CN112129858B - Kit and system based on phospholipid carbon-carbon double bond isomer marker analysis - Google Patents
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Abstract
The invention provides a reagent kit based on phospholipid carbon-carbon double bond isomer marker analysis, and the reagent kit is used for diagnosing brain glioma. By determining the relative content of the isomers with the carbon-carbon double bond of the phospholipid, the brain glioma and the type thereof can be quickly and accurately diagnosed, and a foundation is laid for scientific research and clinical diagnosis and analysis of the brain glioma.
Description
Technical Field
The present invention relates to the field of disease diagnosis. In particular, the invention relates to kits and systems based on phospholipid carbon-carbon double bond isomer marker assays.
Background
Gliomas are the most common primary brain tumors, accounting for approximately 27% of all primary brain and other central nervous system tumors, and approximately 81% of all malignant brain tumors. Isocitrate Dehydrogenase (IDH) gene mutations are present in the vast majority of gliomas II and III classified by the world health organization, i.e., IDH mutant gliomas. In addition, clinical studies have shown that patients who have had their greatest resection of an IDH mutant glioma site during surgery have better prognostic performance than IDH wild-type gliomas (i.e., gliomas that are not caused by IDH mutations). Therefore, identification of the mutation type of brain glioma is of great significance for surgical protocol selection, prognosis protocol formulation and development of novel therapeutic approaches.
Genomics analysis is currently the most commonly used molecular typing method for brain gliomas, such as IDH mutation sanger sequencing and IHC staining of the R132H antibody to identify IDH mutations. In addition, due to the abnormal accumulation of 2-hydroxyglutaric acid (2-HG) in glioma caused by IDH mutation, the judgment of IDH mutant glioma can be carried out by detecting 2-HG by magnetic resonance spectrum or mass spectrum.
Phospholipids are the main components of cell membranes, are more abundant in brain tissues, and play an important role in the functions of intercellular signal transmission and the like in addition to the material basis of the nervous system.
However, there is currently no study of phospholipids as markers for the determination of gliomas.
Disclosure of Invention
The present invention aims to solve, at least to some extent, the technical problems of the prior art. Therefore, the invention provides a kit and a system based on phospholipid carbon-carbon double bond isomer marker analysis, the application of the phospholipid carbon-carbon double bond isomer as a marker and the application of a reagent for detecting the relative content of the phospholipid carbon-carbon double bond isomer in the preparation of the kit.
In one aspect of the invention, the invention provides a kit based on analysis of phospholipid carbon-carbon double bond isomer markers. According to an embodiment of the present invention, the kit is used for diagnosing brain glioma. The inventor finds that the brain glioma and the type thereof can be quickly and accurately diagnosed by detecting the relative content of the isomers with the carbon-carbon double bond of the phospholipid, and lays a foundation for scientific research and clinical diagnosis and analysis of the brain glioma.
According to an embodiment of the present invention, the above kit based on phospholipid carbon-carbon double bond isomer marker analysis may further have the following additional technical features:
according to an embodiment of the invention, the kit includes, but is not limited to: a kit based on photochemical derivatization and mass spectrometry, a kit based on peroxide derivatization and mass spectrometry, a kit based on ozone-induced resolution mass spectrometry and a kit based on ultraviolet light dissociation mass spectrometry. Therefore, the kit can be used for quickly and accurately diagnosing the brain glioma and the type thereof.
According to an embodiment of the invention, the isomers of phospholipid carbon-carbon double bonds are selected from phospholipids containing 16:1 or 18:1 fatty acid chains, the phospholipids containing 16:1 or 18:1 fatty acid chains comprising: PC 16: 0-16: 1 (. DELTA.6), PC 16:1 (. DELTA.6) -16: 1 (. DELTA.6), PC 16: 0-18: 1 (. DELTA.8), PC 17: 0-18: 1 (. DELTA.8) or PE 18: 0-18: 1 (. DELTA.8). The inventor finds that the relative contents of the phospholipid carbon-carbon double bond isomers are remarkably different between normal tissues and brain glioma tissues, so that the brain glioma can be accurately diagnosed by determining the relative contents of the phospholipid carbon-carbon double bond isomers.
It should be noted that the "relative content" described in the present invention refers to the relative content of the amount of the phospholipid having a specific double bond position in the isomers having a carbon-carbon double bond of the phospholipid, based on the total amount of the phospholipid. For example, "relative content of PC 16:0_16:1(Δ 6)" refers to the relative content of phospholipid having Δ 6 carbon-carbon double bond in PC 16:0_16:1 to the sum of all carbon-carbon double bond isomers (Δ 6+ Δ 7+ Δ 9) in PC 16:0_16:1, i.e., the relative content is PC 16:0_16:1(Δ 6)/[ PC 16:0_16:1(Δ 6) + PC 16:0_16:1(Δ 7) + PC 16:0_16:1(Δ 9) ].
