WO2020082642A1

WO2020082642A1 - Grading model for detecting benign and malignant degree of pancreatic tumor, and use thereof

Info

Publication number: WO2020082642A1
Application number: PCT/CN2019/074351
Authority: WO
Inventors: 成彤; 周宁
Original assignee: 立森印迹诊断技术有限公司; 李星
Priority date: 2018-10-26
Filing date: 2019-02-01
Publication date: 2020-04-30
Also published as: CN111100930B; CN111100930A

Abstract

A grading model for detecting a pancreatic tumor, and the use thereof. Said model grades changes in imprinted genes in the pancreatic tumor by calculating the imprinted gene deletion expression, abnormal imprinted gene copy number expression, and total imprinted gene expression. The grading model and device for detecting a pancreatic tumor shows, in an intuitive method, the expression of imprinted gene deletion on tissues and cell samples of a patient with a pancreatic tumor, objectively, intuitively, early, and accurately detecting changes in imprinted genes by means of an in-situ imprint gene marking method, providing a quantified model, and making a huge contribution to the diagnosis of pancreatic tumors.

Description

A grading model for detecting benign and malignant degree of pancreatic tumor and its application

Technical field

The present disclosure relates to the field of biotechnology, for example, the field of gene diagnosis, such as a grading model and its application, for example, a grading model for detecting the degree of pancreatic tumors and its application, for example, a group of imprinted genes in the detection of benign and malignant pancreatic tumors The grading model in the degree and the device it constitutes.

Background technique

Pancreatic cancer is a malignant tumor with a high degree of malignancy and a 5-year survival rate of less than 5%. It is one of the malignant tumors with the fastest progress and the worst prognosis. Every year, about 383,000 people in the world are diagnosed with pancreatic cancer, with 33 million deaths. There are 66,000 new cases and 64,000 deaths in China every year, and the incidence rate is still increasing year by year.

There are more difficulties in the early diagnosis of pancreatic cancer. 80% of pancreatic cancer patients are already in advanced stage at the time of treatment and cannot be operated on. The symptoms of early pancreatic cancer are similar to pancreatitis, not typical, and are often easily overlooked by patients. Due to the special anatomical location of the pancreas, ultrasound examination is difficult. At the same time, the pancreas does not have a direct conduit to the outside of the body and cannot be examined by endoscopy. Current pancreatic cancer tumor markers still have the problem of low sensitivity and specificity. Therefore, a more sensitive and accurate detection method is urgently needed to improve the early diagnosis rate of pancreatic cancer.

The diagnosis of benign and malignant cells by traditional pathology is based on the relationship between cell size, morphology, infiltration and surrounding cell tissue. It has great limitations on the discovery of early changes in cells (cancers), so cancer diagnosis methods at the molecular level have once become a research hotspot. As people continue to conduct in-depth research in the field of molecular biology, more and more molecular detection techniques are used in cancer diagnosis.

Cancer is caused by uncontrolled cell growth / division caused by epigenetic changes and gene mutations that accumulate over time. Traditional pathological diagnosis is based on variations in the size, morphology, and structure of cells and tissues to make a diagnosis of benign and malignant pancreatic tumors. With the development and deepening of molecular biology, more and more molecular detection techniques are applied to the detection of pancreatic cancer. From the analysis of the development of cancer, the changes at the molecular level (epigenetics and genetics) are much earlier than the changes in cell morphology and tissue structure. Therefore, molecular biology tests are more sensitive to early detection of cancer.

Based on the above reasons, the current pancreatic cancer diagnosis requires a new detection system and detection model. Based on the biopsy samples of the patient, the molecular marker changes at the cell level of pancreatic cancer are analyzed to provide more accurate pre-diagnosis and diagnosis information.

Summary of the invention

In view of the deficiencies of the existing technology and actual needs, the present disclosure provides a grading model for detecting the degree of pancreatic tumor benign and malignant and its application. To achieve the above objectives, the present invention adopts the following technical solutions:

In the first aspect, the present disclosure provides a imprinted gene grading model for pancreatic tumors, which calculates the total expression of imprinted genes, the expression of imprinted genes, and the abnormal expression of imprinted gene copy number in pancreatic cancer. Changes classify the expression of imprinted genes;

Wherein, the imprinting gene is any one or a combination of at least two of Z1, Z5, Z10, Z11, or Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z5 is Mest, and the imprinting gene Z10 is Gatm The imprinting gene Z11 is Grb10, and the imprinting gene Z16 is Snrpn / Snurf.

The deletion of the imprint (trace) refers to the activation (demethylation) of the allele in the imprint (trace) gene that was originally in a silent state, which is the most common and early epigenetic change in cancer, and This feature can be used as a pathological marker. Relatively speaking, in the detection of healthy cells, the percentage of imprinted deletion is very low. The imprinted gene and imprinted gene have the same concept at the same time, meaning the same meaning and can be replaced.

The inventors found that by calculating the expression level of the deletion of the imprinted gene and the abnormal expression of the imprinted gene copy number of any imprinted gene of Z1, Z5, Z10, Z11 and Z16 in pancreatic tumors, the diagnostic sensitivity for pancreatic cancer can reach more than 86.7% .

