CN102662012B - Kit for screening colorectal adenomas (CRA) - Google Patents
Kit for screening colorectal adenomas (CRA) Download PDFInfo
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- CN102662012B CN102662012B CN201210141055.5A CN201210141055A CN102662012B CN 102662012 B CN102662012 B CN 102662012B CN 201210141055 A CN201210141055 A CN 201210141055A CN 102662012 B CN102662012 B CN 102662012B
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Abstract
The invention discloses a kit for screening colorectal adenomas (CRA). The kit for screening or screening CRA in an aided manner comprises a device for detecting the contents of phospholipids and a comparison card established by the contents of phospholipids, wherein the phospholipids include 16:0SM, 18:0SM, 14:0LPC, 16:0LPC, 18:2LPC, 18:1LPC, 18:0LPC, 20:4LPC, 20:0LPC, 22:6LPC and 22:0LPC. A molecular model for distinguishing healthy controls from CRA patients is established by taking saturated LPC, 20:4LPC and SM as markers, and the sensitivity and specificity of the model in distinguishing the CRA are respectively 89% and 80%.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of kit of examination knot adenomas.
Background technology
Knot adenomas (CRA) is a kind of benign lesion with canceration potential, is the one of colorectal polypus.Colorectal polypus refers to above mucous membrane, dashes forward to the neoplasm of enteric cavity, can have the base of a fruit, can be also that wide base is without the base of a fruit.The colorectal polypus of clinical diagnosis comprises tumprigenicity polyp and Non-neoplastic polyp on pathology, and knot adenomas belongs to tumprigenicity polyp.
New elaboration according to WHO about colorectal cancer definition, knot adenomas also can be described as knot rectum intraepithelial neoplasia (cin), refer to that Colorectal mucosa epithelium has the atypia on institutional framework and cytology, but this change does not break through muscularis mucosae, entering submucosa once break through mucous membrane flesh, is colorectal cancer (CRC).Most of colorectal cancers (>80%) cancerate from knot adenomas polyp clinically.Knot adenomas is generally admitted as the precancerous lesion of colorectal cancer (CRC), and the sequential theory of adenoma-cancer in this colorectal cancer process is widely accepted.Bibliographical information is also higher in the higher national colorectal cancer incidence rate of some knot adenomas incidences of disease, finds in time knot adenomas and extractd to make the incidence of colorectal cancer reduce by 85%.Other bibliographical informations, comprehensively examination excise the crowd of the above adenoma of 5mm, its colorectal cancer (CRC) incidence of disease reduction 76%-90%.Therefore, correctly diagnose and process knot adenomas there is important clinical meaning.
Knot adenomas conventional screening method clinically has: 1. ight soil occult blood examination, and the susceptibility of diagnosis CRC and polyp is 37.1% ~ 79.4%, specificity is 87.5%; 2. ight soil immunochemistry checks, the susceptibility of diagnosis CRA is 94.1%, and specificity is 87.5%; 3. faeces DNA is measured, and this technology is the DNA that checks adenoma and CRC cast-off cells in ight soil.Available reagent box can detect the DNA of kind more than 20 variation, as K-ras etc.But do not have a kind of independent genetic mutation to appear in all adenoma and CRC, so commercial kit can not be diagnosed whole adenomas and CRC.4. sigmoidoscopy, the easiest, but inspection is not comprehensive, less application; 5. total colonoscopy, application is at present maximum, but intestines mirror is not flawless, and >10mm adenoma rate of missed diagnosis can reach 6%-12%, colorectal cancer rate of missed diagnosis approximately 5%; 6. dual gas barium radiography, can check whole knot rectum, and diagnosis CRA susceptibility is 85%-97%.7. CT colon laminagraphy (CTC), the susceptibility that detects >6mm adenoma is 89%, and the elderly (more than 70 years old) because of the age increase can reduce radioactive susceptibility.
