床，室温80rpm，清洗3次，每次10min。完成后用ddH2O清洗2次，每次10min；6) Cleaning: Take out the chip (be careful not to touch or scratch the upper surface of the chip), place it in a chip cleaning box with cleaning solution, use a horizontal shaker, room temperature 80rpm, and clean 3 times, 10 minutes each time. After completion, wash with ddH2O twice, 10 minutes each time;

7)干燥；7) Drying;

8)扫描：使用晶芯LuxScan 10K微阵列芯片扫描仪扫描；8) Scan: Use Jingxin LuxScan 10K microarray chip scanner to scan;

9)数据提取：将对应GAL文件(记录了芯片中蛋白所在位置)打开，将芯片图像和GAL文件每个阵列整体对齐，按下自动对齐按钮，提取数据并保存。9) Data extraction: Open the corresponding GAL file (recording the location of the protein in the chip), align the chip image and each array of the GAL file as a whole, press the automatic alignment button, extract the data and save it.

四、结果4. Results

肺癌患者血浆外泌体中的CETP的平均表达水平为1.02(蛋白相对定量比值)，健康对照血浆外泌体中CETP的平均表达水平为0.56。肺癌组与肺部良性疾病组(p<0.01)相比均有统计学意义(图1)。肺癌组与健康对照ROC分析结果特异性为97.5％，敏感性为27.5％(图2)；表明血浆外泌体中CETP可特异地区分肺癌与肺部良性疾病。The average expression level of CETP in plasma exosomes of lung cancer patients was 1.02 (relative quantitative protein ratio), and the average expression level of CETP in plasma exosomes of healthy controls was 0.56. There were statistically significant differences between the lung cancer group and the benign lung disease group (p<0.01) (Figure 1). The ROC analysis results of the lung cancer group and healthy controls had a specificity of 97.5% and a sensitivity of 27.5% (Figure 2); indicating that CETP in plasma exosomes can specifically distinguish lung cancer from benign lung diseases.

由以上结果可知，肺癌患者与非肺癌患者的血浆外泌体中CETP的水平差异显著，通过检测血浆外泌体中CETP的水平，能够达到肺癌筛查的目的。From the above results, it can be seen that the level of CETP in plasma exosomes of lung cancer patients and non-lung cancer patients is significantly different. By detecting the level of CETP in plasma exosomes, the purpose of lung cancer screening can be achieved.

实施例2本发明的检测试剂盒的组成及其使用方法Example 2 The composition of the detection kit of the present invention and its use method

一、试剂盒组成1. Composition of kit

检测试剂盒(14人份)：Test kit (for 14 people):

制备血浆外泌体蛋白的试剂需要自备，可以是市售血浆外泌体蛋白提取试剂盒。Reagents for preparing plasma exosome proteins need to be prepared by yourself, which can be commercially available plasma exosome protein extraction kits.

二、试剂盒使用方法2. How to use the kit

同实施例1第三部分。Same as the third part of Example 1.

本发明的试剂盒可以通过检测血浆外泌体中CETP的水平，可以筛查待检人群患肺癌的风险：若CETP水平高(相对于健康对照而言)，则患肺癌的风险高，若CETP水平低，则患肺癌的风险低。可用于临床肺癌的辅助诊断，为患者采取相关的治疗措施或者决策提供有效的依据，临床应用前景良好。The kit of the present invention can screen the risk of lung cancer in the population to be tested by detecting the level of CETP in plasma exosomes: if the CETP level is high (relative to healthy controls), the risk of lung cancer is high, and if the CETP level is high, the risk of lung cancer is high. Low levels are associated with a low risk of lung cancer. It can be used for auxiliary diagnosis of clinical lung cancer, providing effective basis for patients to take relevant treatment measures or decision-making, and has good clinical application prospects.

SEQUENCE LISTINGSEQUENCE LISTING

<110> 四川大学华西医院<110> West China Hospital of Sichuan University

<120> GYKH1094-2020P0111926CC20JS044<120> GYKH1094-2020P0111926CC20JS044

<130> CETP检测试剂在制备肺癌筛查试剂盒中的用途<130> Use of CETP detection reagents in preparing lung cancer screening kits

<160> 1<160> 1

<170> PatentIn version 3.5<170> PatentIn version 3.5

<210> 1<210> 1

<211> 10<211> 10

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 1<400> 1

Ala Ser Tyr Pro Asp Ile Thr Gly Glu LysAla Ser Tyr Pro Asp Ile Thr Gly Glu Lys

1 5 101 5 10

Claims (4)

Translated fromChinese

1.检测CETP蛋白的试剂在制备肺癌筛查试剂盒中的用途，所述检测CETP蛋白的试剂是检测人血浆外泌体中CETP蛋白的试剂。1. Use of a reagent for detecting CETP protein in the preparation of a lung cancer screening kit. The reagent for detecting CETP protein is a reagent for detecting CETP protein in human plasma exosomes.2.如权利要求1所述的用途，其特征在于，所述检测CETP蛋白的试剂为酶联免疫吸附试验用试剂。2. The use according to claim 1, characterized in that the reagent for detecting CETP protein is an enzyme-linked immunosorbent assay reagent.3.如权利要求1所述的用途，其特征在于，所述检测CETP蛋白的试剂为western blot试剂。3. The use according to claim 1, characterized in that the reagent for detecting CETP protein is a western blot reagent.4.如权利要求1所述的用途，其特征在于，所述检测CETP蛋白的试剂为蛋白芯片检测方法用试剂。4. The use according to claim 1, characterized in that the reagent for detecting CETP protein is a reagent for protein chip detection method.