In an embodiment of this document, if only one imprinting gene is detected in the preliminary detection, any one of Z1, Z5, Z10, Z11, and Z16 may be detected.

In an embodiment of this document, if only one imprinting gene is detected in the preliminary detection, any one of Z1, Z11, or Z16 may be detected.

In an embodiment of this document, if only one imprinted gene is detected in the preliminary detection, Z11 or Z16 may be detected.

The inventor found that if a Z1 imprinting gene is detected alone, the diagnostic sensitivity for pancreatic cancer can reach 88.9%, if a Z5 imprinting gene is detected alone, the diagnostic sensitivity for pancreatic cancer can reach 86.7%, if a Z10 imprint is detected alone Gene, the diagnostic sensitivity of pancreatic cancer can reach 86.7%, if a single Z11 imprinting gene is detected, the diagnostic sensitivity of pancreatic cancer can reach 91.1%, if a single Z16 imprinting gene is detected, the diagnostic sensitivity of pancreatic cancer can be Reached 91.1%.

In an embodiment of this document, the method for calculating the imprinted gene by the model is: if the combination of two imprinted genes of the imprinted gene is detected, the combination may be any two of Z1, Z5, Z10, Z11, and Z16, preferably It is the combination of Z1 and Z10, the combination of Z5 and Z11, the combination of Z10 and Z16 or the combination of Z11 and Z16.

The inventor found that by calculating the total expression level of two or more imprinted genes, the expression level of the imprinted gene deletion and the abnormal expression of the imprinted gene copy number, the sensitivity can be further improved, and the imprinted genes Z1, Z5, Z10, Z11 and Z16 can be detected The combination of any two imprinted genes in pancreatic cancer can achieve a diagnostic sensitivity of more than 93.3%. When detecting the combination of Z1 and Z10, and the combination of Z11 and Z16, the diagnostic sensitivity of pancreatic cancer can reach 97.8%. When the combination of Z5 and Z11, and the combination of Z10 and Z16, the diagnostic sensitivity for pancreatic cancer can reach more than 99.0%.

In an embodiment herein, the imprinting gene further includes any one or a combination of at least two of Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14, or Z15; wherein, the imprinting gene Z2 Is Igf2, the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z6 is Plagl1, the imprinting gene Z8 is Dcn, the imprinting gene Z9 is Dlk1, and the imprinting gene Z12 is Peg3 The imprinting gene Z13 is Sgce, the imprinting gene Z14 is Slc38a4, and the imprinting gene Z15 is Diras3.

The inventors found that adding Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14, Z15 genes to the combined diagnosis using the Z1, Z5, Z10, Z11, and Z16 gene tests will not only help increase The accuracy of the detection, and the addition of other probes to assist in diagnosis can further avoid the occurrence of false positives, and can further improve the detection accuracy, so that all pancreatic tumor samples can be accurately graded and judged.

In an embodiment of the present invention, the method for calculating the imprinting genes by the model is: calculating the combination of imprinting genes, calculating Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Combination of Z15 and Z16 genes.

The deletion of the imprinted gene is that there are two red / brown markers in the nucleus of the cell after hematoxylin staining. The abnormal number of the imprinted gene is that there are more than two red / brown markers in the nucleus of the cell after hematoxylin is stained. The abnormal copy number is due to the abnormal gene replication of cancer cells, which results in the expression of this gene as a triploid or even higher polyploid.

The imprinted gene and the imprinted gene have the same concept at the same time, which means the same meaning and can be replaced.

In an embodiment of this document, the formula for calculating the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression level of the imprinted gene copy number is as follows:

Total expression level = (b + c + d) / (a + b + c + d) × 100%;

Normal imprinted gene expression = b / (b + c + d) × 100%;

Imprinted gene deletion gene expression (LOI) = c / (b + c + d) × 100%;

Imprinted gene copy number abnormal gene expression (CNV) = d / (b + c + d) × 100%;

Wherein, a is a cell nucleus after hematoxylin staining, there is no marker in the nucleus, and the imprinted gene is not expressed; b is a red / brown mark in the nucleus after the cell is hematoxylin stained, and the imprinting gene is present Cell nuclei; c is two red / brown markers in the nucleus of the cell after hematoxylin staining, marking the nuclei where the gene is missing; d is more than two red / brown markers in the nucleus of the cell after hematoxylin staining , Imprinting the nucleus with abnormal gene copy number.

In one example herein, the hematoxylin-stained marker is selected from but not limited to red or brown. Staining markers with other colors can also be used for imprinted gene expression, imprinted gene deletion expression and imprinted gene copy number abnormal expression. Calculation.

In an embodiment of this article, the probe is hybridized in situ and stained with Hemotoxy (hematoxylin) nuclei to amplify the signal. Under a 40 × or 60 × microscope, the presence or absence of imprinted genes in each nucleus is determined. If the copy number is abnormal, the degree of benign and malignant tumors in this sample can be determined by calculating the expression level of the imprinted gene deletion gene and the gene expression of the imprinted gene copy number. Because the slice is only 10 μm, about 20% of the cell nucleus seen under the microscope is an incomplete cell nucleus, which means that there is a possibility of partial false negatives.