According to an embodiment of the present invention, the kit is used for diagnosing normal tissue or brain glioma tissue, or IDH mutant glioma tissue or IDH wild type glioma tissue.
According to an embodiment of the present invention, the diagnosis includes preoperative diagnosis, intraoperative tissue type differentiation, and prognostic diagnosis.
According to the embodiment of the invention, when the relative content of PC 16: 0-16: 1 (delta 6) in the sample to be tested is larger than that of the normal tissue, the sample to be tested is an indication that the sample to be tested is derived from IDH mutant glioma tissue; when the relative content of PC 16: 1-16: 1 (delta 6) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue; when the relative content of PC 16: 0-18: 1 (delta 8) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue; when the relative content of PC 17: 0-18: 1 (delta 8) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue; when the PE 18 in the sample to be tested is in a ratio of 0 to 18: the relative content of 1 (delta 8) is greater than that of normal tissues, and is an indication that the sample to be tested is derived from IDH mutant glioma tissues.
According to an embodiment of the present invention, when the PC 16: 0-18: 1(Δ 9/Δ 11) in the test sample is lower than that in the normal tissue, it is an indication that the test sample is derived from glioma tissue; when PC 18: 0-18: 1 (delta 9/delta 11) in the test sample is lower than that in the normal tissue, the result is an indication that the test sample is derived from glioma tissue; when PI 18: 0-18: 1 (delta 9/delta 11) in the test sample is lower than that in the normal tissue, the result is an indication that the test sample is derived from glioma tissue.
Specifically, when the relative content of PC 16: 0-18: 1 (delta 9/delta 11), PC 18: 0-18: 1 (delta 9/delta 11) and/or PI 18: 0-18: 1 (delta 9/delta 11) in the test sample is lower than that in the normal tissue, the test sample is determined to be from the glioma tissue. On the basis, the glioma tissues are further determined to be IDH wild type or IDH mutant glioma tissues, which are specifically as follows: the measurement of the ratio of PC 16: 0-16: 1 (. DELTA.6), PC 16: 1-16: 1 (. DELTA.6), PC 16: 0-18: 1 (. DELTA.8), PC 17: 0-18: 1 (. DELTA.8) and PE 18: 0-18: 1 (delta 8), when the relative content of PC 16: 0-16: 1 (delta 6), PC 16: 1-16: 1 (delta 6), PC 16: 0-18: 1 (delta 8), PC 17: 0-18: 1 (delta 8) and/or PE 18: 0-18: 1 (delta 8) in the sample to be detected is greater than that of normal tissue, determining that the sample to be detected is from IDH mutant glioma tissue, and if the relative content is less than or equal to that of the normal tissue, determining that the sample to be detected is from IDH wild type glioma tissue.
In another aspect of the invention, the invention features a system based on analysis of phospholipid carbon-carbon double bond isomer markers. According to an embodiment of the invention, the system comprises: an extraction device for extracting lipids in brain tissue; the measuring device is connected with the extracting device and is used for measuring the relative content of isomers with carbon-carbon double bonds in the phospholipid in the lipid; and the determining device is connected with the measuring device and is used for determining whether the phospholipid carbon-carbon double bond isomer is derived from the brain glioma or not and determining the type of the brain glioma based on the relative content of the phospholipid carbon-carbon double bond isomer. Therefore, whether the sample to be detected is from the brain glioma and the type of the brain glioma can be quickly and accurately determined by using the system provided by the embodiment of the invention.
In yet another aspect of the invention, the invention proposes the use of isomers of phospholipid carbon-carbon double bonds as markers. According to an embodiment of the present invention, the marker is used for determining whether the sample to be tested is derived from brain glioma. The inventor finds that the phospholipid carbon-carbon double bond isomer is used as a marker, and whether a sample to be detected is from the brain glioma and the type of the brain glioma can be quickly and accurately determined by determining the phospholipid carbon-carbon double bond isomer, so that a foundation is laid for scientific research and clinical diagnosis and analysis of the brain glioma.
