阅读说明：本技术 透明肾细胞癌代谢标志物在肾细胞癌早期筛查和诊断产品中的应用 (Application of transparent renal cell carcinoma metabolic marker in renal cell carcinoma early screening and diagnosis product ) 是由 孙伟 刘晓燕 孙海丹 刘响 郭正光 李京 汤晓悦 于 2020-05-29 设计创作，主要内容包括：本发明公开了透明肾细胞癌代谢标志物在肾细胞癌早期筛查和诊断产品中的应用,所述代谢标志物为12,13-双羟基十八碳烯酸或5-L-谷酰基-丙氨酸中的一种或全部。所述早期筛查标志物可以结合标准品用其进行人群中这两种代谢物基线的建立,可以基于正常对照的含量范围,对透明肾细胞癌患者进行早期诊断,其对后续临床应用研究的开展具有指导意义。(The invention discloses application of a metabolic marker of clear renal cell carcinoma in a renal cell carcinoma early screening and diagnosis product, wherein the metabolic marker is one or all of 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine. The early screening marker can be combined with a standard substance to establish a baseline of the two metabolites in a population, can be used for carrying out early diagnosis on a patient with the renal cell carcinoma cell based on the content range of a normal control, and has guiding significance for the development of subsequent clinical application research.)

1. The application of the metabolic marker of the clear renal cell carcinoma in the products for screening and diagnosing the early renal cell carcinoma is characterized in that the metabolic marker is one or all of 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine.

2. The use of claim 1, wherein the metabolic marker is a combined metabolic marker of 12, 13-bishydroxyoctadecenoic acid and 5-L-glutamyl-alanine.

3. The use of claim 1, wherein the metabolic marker 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine is significantly downregulated (below the level of healthy controls) in patients with clear renal cell carcinoma.

4. The use according to any one of claims 1 to 3, wherein the step of screening and diagnosing clear renal cell carcinoma using the metabolic markers is: (1) obtaining a subject plasma sample; (2) detecting the concentration of the one or more metabolic markers in a sample from the subject; (3) comparing the subject metabolite concentration to the metabolite concentration of a healthy control; (4) a decrease in the level of the metabolic marker and a decrease in the combined level thereof as compared to a healthy control indicates that the subject has clear renal cell carcinoma.

5. The use of claim 4, wherein the means for detecting the concentration of said one or more metabolic markers in the sample of the subject comprises mass spectrometry, nuclear magnetic resonance analysis, enzymatic assays.

6. The use of claim 5, wherein the method of detecting the concentration of said one or more metabolic markers in a sample from a subject is mass spectrometry, and said mass spectrometry is liquid chromatography-high resolution mass spectrometry.

7. The use of claim 6, wherein when determining metabolite levels using mass spectrometry, the step of obtaining a plasma sample may be followed by a metabolite extraction, protein removal step.

8. The use of claim 1, wherein the early screening and diagnostic product comprises a diagnostic formulation, kit or chip.

9. A kit or chip for assisting in early screening and diagnosis of renal cell carcinoma, wherein the kit or chip comprises a reagent for detecting the concentration of a marker group of 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine.

10. The kit or chip of claim 9, wherein the kit or chip comprises:

(1) and (3) standard substance: 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine, said standard specifically recognizing an antibody to 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine, respectively;

(2) plasma sample treatment fluid: the plasma sample processing solution is used for pretreating a plasma sample from a subject and comprises a methanol-chloroform mixed solution, dichloromethane added with 10% ethyl acetate or n-hexane solution.

Technical Field

The invention relates to the field of biological detection, in particular to application of a transparent renal cell carcinoma metabolic marker in renal cell carcinoma early screening and diagnosis products.

Background

Renal carcinoma (RCC) is the second most lethal urological tumor. Clinically, kidney cancer is diagnosed mainly by detection techniques such as ultrasound, computed tomography, and magnetic resonance imaging [1 ]. At present, the cost of the diagnosis means is high, and the diagnosis accuracy is low for smaller lesions. New diagnostic markers are yet to be developed.

Plasma is widely used as a diagnostic body fluid commonly used in clinic and is widely applied to research of kidney cancer markers. Plasma metabonomics is a hotspot for researching kidney cancer diagnosis markers at present due to the characteristics of high flux and high accuracy. There have been numerous studies to apply plasma metabolomics to the discovery of biomarkers in kidney cancer [2 ]. In 2016, plasma samples of 68 healthy subjects and 58 renal cancer patients are collected by Hong Zheng et al, and a nuclear magnetic resonance technology is adopted to characterize metabolome in the plasma, so that an early renal cancer diagnosis method based on a biomarker cluster is established. It is determined that the prediction accuracy of the combination of 7 metabolites (alanine, creatine, choline, isoleucine, lactic acid, leucine and valine) for healthy subjects can reach 91.30%, and the prediction accuracy of renal cancer patients can reach 94.74% [3 ]. Additional studies have shown that renal cell carcinogenesis is associated with dysregulation of lipid metabolism [4 ]. In 2017, Zhang, Y and the like research lipidomics of 45 primary clear renal cell carcinoma tissues, and observe intratumoral and intrasynovial heterogeneity of lipid accumulation in the same tumor grade, and as a result, the lipid accumulation of most invasive tumors is found to be low [5 ]. Previous studies used relatively small sample volumes, requiring the use of larger sample volumes for further validation of potential plasma markers. This is of great significance for the early diagnosis of renal cell carcinoma.