In an embodiment of this document, the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression level of the imprinted gene copy number are divided into five different levels, and each probe pairs at least 1200 Cells were counted, and the expression levels of the imprinted genes for the fifteen imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 were missing, the imprinted genes The copy number abnormal expression level and the total expression level of the imprinted genes are divided into five different levels.

In an embodiment of this document, the five different levels of the expression level of the imprinted gene deletion, the abnormal expression of the imprinted gene copy number, and the total expression level for Z1 and Z11 are:

Level 0: The expression level of the imprinted gene deletion of the imprinted genes Z1 and Z11 is less than 15%, the abnormal expression of the imprinted gene copy number of the imprinted genes Z1 and Z11 is less than 1.5%, or the total expression of the imprinted genes Z1 and Z11 Less than 25% of any one or a combination of at least two;

Level I: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is 1.5-3% or the imprinting genes Z1 and Z11 The total expression level is 25-35% of any one or a combination of at least two;

Level II: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is 3-5%, or the imprinting genes Z1 and Z11 The total expression level is 35-45% of any one or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is 25-30%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is 5-8% or the imprinting genes Z1 and Z11 The total expression level of 45-55% is any one or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is greater than 30%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is greater than 8%, or the total expression level of the imprinting genes Z1 and Z11 Any one or a combination of at least two of more than 55%;

The imprinted gene deletion expression levels, imprinted gene copy number abnormal expression levels and total expression levels of the imprinted genes Z1 and Z11 are independent of each other.

The expression levels of the missing expression of imprinted genes, abnormal expression of copy number of imprinted genes and total expression of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 The different levels are:

Level 0: the imprinting genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15, and Z16 are less than 11% of the expression level of the imprinting genes, the imprinting genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression is less than 1.5% or the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9 , Z10, Z12, Z13, Z14, Z15 and Z16 total expression is less than 20% of any one or a combination of at least two;

Level I: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are 11-15%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression level is 1.5-2.5% or the imprinted genes Z2, Z3, Z4, Z5, Z6 , Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, the total expression is 20-30% of any one or a combination of at least two;

Level II: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are 15-20%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression level is 2.5-4% or the imprinted genes Z2, Z3, Z4, Z5, Z6 , Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, the total expression level is any one or a combination of at least two of 30-40%;

Level III: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are 20-25%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression level is 4-6% or the imprinted genes Z2, Z3, Z4, Z5, Z6 , Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, the total expression level is any one of 40-50% or a combination of at least two;

Level IV: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are greater than 25%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression is greater than 6% or the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9 , Z10, Z12, Z13, Z14, Z15 and Z16 total expression is greater than 50% of any one or a combination of at least two;

The imprinted gene deletion expression level, imprinted gene copy number abnormal expression level and total expression level of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are independent of each other of.

In an embodiment of this document, the present disclosure provides a device for detecting the degree of benign and malignant pancreatic tumors, which uses the above imprinted gene grading model and includes the following units:

(1) Sampling unit: Obtain the sample to be tested;

(2) Probe design unit: design specific primers based on the imprinted gene sequence;

(3) Detection unit: in situ hybridization of the probe of step (2) and the sample to be tested;

(4) Analysis unit: Microscope imaging analyzes the expression of imprinted genes;

Wherein, the analysis unit calculates the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression of the imprinted gene copy number, and through the imprinted gene classification model, the imprinted gene deletion expression and the imprinted gene copy number are abnormal The level of expression and total expression determine the degree of benign and malignant pancreatic tumors.

The deletion of the imprinted gene is that after the cells are hematoxylin-stained, there are two red / brown-marked nuclei in the nucleus, and the abnormal copy number of the imprinted gene is that after the cells are hematoxylin-stained, there are more than two red / brown-marked in the nucleus In the nucleus of the nucleus, the abnormal copy number is due to the abnormal replication of the gene by the cancer cells, resulting in the case that this gene is expressed as a triploid or even higher polyploid.

The hematoxylin-stained marker is selected from but not limited to red or brown. Staining markers with other colors can also be used to calculate the total expression level of imprinted genes, the expression level of imprinted gene deletions, and the abnormal expression of imprinted gene copy number.

The detection device is used to visually observe the changes of the imprinting (trace) genes of pancreatic tumors at the cell and tissue level early to judge the benign and malignant degree of the tumor, and provide the most favorable treatment opportunities for patients with early pancreatic tumors.

In an embodiment of this document, the present disclosure provides a method for detecting the degree of benign and malignant pancreatic tumors, characterized in that the imprinting gene grading model described above includes the following steps:

(1) Obtain the sample to be tested;

(2) Design specific primers based on the imprinted gene sequence;

(3) In situ hybridization of the probe of step (2) and the sample to be tested;

(4) Microscopic imaging analysis of the expression of imprinted genes;

Wherein, the analysis unit calculates the expression level of the imprinted gene deletion, the abnormal expression level of the imprinted gene copy number and the total expression amount, and passes the imprinted gene grading model to obtain the expression level of the imprinted gene deletion expression and the imprinted gene copy number abnormal expression And the total expression level to diagnose the degree of benign and malignant pancreatic tumors.