In another aspect of the invention, the invention provides the use of a reagent for detecting the relative content of isomers with carbon-carbon double bonds in phospholipid in the preparation of a kit. According to an embodiment of the present invention, the kit is used for diagnosing brain glioma. The inventor finds that the relative content of the isomers of the phospholipid carbon-carbon double bond is used as a marker, and whether a sample to be detected is from the brain glioma and the type of the brain glioma can be quickly and accurately determined by determining the relative content of the isomers of the phospholipid carbon-carbon double bond, so that the method lays a foundation for scientific research and clinical diagnosis and analysis of the brain glioma.
The invention has the advantages that:
1. the biomarker, namely the phospholipid carbon-carbon double bond isomer, disclosed by the invention is rich in content in human brain and easy to detect, and the relative content of the isomer can be used for typing (IDH mutant glioma or IDH wild glioma) and diagnosing brain glioma.
2. The biomarker, namely the phospholipid carbon-carbon double bond isomer, disclosed by the invention does not need to carry out immunoassay or gene analysis, and can carry out rapid analysis by means of mass spectrometry and the like.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a graph of distribution of different classes of phospholipids in normal brain tissue;
FIG. 2 is a mass spectrum for analysis of the composition of phospholipid chains and carbon-carbon double bond positions in glioma tissues;
FIG. 3 is a graph showing the relative content of the 16:1 (. DELTA.6) isoforms in normal brain tissue, IDH wild type and IDH mutant glioma tissues in the corresponding 16:1 lipid-containing lipid; p values were calculated by student t-test, representing P <0.05, P <0.01, Normal tissue, and IDH wild-type glioma tissue;
FIG. 4 is a graph showing the relative content of the 18:1 (. DELTA.8) -containing isoform in normal brain tissue, IDH wild-type and IDH mutant glioma tissues in the corresponding 18: 1-containing lipid; p values were calculated by student's t test, representing P <0.05, P <0.01, P < 0.001;
FIG. 5 is a graph of the ratio of the 18:1 (. DELTA.9) isomer to the 18:1 (. DELTA.11) isomer in normal brain tissue, IDH wild type and IDH glioma tissue; p values were calculated by student t-test, representing P <0.05, P <0.01, P < 0.001.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
1. Extraction of full lipids from brain tissue
70mg of the tissue sample was placed in a 10mL centrifuge tube and 1mL of deionized water was added. Tissue samples were homogenized by a hand-held homogenizer at 40000Hz for 5 minutes, then mixed with 1mL methanol, 1mL water and 2mL chloroform for liquid-liquid extraction, and after vortexing for about 5 minutes, the mixture was centrifuged at 11269g for 8 minutes. The centrifuged bottom solution was collected and transferred to a new 10mL centrifuge tube. The above extraction process was repeated once. The chloroform layers from the two extractions were combined and dried under a stream of nitrogen. Finally, the extract was redissolved in 1mL of methanol solution, shaken well and stored at-20 ℃ for analysis.
2. Determining distribution information of phospholipids in lipid extract of brain tissue
And (3) performing liquid chromatography-mass spectrometry analysis on the extracted lipid sample, and determining the phospholipid subclass information, the total number of lipid chain carbon atoms and the unsaturation degree through neutral loss scanning or precursor ion scanning. The results are shown in FIG. 1.
3. Determining fatty acid chain composition information of phospholipids in lipid extracts of brain tissue
And performing liquid chromatography-mass spectrometry on the extracted lipid sample, and obtaining fragment information of the fatty acid chains by collision induced analysis in a negative ion mode.
4. Determining the position information of the carbon-carbon double bond of phospholipid in brain tissue lipid extract and the relative content of isomers
And analyzing the carbon-carbon double bond information of the phospholipid in the brain tissue lipid extract by adopting an online photochemical derivatization-liquid chromatography-tandem mass spectrometry method. And analyzing in a positive ion mode, and obtaining fragment information related to the carbon-carbon double bond through collision induced analysis. The relative isomer content was calculated from the fragment intensity information. The results are shown in FIG. 2.
FIG. 3 is a graph showing the relative content of phospholipid C-C double bond isomers in normal brain tissue, IDH wild type and IDH mutant brain glioma tissue. The relative content of the isomers at the positions of the carbon-carbon double bonds of the phospholipid is obviously different between normal brain tissue-IDH mutant brain tissue and IDH wild brain tissue-IDH mutant brain tissue by student t detection analysis of the obtained data. The relative content of these isoforms can be used to identify IDH mutant gliomas.