Phosphatide is not only and is formed biomembranous important component, some phospholipid molecules itself or its metabolic product are still as important signaling molecule, wherein lysophosphatidyl choline (lysophosphatidylcholine, LPC), Sphingosylphosphocholine (sphingosylphosphorylcholine, SPC), lysophosphatidic acid (lysophosphatidic acid, LPA), sphingosine-1-phosphate (sphingosine-1-phosphate, and sphingomyelins (sphingomyelin S1P), etc. SM) can be by the receptors bind of himself or metabolic product and cell surface, activate downstream signal path, participate in cell proliferation, migration, there are and be adsorbed on the regulation and control of interior multiple vital movement in form.These lipid molecules and metabolic product thereof and multiple physiology and pathology process are closely bound up.For example SPC and SM all can be converted into S1P, and LPC can generate LPA by ATX catalysis, and S1P and LPA are very remarkable to tumorigenic regulatory function as functional activity lipid molecule.Along with biologically active lipid molecule and cancer-related understanding deeply and the progress of lipid molecule detection means, some biologically active lipid molecules are considered to serve as the biomarker of early diagnosis of tumor.In view of important physiology and the pathology sense of biologically active phosphatide molecule, the present invention utilizes mass-spectrometric technique to detect the content of a series of biologically active phosphatide molecules in normal person and knot adenomas patient blood plasma, has set up the knot adenomas examination technology based on lipidomics.
Summary of the invention
The object of this invention is to provide the kit of a kind of evaluation, assistant identification, examination or auxiliary examination knot adenomas.
Kit provided by the invention, comprises that the comparison of setting up for detection of the device of content of phospholipid with by described content of phospholipid blocks;
Described phosphatide is 16:0SM, 18:0SM, 14:0LPC, 16:0LPC, 18:2LPC, 18:1LPC, 18:0LPC, 20:4LPC, 20:0LPC, 22:6LPC and 22:0LPC.
In mentioned reagent box, on the described comparison card of being set up by content of phospholipid, be provided with straight line, horizontal ordinate and ordinate;
Described straight line is 0.0093 × saturated LPC Zong Liang – 1225 × 20:4LPC%+0.02 × SM total amount=3.3;
Described horizontal ordinate is 0.0093 × saturated LPC Zong Liang –, 1225 × 20:4LPC%;
Described ordinate is SM total amount;
Described 20:4LPC% is the number percent that described 20:4LPC content accounts for unsaturated LPC total amount.
The above-mentioned comparison card of being set up by content of phospholipid also can be thought and be made up of straight line, horizontal ordinate and ordinate;
In mentioned reagent box, described SM total amount is the summation of described 16:0SM and described 18:0SM content;
Described saturated LPC total amount is the summation of described 14:0LPC, described 16:0LPC, described 18:0LPC, described 20:0LPC and described 22:0LPC content;
Described unsaturated LPC total amount is the summation of described 18:2LPC, described 18:1LPC, described 20:4LPC and described 22:6LPC content.
In mentioned reagent box, the described device for detection of content of phospholipid is LC-MS instrument.
In mentioned reagent box, the mobile phase that described detection adopts is that volume ratio is methyl alcohol, water, the ammonia water mixture of 90:10:0.1;
Described content of phospholipid is content of phospholipid in Blood plasma in vitro.
In mentioned reagent box, described kit is made up of the described device for detection of content of phospholipid and the described comparison card of being set up by described content of phospholipid.
The application of above-mentioned kit in the product of characterization, assistant identification, examination or auxiliary examination knot adenomas is also the scope of protection of the invention.
Of the present inventionly experiment showed, that a series of phospholipid molecules that contain choline group can be used as the molecular marker of distinguishing healthy population and knot adenomas patient, for example LPCs, 16:0SM and 18:0SM.Utilize saturated LPC, 20:4LPC, 16:0SM and 18:0SM to set up molecular model for mark, can distinguish normal healthy controls and knot adenomas patient, sensitivity and specificity reach respectively 89% and 80%.
Accompanying drawing explanation
Fig. 1 is saturated LPC, unsaturated LPC, total LPC and total SM(16:0SM+18:0SM) content of content in normal healthy controls group and knot adenomas patient blood plasma
Fig. 2 is for utilizing LPC and (16:0,18:0) SM to distinguish normal healthy controls and the model of tying adenomas patient in Chinese population
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The structure of the kit of embodiment 1, examination knot adenomas
One, the foundation of molecular model
1, blood collection, lipid molecule extract and detect
1) research object
All sample collections are in May, 2007-2010 year May, and sample is from hospital general of the Second Artillery Force of the entire PLA, Beijing Hospital, the second affiliated hospital of Peking University.Get patient blood on an empty stomach morning and carry out pre-service.This project is examined through Institutional Review Board, all subjects signed Informed Consent Forms.