In an embodiment of this document, the sample to be tested in step (1) comes from human tissues and / or cells.

The sample to be tested is feasible as long as the RNA is fixed in time, and those skilled in the art can make a selection according to needs, which is not particularly limited here. The sample to be tested in the present invention includes tissue paraffin sections and biopsy samples Any one of them or a combination of at least two of them.

The specific operation procedure of the paraffin section of the tissue is to obtain a human tumor tissue sample, which is fixed with 10% neutral formalin in time, embedded in paraffin, cut to a thickness of 10 μm, and made into a tissue slice with a positively charged glass slide; because It is only 10 μm thick, so part of the nucleus seen under the microscope is an incomplete nucleus, so a partial false negative gene deletion will occur.

The specific operation procedure of the puncture biopsy sample is to obtain human cells through puncture and fix it with 10% neutral formalin in time.

Because the puncture biopsy is less harmful to the patient, the sampling process is simple, and the puncture biopsy can also be positioned compared to the blood circulation characteristics. The puncture biopsy has its special advantages as an experimental sample.

In an embodiment herein, the sample to be tested is a paraffin section of the tissue and / or a biopsy sample.

In an embodiment herein, the imprinting genes are Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16, and the imprinting gene Z1 is Gnas, The imprinting gene Z2 is Igf2, the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z5 is Mest, the imprinting gene Z6 is Plagl1, the imprinting gene Z8 is Dcn, the The imprinting gene Z9 is Dlk1, the imprinting gene Z10 is Gatm, the imprinting gene Z11 is Grb10, the imprinting gene Z12 is Peg3, the imprinting gene Z13 is Sgce, the imprinting gene Z14 is Slc38a4, the imprinting gene Z15 is Diras3, and the imprinting gene Z16 is Snrpn / Snurf.

The imprinting genes Z1 (Gnas), Z2 (Igf2), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z9 (Dlk1), Z10 (Gatm), Z11 (Grb10), Z12 (Peg3), Z13 (Sgce), Z14 (Slc38a4), Z15 (Diras3), Z16 (Snrpn / Snurf) have different levels of expression in normal tumor cell tissues, when malignant lesions occur, the expression level And the state of the imprint will change significantly.

The design probe is based on imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16, namely Gnas, Igf2, Peg10, Igf2r, Mest, Plagl1, Dcn, Dlk1, Gatm, Grb10, Peg3, Sgce, Slc38a4, Diras3 and Snrpn / Snurf were designed. A specific sequence was selected as the probe within the inner voron of each gene. The specific probe was selected by Advanced Cell Diagnostics design.

In an embodiment herein, the in situ hybridization uses the RNAscope in situ hybridization method.

In one embodiment herein, the RNAscope in situ hybridization method uses a single-channel or multi-channel color kit or a single-channel or multi-channel fluorescent kit, preferably a single-channel red / brown color kit or multi-channel Fluorescent kit.

The multi-channel color rendering kit or multi-channel fluorescent kit includes two or more channels of color rendering kit or fluorescent kit. The two-channel color rendering kit or multi-channel fluorescent kit can use two One imprinted gene probe or the combined expression of imprinted genes and other genes or even the comprehensive expression of multiple imprinted genes and non-imprinted genes.

In an embodiment of this document, the formulas for calculating the total expression of imprinted genes, the expression of imprinted genes, and the abnormal expression of imprinted genes in the model are as follows:

Total expression level = (b + c + d) / (a + b + c + d) × 100%;

Normal imprinted gene expression = b / (b + c + d) × 100%;

Imprinted gene deletion gene expression (LOI) = c / (b + c + d) × 100%;

The hematoxylin-stained marker is selected from but not limited to red or brown. Staining markers with other colors can also be used to calculate the total expression level of imprinted genes, the expression level of imprinted gene deletions, and the abnormal expression level of imprinted gene copy number.

The probe was hybridized in situ and stained with Hemotoxy (hematoxylin) cell nucleus to amplify the signal. Under a 40 × or 60 × microscope, the presence or absence of imprinted genes in each nucleus was determined, and the imprinted gene was abnormal. The total gene expression level, the imprinted gene deletion gene expression level and the imprinted gene copy number abnormal gene expression level are used to determine the degree of tumor in this sample. Since the slice is only 10 microns, about 20% of the cell nucleus seen under the microscope is an incomplete cell nucleus, which means that there is a possibility of partial false negatives.

In an example herein, the expression level of the imprinted gene deletion, the abnormal expression level of the imprinted gene copy number, and the total expression level are divided into five different levels.

The five different levels are for counting at least 1200 cells in the most positively expressed region of each probe in the sample, for Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, The fifteen imprinted genes of Z13, Z14, Z15, and Z16 were divided into three categories: imprinted gene deletion expression, imprinted gene copy number abnormal expression, and total expression.