FIG. 4 is a ratio analysis of phospholipid C-C double bond isomers 18:1 (. DELTA.9)/18: 1 (. DELTA.11) in normal brain tissue, IDH mutant and IDH glioma tissue. The ratio of isomers at the position of the carbon-carbon double bond of the phospholipid is obviously different between normal brain tissue-IDH mutant brain tissue and normal brain tissue-IDH wild brain tissue by student t detection analysis of the obtained data. The relative amounts of these isomers can be used to identify glioma tissues.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (4)
1. A system based on analysis of phospholipid carbon-carbon double bond isomer markers, comprising:
an extraction device for extracting lipids in brain tissue;
the measuring device is connected with the extracting device and is used for measuring the relative content of isomers with carbon-carbon double bonds in the phospholipid in the lipid;
a determining device connected with the measuring device and used for determining whether the phospholipid carbon-carbon double bond isomer is originated from the brain glioma or not and determining the type of the brain glioma based on the relative content of the phospholipid carbon-carbon double bond isomer;
the isomers of the phospholipid carbon-carbon double bond are selected from phospholipids containing 16:1 or 18:1 fatty acid chains, and the phospholipids containing the 16:1 or 18:1 fatty acid chains comprise: PC 16: 0-16: 1(Δ 6), PC 16:1(Δ 6) _16:1(Δ 6), PC 16: 0-18: 1(Δ 8), PC 17: 0-18: 1(Δ 8), or PE 18: 0-18: 1(Δ 8);
when the relative content of PC 16: 0-16: 1 (delta 6) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the relative content of PC 16: 1-16: 1 (delta 6) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the relative content of PC 16: 0-18: 1 (delta 8) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the relative content of PC 17: 0-18: 1 (delta 8) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the PE 18 in the sample to be tested is in a ratio of 0 to 18:1(Δ 8) is greater than normal tissue and is indicative that the test sample is derived from IDH mutant glioma tissue;
when the PC 16: 0-18: 1 (delta 9/delta 11) in the sample to be tested is lower than that in the normal tissue, the indication that the sample to be tested is derived from the glioma tissue is given;
when PC 18: 0-18: 1 (delta 9/delta 11) in the test sample is lower than that in the normal tissue, the result is an indication that the test sample is derived from glioma tissue;
when PI 18: 0-18: 1 (delta 9/delta 11) in the test sample is lower than that in the normal tissue, the result is an indication that the test sample is derived from glioma tissue;
the relative content is the relative content of the number of phospholipids with a certain double bond position in the isomers of the phospholipid carbon-carbon double bonds in the total number of the phospholipids.
2. The application of the reagent for detecting the relative content of the phospholipid carbon-carbon double bond isomer in the preparation of the kit is characterized in that the kit is used for diagnosing brain glioma;
the isomers of the phospholipid carbon-carbon double bond are selected from phospholipids containing 16:1 or 18:1 fatty acid chains, and the phospholipids containing the 16:1 or 18:1 fatty acid chains comprise: PC 16: 0-16: 1(Δ 6), PC 16:1(Δ 6) _16:1(Δ 6), PC 16: 0-18: 1(Δ 8), PC 17: 0-18: 1(Δ 8), or PE 18: 0-18: 1(Δ 8);
when the relative content of PC 16: 0-16: 1 (delta 6) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the relative content of PC 16: 1-16: 1 (delta 6) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the relative content of PC 16: 0-18: 1 (delta 8) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the relative content of PC 17: 0-18: 1 (delta 8) in the sample to be detected is larger than that of the normal tissue, the relative content is an indication that the sample to be detected is derived from IDH mutant glioma tissue;
when the PE 18 in the sample to be tested is in a ratio of 0 to 18:1(Δ 8) is greater than normal tissue and is indicative that the test sample is derived from IDH mutant glioma tissue;
when the PC 16: 0-18: 1 (delta 9/delta 11) in the sample to be tested is lower than that in the normal tissue, the indication that the sample to be tested is derived from the glioma tissue is given;
when PC 18: 0-18: 1 (delta 9/delta 11) in the test sample is lower than that in the normal tissue, the result is an indication that the test sample is derived from glioma tissue;
when PI 18: 0-18: 1 (delta 9/delta 11) in the test sample is lower than that in the normal tissue, the result is an indication that the test sample is derived from glioma tissue;
the sample to be detected is brain tissue;
the relative content is the relative content of the number of phospholipids with a certain double bond position in the isomers of the phospholipid carbon-carbon double bonds in the total number of the phospholipids.
3. The use according to claim 2, wherein the kit comprises but is not limited to: a kit based on photochemical derivatization and mass spectrometry, a kit based on peroxide derivatization and mass spectrometry, a kit based on ozone-induced resolution mass spectrometry and a kit based on ultraviolet light dissociation mass spectrometry.
4. The use according to claim 2, wherein the diagnosis comprises preoperative diagnosis, intraoperative tissue type differentiation and prognostic diagnosis.
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