Table 1 has been summarized the basic condition of research object: 240 experimenters are Chinese, and 65.4% is the male sex (157/240), and the ratio that the male sex accounts in normal healthy controls and adenoma patient is respectively 68% and 63%.Normal healthy controls, adenoma patient's age is respectively: 55.2+7.5,54.5+14.2, the sampling age does not have significant difference.
The basic condition of table 1 research object
2) plasma sample preparation, lipid molecule extract and detect
In the pipe that contains EDTA, collect above-mentioned 1) the in vitro whole blood of sample, room temperature (25 ℃) 1, centrifugal 15 minutes of 750g, is distributed into Blood plasma in vitro on the pipe of silicidation ,-80 degree Refrigerator stores, avoid multigelation.
The extraction of LPC, SPC and SM: add 0.5ml PBS(Hyclone company in glass centrifuge tube, SH30256.01B), add 100 μ l 12:0LPC (avanti polar lipids 855475P, 100mM), add 3ml methyl alcohol/chloroform (volume ratio is 2:1).In above-mentioned solution, add 10 μ l Blood plasma in vitro obtained above, after whirlpool mixes, place on ice 10 minutes.Add 1ml chloroform, 1.3ml distilled water, mixes, room temperature (25 ℃) 1, and centrifugal 10 minutes of 750g, takes off a layer organic phase, and nitrogen dries up, and dissolves by 1ml methanol/water (9:1), gets machine testing LPC, SPC and SM and each subclass content on 200 μ l.
Above-mentioned upper machine testing all adopts Mass Spectrometer Method system to be: Applied Biosystems Sciex 3200 QtrapTMmassspectrometer, Applied Biosystems Sciex, Ontario, Canada.Mobile phase is: methanol/water/ammoniacal liquor (90:10:0.1), the flow velocity of described detection content of phospholipid mobile phase is 0.2ml/min.
The standard items of a series of phosphatide are all purchased from Avanti Polar Lipids company, and the catalog number of portioned product is as shown in table 2 below:
The standard items of table 2 lipid
| Name | Catalogue N0. |
| 12：0LPC | 855475P |
| 14：0LPC | 855575P |
| 16：0LPC | 855675P |
| 18：2LPC | 790648P |
| 18：1LPC | 845875C |
| 18：0LPC | 855775C |
| 20：0LPC | 855777C |
| 20:4LPC | Synthesized by the said firm |
| 22：0LPC | 855779C |
| 22:6LPC | Synthesized by the said firm |
| SPC | 860600P |
| lyso-PAF | 878119C |
| 14：0LPA | 857120X |
| 16：0LPA | 857123P |
| 18：1LPA | 857130C |
| 18：0LPA | 857128X |
| S1P | 860492P |
| 16：0SM | 860061C |
| 18：0SM | 860062C |
2, experimental result
1) impact of sex on content of phospholipid in blood plasma
Due to masculinity proportion higher (65.4%) in whole 240 samples, the relation between our content to phospholipid molecule in blood plasma (μ M) and sex detects.Testing result is as shown in table 3, if carry out t check using p=0.01 as standard, in all blood plasma, the content of detected phospholipid molecule does not all have significant difference between masculinity and femininity.
The gender differences of content of phospholipid in table 3 blood plasma
2) mensuration of phospholipid molecule content in normal healthy controls and knot adenomas patient blood plasma
Detect the content (μ M) of following phospholipid molecule in whole 240 samples and subclass thereof: SPC, lyso-PAF, 14:0LPC, 16:0LPC, 18:2LPC, 18:1LPC, 18:0LPC, 20:4LPC, 20:0LPC, 22:6LPC, 22:0LPC, 16:0SM and 18:0SM.
Result is as shown in Fig. 1 and table 4, compared with normal healthy controls group, most LPC hypotypes, comprise 14:0LPC, 16:0LPC, 18:2LPC, 18:1LPC, 18:0LPC, 20:0LPC, 22:6LPC and 22:0LPC, content in knot adenomas patient blood plasma all significantly reduces, but the content of 22:4LPC raises in knot adenomas patient blood plasma.Saturated LPC, unsaturated LPC and total LPC content all significantly decline in knot adenomas patient blood plasma.In addition 16:0SM, 18:0SM and total SM content also all significantly decline (table 4, Fig. 1) in knot adenomas patient blood plasma.