The five different levels of the expression level of the imprinted gene deletion, the abnormal expression of the imprinted gene copy number, and the total expression level for Z1 and Z11 are:

Level I: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are 11-15%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression level is 1.5-2.5% or the imprinted genes Z2, Z3, Z4, Z5, Z6 , Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, the total expression level is any one or a combination of at least two of 20-30%;

Level IV: The imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 25% of the expression level of the imprinted gene deletion, the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression is greater than 6% or the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9 , Z10, Z12, Z13, Z14, Z15 and Z16 total expression is greater than 50% of any one or a combination of at least two;

In an embodiment of this document, the degree of benign and malignant diagnosis of pancreatic tumors is divided into benign tumors, pancreatic cancer potential, early pancreatic cancer, intermediate pancreatic cancer, and advanced pancreatic cancer;

In an example herein, the result of diagnosing the degree of benign and malignant pancreatic tumors is the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level and imprinted gene copy number abnormal expression level are less than those of grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 No more than 1 imprinting gene has an imprinting gene deletion expression level of 1 and imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The abnormal expression of the imprinted gene copy number of no more than 1 imprinted gene is grade I, it is a benign tumor;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade I, imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinting genes with abnormal copy number expression of grade I or imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 A copy of the imprinting gene with a level II deletion and no more than one imprinting gene among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade II, which is the potential of pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade II, at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted genes with abnormal copy number expression of grade II or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 A copy of the imprinting gene with a level III deletion and imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 of no more than 1 imprinting gene The abnormal expression of the number is any of the cases of grade III, which is early pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade III, imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted genes with abnormal copy number expression of grade III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 A copy of the imprinted gene with an expression level of IV and no more than one imprinted gene among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade IV, which is mid-stage pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is at least two imprinted gene copies of imprinted genes of grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The abnormal expression level is grade IV, which is advanced pancreatic cancer.

In an embodiment of this document, the present disclosure provides the imprinted gene grading model or the device for pancreatic tumor detection.

In an embodiment of this document, the present disclosure provides a use of the imprinted gene grading model or the device for preparing drugs or devices for treating pancreatic tumors.

In an embodiment of this document, the degree of benign and malignant diagnosis of pancreatic tumors is divided into benign tumors, pancreatic cancer potential, early pancreatic cancer, intermediate pancreatic cancer, and advanced pancreatic cancer.

In one example herein, the results of diagnosing the degree of benign and malignant pancreatic tumors are the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted gene deletion expression and imprinted gene copy number abnormal expression levels are less than those in grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 No more than 1 imprinting gene has an imprinting gene deletion expression level of 1 and among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is grade I, it is a benign tumor;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade II, at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted genes with abnormal copy number expression of grade II or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 A copy of the imprinted gene with a level III deletion and no more than one imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade III, which is early pancreatic cancer;

Compared with the related art, in the examples herein, the detection model and device are used to intuitively express the performance of the imprinted genes on samples of patients with esophageal tumors and / or gastric tumors. , Objective, intuitive, early, accurately detect changes in imprint (trace) genes, and can provide a quantitative model, making a huge contribution to the diagnosis of molecular pathology.

BRIEF DESCRIPTION

FIG. 1 is a pathological section of pancreatic cancer with hematoxylin-stained nuclei of the present invention, where a is a hematoxylin-stained cell, there is no marker in the nucleus, and the imprinted gene is not expressed; and b is a hematoxylin-stained cell. There is a red / brown mark in the nucleus, the imprinting gene is present; c is the red / brown mark in the nucleus after the cell is stained, and the imprinting gene is missing; d is the nucleus after the cell is hematoxylin staining There are more than two red / brown marks in the memory, and the imprinted gene copy number is abnormal;

Figure 2 (a) is the expression status of 15 genes in pathological sections of grade 0 pancreatic tumors, FIG. 2 (b) is the expression status of 15 genes in pathological sections of grade I pancreatic cancer, and FIG. 2 (c) is grade II The expression status of 15 genes in the pathological section of pancreatic cancer. Figure 2 (d) is the expression status of 15 genes in the pathological section of grade III pancreatic cancer. Figure 2 (e) is the expression status of 15 genes in the pathological section of pancreatic cancer. Gene expression status;

Fig. 3 (a) is the intensity of imprinting genes Z1, Z5, Z10, Z11 and Z16 imprinting loss on pancreatic cancer, and Fig. 3 (b) is the abnormal copy number of imprinting genes Z1, Z5, Z10, Z11 and Z16 on pancreatic cancer 3 (c) is the intensity of the total expression of imprinted genes Z1, Z5, Z10, Z11 and Z16 on pancreatic cancer, and FIG. 3 (d) is the imprinted genes Z2, Z3, Z4, Z6, Z8, Z9, The intensity of Z12, Z13, Z14, and Z15 imprinting loss on pancreatic cancer. Figure 3 (e) shows the abnormal copy number of imprinting genes Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14, and Z15 on pancreatic cancer. Figure 3 (f) is the intensity of the total expression of imprinted genes Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14 and Z15 on pancreatic cancer, where LOI is the expression of the imprinted gene deletion gene , CNV is the gene expression level of imprint gene copy number abnormality, and TE is the total expression level of imprint gene;