Disclosure of Invention

In order to realize early detection, early intervention and diagnosis and screening of renal cell carcinoma, the invention aims to provide application of the transparent renal cell carcinoma metabolic marker in renal cell carcinoma early screening and diagnosis products.

It is still another object of the present invention to provide a kit for assisting in the early screening and diagnosis of renal cell carcinoma.

High throughput plasma metabolomics has helped screen for more sensitive and specific markers of renal cancer. In the invention, 143 clear renal cell carcinoma blood plasma and 204 normal persons with matched age and sex are collected in total, and qualitative and quantitative analysis is carried out on the blood plasma metabolome by adopting a non-targeted combined targeted metabolome method. 29 potential biomarkers were screened by supervised clustering analysis on OPLS-DA, fold difference analysis and T-test analysis. Further data analysis shows that the combination of 2 metabolites has good differentiation on kidney cancer group and good clinical application prospect

In order to achieve the above objects, the present invention firstly provides the use of a metabolic marker of clear renal cell carcinoma, which is one or all of 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine, in a product for screening and diagnosing early renal cell carcinoma.

More preferably, the metabolic marker is a combined metabolic marker of 12, 13-dihydroxyoctadecenoic acid and 5-L-glutamyl-alanine.

In specific embodiments, the metabolic marker 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine is significantly down-regulated (below healthy control levels) in patients with clear renal cell carcinoma. Healthy controls refer to healthy individuals who do not have clear renal cell carcinoma and other diseases.

The metabolic marker screening is determined in a plasma sample of the subject.

Secondly, the steps of screening and diagnosing the clear renal cell carcinoma by using the metabolic markers are as follows: (1) obtaining a subject plasma sample; (2) detecting the concentration of the one or more metabolic markers in a sample from the subject; (3) comparing the subject metabolite concentration to the metabolite concentration of a healthy control; (4) a decrease in the level of the metabolic marker and a decrease in the combined level thereof as compared to a healthy control indicates that the subject has clear renal cell carcinoma.

The subject is a human.

Preferably, the method of detecting the concentration of said one or more metabolic markers in a sample from a subject comprises mass spectrometry, nuclear magnetic resonance analysis, enzymatic assays.

Preferably, the method of detecting the concentration of said one or more metabolic markers in a sample from a subject is mass spectrometry, said mass spectrometry being liquid chromatography-high resolution mass spectrometry.

Preferably, when determining metabolite levels using mass spectrometry, a metabolite extraction, protein removal step may also be included after the step of obtaining a plasma sample.

Preferably, the mass spectrometry is a primary full scan mode combined with a targeted secondary analysis. The mass spectrum full-scanning mode is to simultaneously acquire primary information of all small molecules within a mass range of 100m/z to 1000m/z, screen differential metabolites through multivariate statistical analysis, further perform targeted secondary fragmentation on the differential metabolites, and finally determine the differential molecules by combining a database secondary spectrogram.

Preferably, the early screening and diagnostic product comprises a diagnostic formulation, kit or chip.

Further, the invention also provides a kit or a chip for assisting in early screening and diagnosis of renal cell carcinoma, wherein the kit or the chip comprises a reagent for detecting the concentration of the marker groups of 12, 13-dihydroxyoctadecenoic acid or 5-L-glutamyl-alanine.

In one embodiment, the kit or chip comprises:

(1) and (3) standard substance: 12, 13-bishydroxyoctadecenoic acid, diaminopimelic acid or 5-L-glutamyl-alanine, said standard specifically recognizing an antibody to 12, 13-bishydroxyoctadecenoic acid or 5-L-glutamyl-alanine, respectively;

(2) plasma sample treatment fluid: the plasma sample processing solution is used for pretreating a plasma sample from a subject and comprises a methanol-chloroform mixed solution, dichloromethane added with 10% ethyl acetate or n-hexane solution.

The invention has the following beneficial effects:

the invention discloses a metabolic marker related to renal cell carcinoma, and further proves that the metabolite can be used as an early screening marker for detecting the renal cell carcinoma. The early screening marker can be combined with a standard substance to establish a baseline of the two metabolites in a population, can be used for carrying out early diagnosis on a patient with the renal cell carcinoma cell based on the content range of a normal control, and has guiding significance for the development of subsequent clinical application research.

Drawings

FIG. 1: clear renal cell carcinoma and control group plasma metabolism profile PCA classification.

FIG. 2: clear renal cell carcinoma and control group plasma metabolism profile OPLS-DA classification map.

FIG. 3: the combination of the two metabolites predicted the ROC curve for clear renal cell carcinoma in sample set 1.

FIG. 4 ROC curve for clear renal cell carcinoma samples of sample group 2, which are differentiated by the combination of two metabolites.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available.

However, it should be understood that these are exemplary only and not intended to limit the present invention, and that materials that are the same as or similar to the type, model, quality, nature, or function of the following reagents and instruments may be used in the practice of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

We used liquid chromatography-high resolution mass spectrometry (LC-MS) to detect metabolites in plasma by full scan mode and screened metabolites associated with clear renal cell carcinoma by multivariate statistical analysis. The identification of the marker is carried out by matching or resolving the secondary fragments by using a secondary targeting analysis method.