The content of phospholipid molecule in table 4 normal healthy controls and knot adenomas patient blood plasma
3, set up the model of distinguishing normal healthy controls and knot adenomas patient
Detect the difference in group based on above-mentioned phospholipid molecule in difference, set up the molecular model of distinguishing normal healthy controls and knot adenomas patient:
Because the content of 16:0SM and 18:0SM in knot adenomas patient blood plasma all significantly declines (Fig. 1), therefore SM will become the important indicator of distinguishing adenoma and normal control or adenoma and tumor patient.
The formula of model: [0.0093 × saturated LPC Zong Liang – 1225 × 20:4LPC%+0.02 × SM total amount=3.3]
SM total amount is the summation of 16:0SM and 18:0SM content;
Saturated LPC total amount is the summation of 14:0LPC, 16:0LPC, 18:0LPC, 20:0LPC and 22:0LPC content;
20:4LPC% is the number percent that 20:4LPC content accounts for unsaturated LPC total amount.
Unsaturated LPC total amount is the summation of 18:2LPC, 18:1LPC, 20:4LPC and 22:6LPC content.
Two, the structure of examination knot adenomas kit
1) set up comparison card
Comparison card is made up of formula (straight line), horizontal ordinate and the ordinate of model;
Horizontal ordinate is 0.0093 × saturated LPC Zong Liang –, 1225 × 20:4LPC%;
Ordinate is SM total amount;
2) structure of kit
Kit comprises comparison card and LC-MS instrument Applied Biosystems Sciex 3200QtrapTM massspectrometer, Applied Biosystems Sciex, Ontario, Canada.
The application of the kit of embodiment 2, examination knot adenomas
1, sensitivity and the specific detection of the kit of examination knot adenomas
Get 120 knot adenomas (all passing through clinical identification) and 120 routine colorectal carcinoma patients' (all making a definite diagnosis through clinical identification) blood; According to embodiment 1 one 1 2) method carry out Blood plasma in vitro preparation, lipid extraction, LC-MS instrument and detect, obtain 16:0SM, 18:0SM, 14:0LPC, 16:0LPC, 18:2LPC, 18:1LPC, 18:0LPC, 20:4LPC, 20:0LPC, 22:6LPC and 22:0LPC content;
Take 0.0093 × saturated LPC Zong Liang –, 1225 × 20:4LPC% as horizontal ordinate, take SM total amount as ordinate, make straight line with the formula [0.0093 × saturated LPC Zong Liang – 1225 × 20:4LPC%+0.02 × SM total amount=3.3] of model, map.
Result is as shown in the right figure of Fig. 2, and in the blood plasma for each adenoma patient (a) and normal healthy controls (h), (0.0093 × saturated LPC total amount-1225 × 20:4LPC%) maps to SM total amount.Figure cathetus is: [0.0093 × saturated LPC Zong Liang – 1225 × 20:4LPC%+0.02 × SM total amount=3.3].It is adenoma patient that straight line left side is determined, and right side is judged to be health.Amount to 120 normal healthy controls and 120 knot adenomas patients.20:4LPC% scope in formula is 0-100%, represents that 20:4LPC content accounts for the number percent of all unsaturated LPC total amounts.
By analysis, have 134 samples in straight line left side, wherein 107 samples derive from knot adenomas patient (120), and 27 samples derive from normal healthy controls (120); 107 patients in 120 colorectal cancer patients are by correct diagnosis (in the left side of straight line, and survey and make a definite diagnosis through intestines microscopy).Therefore, the sensitivity of this model is the number 107/ adenoma patient sum 120=89% being diagnosed in 89%(adenoma patient) and specificity be the number 134=80% that number 107/ that 80%(is diagnosed as actual adenoma patient in adenoma patient is diagnosed as adenoma patient).
The left figure of Fig. 2 serve as reasons differentiation knot normal healthy controls that right figure sets up and knot adenomas patient's ROC curve.
If the sensitivity of this model is set in to 85%, specificity can be upgraded to 82%.Utilize this formula, in 120 adenoma patients, 18 patients are misdiagnosed as health, and 22 normal healthy controls are misdiagnosed as adenoma patient.That is, can set up and distinguish normal healthy controls and the model of tying adenomas patient in Chinese population as mark take LPC and SM, this model has good sensitivity and specificity.