Figure 4 (a) is the intensity of imprinting gene Z1 imprinting deletion, abnormal copy number and total expression level, Figure 4 (b) is the intensity of imprinting gene Z5 imprinting deletion, copy number abnormality and total expression level, and Figure 4 (c) is Imprinting gene Z10 imprinting deletion, copy number abnormality and intensity of total expression, Figure 4 (d) is imprinting gene Z11 imprinting deletion, copy number abnormality and total expression intensity, Figure 4 (e) is imprinting gene Z16 imprinting loss, Abnormal copy number and intensity of total expression, Figure 4 (f) is the imprinting gene Z2 imprinting deletion, copy number abnormality and total expression intensity, Figure 4 (g) is imprinting gene Z3 imprinting deletion, copy number abnormality and total expression Figure 4 (h) is the intensity of imprinting gene Z4 imprinting deletion, copy number abnormality and total expression level, Figure 4 (i) is the imprinting gene Z6 imprinting deletion, copy number abnormality and total expression intensity, figure 4 (j) is the intensity of the imprinting gene Z8 imprinting deletion, abnormal copy number and total expression level, Figure 4 (k) is the imprinting gene Z9 imprinting deletion, copy number abnormality and total expression intensity, and Figure 4 (l) is the imprinting gene Z12 imprinting deletion, abnormal copy number and intensity of total expression, Figure 4 (m) is imprinting gene Z 13 Imprint deletion, abnormal copy number and intensity of total expression, Figure 4 (n) is the imprinting gene Z14 imprint deletion, copy number abnormality and total expression intensity, Figure 4 (o) is imprinting gene Z15 imprint deletion, copy number Abnormality and intensity of total expression, where LOI is the expression level of the imprinted gene deletion gene, CNV is the gene expression level of the imprinted gene copy number abnormality, and TE is the total expression level of the imprinted gene;

Figure 5 (a) shows the imprinting gene Z1 applied to 45 cases of pancreatic cancer pathological slices, the distribution range and grading criteria of imprinting deletion and copy number abnormality, and FIG. 5 (b) shows the imprinting gene Z5 applied to 45 cases of pancreatic cancer pathological slices. , The distribution range and grading criteria of imprinting deletion and copy number abnormality, Figure 5 (c) is the imprinting gene Z10 applied to 45 cases of pancreatic cancer pathological sections, the distribution range and grading criteria of imprinting deletion and copy number abnormality, Figure 5 (d ) Is the imprinting gene Z11 applied to 45 cases of pancreatic cancer pathological slices, the distribution range and grading criteria of imprinting deletion and copy number abnormality. Figure 5 (e) is the imprinting gene Z16 applied to 45 cases of pancreatic cancer pathological slices. The distribution range and grading standard of copy number abnormality. Figure 5 (f) is the imprinting gene Z2 applied to 45 cases of pancreatic cancer pathological sections. The distribution range and grading standard of imprinting deletion and copy number abnormality is shown in FIG. 5 (g). Z3 was applied in 45 cases of pancreatic cancer pathological slices, and the distribution range and grading criteria of imprinting loss and copy number abnormality. Figure 5 (h) is the imprinting gene Z4 applied to 45 cases of pancreatic cancer pathological slices. Distribution range and grading standard. Figure 5 (i) shows the imprinting gene Z6 applied to 45 cases of pancreatic cancer pathological sections. The distribution range and grading standard of imprinting deletion and copy number abnormality are shown in Figure 5 (j). In the pancreatic cancer pathological section, the distribution range and grading criteria of the imprinting deletion and copy number abnormality are shown in Figure 5 (k). The imprinting gene Z9 is applied to 45 cases of pancreatic cancer pathological slices. Standard, Figure 5 (l) shows the imprinting gene Z12 applied to 45 cases of pancreatic cancer pathological sections, the distribution range and grading criteria of imprinting deletion and copy number abnormality, and Figure 5 (m) shows the imprinting gene Z13 applied to 45 cases of pancreatic cancer pathology. The distribution range and grading criteria of the missing imprints and copy number abnormalities in the slices. Figure 5 (n) shows the distribution range and grading criteria of the imprinting deletion and copy number abnormalities in the 45 cases of pancreatic cancer pathological slices. (o) is the imprinting gene Z15 applied to 45 cases of pancreatic cancer pathological sections, the distribution range and grading criteria of imprinting deletion and copy number abnormality, where LOI is the expression level of the imprinting gene deletion gene and CNV is the imprinting gene copy Gene expression level with abnormal shell number, TE is the total expression level of imprinted genes;

detailed description

In order to further illustrate the technical means adopted by the present invention and its effects, the technical solutions of the present invention will be further described in conjunction with the accompanying drawings and specific implementation manners below, but the present invention is not limited to the scope of the embodiments.

Genomic imprinting is a method of gene regulation in epigenetics. It is characterized by methylating alleles from a specific parent, so that only one allele of a certain gene is expressed, while the other is in a gene silencing state. Genes of this kind are called imprinted genes. Imprinting deletion is an epigenetic change in which demethylation of imprinted genes results in the activation of silent alleles and the start of gene expression. A large number of studies have shown that this phenomenon (deletion of imprinting) is common in various types of cancer and occurs earlier than changes in cell and tissue morphology. At the same time, in healthy cells, the percentage of blotting loss is extremely low, in stark contrast to cancer cells. Therefore, the methylation status of imprinted genes can be used as a pathological marker to analyze the abnormal state of cells through specific molecular detection techniques.

The detection model and device described in the present disclosure intuitively express the performance of the missing imprint on the samples of patients with pancreatic tumors. The in situ labeling of the imprinted genes can objectively, intuitively, early and accurately detect the imprint (trace) ) Gene changes, and can provide a quantitative model, make a great contribution to the diagnosis of pancreatic tumors;

The detection device of the present disclosure can determine the degree of benign and malignant pancreatic tumors by puncturing the cells before surgery for patients with pancreatic tumors, thereby providing a basis for surgery and precise treatment. This is a revolutionary breakthrough in the diagnosis of pancreatic tumors in the field of cellular molecules;

The present disclosure can detect early pancreatic cancer very sensitively, and can detect precancerous pancreas such as pancreatic intraepithelial neoplasia, chronic pancreatitis, intraductal papillary mucinous tumor (IPMN), mucinous cystic tumor, etc. through the combination of imprinted genes. Samples that have begun to develop into cancer have been detected in the diseased patients, and pancreatic puncture has little harm to the patient. The sampling process is simple. It is used in early screening and cancer postoperative follow-up, especially for the follow-up follow-up of suspected relapse patients, which can buy time to save the patient. Make a significant contribution to life;

The technology of the present disclosure can be used not only to detect pancreatic exocrine tumors, but also to detect benign and malignant pancreatic islet neuroendocrine tumors, and has important guiding significance for accurate judgment of pancreatic cancer type and selection of treatment methods, greatly reducing pancreas Postoperative recurrence of cancer.

The detection method of the present disclosure is different from the immunohistochemical method, which reduces false positives and other negative effects. Not only that, the targeted drugs or technologies that cause the silencing, deletion, and rearrangement of the gene of the pancreatic tumor-related imprinting gene deletion site The method can be used to guide the later treatment and medication.

Example 1 Imprinted gene analysis of pancreatic cancer

The method for detecting imprinted genes includes the following steps:

(1) Obtain pancreatic cancer tissue cell sections (10 microns), put in 10% neutral formalin solution for fixation to prevent RNA degradation, fixation time is 24 hours, paraffin embedding (FFPE), the Slides need to be unloaded with positive charge, and the slices are baked in a 40 ° C oven for more than 3h;

(2) Perform dewaxing treatment according to the RNASCope sample processing method, block the endogenous peroxidase activity in the sample, enhance permeability and expose RNA molecules;

(3) Design probe: design specific primers based on the imprinted gene sequence;

The design probe is based on imprinting genes Z1 (Gnas), Z2 (Igf2), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z9 (Dlk1), Z10 (Gatm), Z11 (Grb10), Z12 (Peg3), Z13 (Sgce), Z14 (Slc38a4), Z15 (Diras3) and Z16 (Snrpn / Snurf) were designed, specifically select a segment within the inner voron of each gene The sequence is used as a probe, and the specific probe is designed by Advanced Cell Diagnostics.

(4) In situ hybridization of the probe of step (3) and the sample to be tested through the kit by RNA SCope;

(5) Signal amplification and hematoxylin staining, and analysis of the expression of imprinted genes by microscope imaging;

The formulas for calculating the total expression level of imprinted genes, the expression level of imprinted gene deletion and the abnormal expression of imprinted gene copy number in the model are as follows:

Total expression level = (b + c + d) / (a + b + c + d) × 100%;

Normal imprinted gene expression = b / (b + c + d) × 100%;

Imprinted gene deletion gene expression (LOI) = c / (b + c + d) × 100%;

Among them, a, b, c, and d are as shown in FIG. 1, where a is a cell nucleus after hematoxylin staining, and there is no marker in the nucleus, and the imprinted gene is not expressed; b is after hematoxylin staining of the cell, There is a red / brown mark in the nucleus that marks the nucleus where the gene is present; c is the cell where there are two red / brown markers that mark the gene-deficient nucleus after hematoxylin staining; the d is the hematoxylin that makes the cell After staining, there are more than two red / brown marks in the nucleus, marking the nucleus with abnormal gene copy number.

As can be seen from Figure 2 (a) -Figure 2 (e), in the samples from grade 0 to grade IV, the imprint is missing (two signal points in the nucleus) and the copy number is abnormal (three or more signal points in the nucleus) ) The proportion of cells gradually increases with the increase of malignancy.

Example 2 Imprinting genetic analysis of biopsy samples

The puncture biopsy sample is to remove suspicious diseased tissue by puncture, fix with 10% neutral formalin solution for more than 24h, and other detection methods are the same as in Example 1.

As can be seen from Fig. 3 (a) -Fig. 3 (f), Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16 each gene pair pancreas The sensitivity of cancer response, or the intensity and state of the loss of imprint corresponding to pancreatic cancer expression, are different.