Claims (6)
1. a kit for examination or auxiliary examination knot adenomas, comprises that the comparison of setting up for detection of the device of content of phospholipid with by described content of phospholipid blocks;
Described phosphatide is 16:0 SM, 18:0 SM, 14:0 LPC, 16:0 LPC, 18:2 LPC, 18:1 LPC, 18:0 LPC, 20:4 LPC, 20:0 LPC, 22:6 LPC and 22:0 LPC;
On the described comparison card of being set up by content of phospholipid, be provided with straight line, horizontal ordinate and ordinate;
Described straight line is 0.0093 × saturated LPC Zong Liang – 1225 × 20:4 LPC%+0.02 × SM total amount=3.3;
Described horizontal ordinate is 0.0093 × saturated LPC Zong Liang –, 1225 × 20:4 LPC%;
Described ordinate is SM total amount;
Described 20:4 LPC% is the number percent that described 20:4 LPC content accounts for unsaturated LPC total amount.
2. kit according to claim 1, is characterized in that:
Described SM total amount is the summation of described 16:0 SM and described 18:0 SM content;
Described saturated LPC total amount is the summation of described 14:0 LPC, described 16:0 LPC, described 18:0 LPC, described 20:0 LPC and described 22:0 LPC content;
Described unsaturated LPC total amount is the summation of described 18:2 LPC, described 18:1 LPC, described 20:4 LPC and described 22:6 LPC content.
3. kit according to claim 1 and 2, is characterized in that:
The described device for detection of content of phospholipid is LC-MS instrument.
4. kit according to claim 3, is characterized in that:
The mobile phase that described detection adopts is that volume ratio is methyl alcohol, water, the ammonia water mixture of 90:10:0.1;
Described content of phospholipid is content of phospholipid in Blood plasma in vitro.
5. kit according to claim 1, is characterized in that:
Described kit is made up of the described device for detection of content of phospholipid and the described comparison card of being set up by described content of phospholipid.
6. the application of arbitrary described kit in the product of characterization, assistant identification, examination or auxiliary examination knot adenomas in claim 1-5.
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| WO2016119190A1 (en) * | 2015-01-30 | 2016-08-04 | Bgi Shenzhen | Biomarkers for colorectal cancer related diseases |
| CN106370834B (en) * | 2016-09-07 | 2018-01-19 | 辽宁润生康泰生物医药科技有限公司 | A kind of kit for the carcinoma of the rectum that come to an end using common molecular sieve |
| CN114544826B (en) * | 2020-11-24 | 2023-12-08 | 重庆医科大学 | Application of reagent for detecting histidine in blood plasma in preparation of depression detection kit |
| CN116324413A (en) * | 2022-11-07 | 2023-06-23 | 中精普康(北京)医药科技有限公司 | Biomarkers and methods for detecting colorectal cancer or colorectal adenoma |
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| CN101688239A (en) * | 2007-02-12 | 2010-03-31 | 约翰·霍普金斯大学 | The early detection of colorectal carcinoma and prognosis |
| FR2919062B1 (en) * | 2007-07-19 | 2009-10-02 | Biomerieux Sa | METHOD OF DETERMINING AMINOACYLASE 1 FOR IN VITRO DIAGNOSIS OF COLORECTAL CANCER. |
| US9726670B2 (en) * | 2007-07-19 | 2017-08-08 | Biomerieux | Method for the assay of liver fatty acid binding protein, ACE and CA 19-9 for the in vitro diagnosis of colorectal cancer |
| CN101498700A (en) * | 2009-02-16 | 2009-08-05 | 桑建利 | Production and use of reagent kit for measurement of lipide molecule content in human blood sample and colorectal carcinoma diagnosis |
| FR2944019B1 (en) * | 2009-04-03 | 2011-04-22 | Biomerieux Sa | METHOD FOR DETERMINING PRODEFENSIN-A6 FOR IN VITRO DIAGNOSIS OF COLORECTAL CANCER |
| CN102147396B (en) * | 2010-02-10 | 2014-01-01 | 桑建利 | Preparation and application of kit used for testing content of lipid molecules in human blood sample and diagnosing colorectal adenomas |
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