The specific sensitivity of each imprinted gene to pancreatic cancer is shown in Figure 4 (a)-Figure 4 (o). As can be seen from Figure 4 (a)-Figure 4 (e), the imprinting gene Z1 is missing or has abnormal copy number Increased expression level increased significantly in the stage of malignant potential, slightly increased in the early stage of pancreatic cancer, rapidly increased to a high level in the middle stage of pancreatic cancer, and remained stable in the stage of advanced pancreatic cancer; the imprinting and expression of the imprinting gene Z5 The amount of increase rapidly rises in the stage of malignant potential, maintains stability in the early stage of pancreatic cancer, continues to rise to a high level in the stage of pancreatic cancer in the middle stage, and continues to maintain in the advanced stage of pancreatic cancer. , Maintains stability in the early and middle stages of pancreatic cancer, and gradually rises to a high level in the stage of advanced pancreatic cancer; the deletion of the imprinting gene Z10 begins to appear in the stage of malignant potential, there is no significant increase in early pancreatic cancer, in the middle and late pancreas In cancer, it gradually rises to a high level, and the deletion of imprinting gene Z10, abnormal copy number, and increased expression begin to appear in the stage of malignant potential. During the development of early to advanced pancreatic cancer, it gradually rises to a very high level; the deletion of the imprinting gene Z11, the abnormal copy number and the increase in expression increase rapidly in the stage of malignant potential, slow in the early stage of pancreatic cancer, and slow in the mid-stage pancreas The cancer stage has risen rapidly, maintaining a high level in advanced pancreatic cancer; the deletion of the imprinting gene Z16 and the increase in the expression level begin to appear in the stage of malignant potential, and gradually rise to a high level in early and mid-stage pancreatic cancer. The stage of advanced pancreatic cancer continues to maintain stability, and the imprinting gene Z16 has an abnormal copy number malignant potential stage. It begins to rise rapidly in early pancreatic cancer and slows down in mid-stage and advanced pancreatic cancer, reaching a high level;

As can be seen from Figure 4 (f) -Figure 4 (o), the imprinting gene Z2 deletion and copy number abnormalities began to appear in the mid-stage pancreatic cancer stage, and rapidly increased to a higher level in the advanced pancreatic cancer stage. The increase in expression increased rapidly to a higher level in the middle-stage pancreatic cancer stage and remained stable in the advanced pancreatic cancer stage; the deletion of the imprinting gene Z3 and the increased expression level began to appear in the middle-stage pancreatic cancer stage, and rapidly increased to a higher level in the advanced pancreatic cancer stage High level, abnormal copy number of imprinting gene Z3 begins to appear in the early stage of pancreatic cancer, maintains stability in the middle stage of pancreatic cancer, and rapidly rises to a high level in the stage of advanced pancreatic cancer; the imprinting of the imprinting gene Z4 is missing in the early stage of pancreatic cancer Rapid rise, continued to rise to a higher level in mid- and advanced pancreatic cancer, imprinting gene Z4 copy number abnormalities began to appear in the mid-stage pancreatic cancer stage, and rapidly increased to a higher level in the advanced pancreatic cancer stage, the expression of the imprinting gene Z4 The amount of increase begins to appear in the early stage of pancreatic cancer, and rises rapidly to a very high level in the middle stage of pancreatic cancer. The cancer stage continues to be maintained; imprinting gene Z6 imprinting loss and copy number abnormalities begin to appear in the imprinting missing stage, and gradually rise to a high level during the development of early to advanced pancreatic cancer, and the expression of imprinting gene Z6 increases in the early pancreas The cancer stage began to appear, rapidly rising to a high level in the middle and advanced pancreatic cancer stages; the deletion of the imprinting gene Z8, abnormal copy number and increased expression increased rapidly in the middle pancreatic cancer stage, and remained stable in the advanced pancreatic cancer stage; The missing imprint, abnormal copy number and increased expression of gene Z9 increased rapidly in the mid-stage pancreatic cancer stage and remained stable in the advanced pancreatic cancer stage; the missing imprint of the imprinting gene Z12 began to appear in the early pancreatic cancer stage and rapidly increased in the mid-stage pancreatic cancer stage To a higher level, it remained stable in the advanced pancreatic cancer stage, and the abnormal copy number and expression of the imprinted gene Z12 began to appear in the early stage of pancreatic cancer, and gradually increased to a higher level in the middle and advanced pancreatic cancer stage; the imprinted gene Z13 Imprinting loss and increased expression start at the early stage of pancreatic cancer Appeared, rapidly increased to a high level in the mid-stage pancreatic cancer stage, maintained stable in the advanced pancreatic cancer stage, the copy number abnormality of the imprinting gene Z13 began to appear in the early pancreatic cancer stage, and gradually increased to a higher level in the mid- and advanced pancreatic cancer stage Level; the imprinting loss and copy number abnormality of imprinting gene Z14 began to appear in the stage of malignant potential, rapidly increased in the early stage of pancreatic cancer, slowed down in the middle stage of pancreatic cancer, and rapidly increased to a higher level in the advanced pancreatic cancer stage. The increased expression of the imprinting gene Z14 begins to appear in the early stage of pancreatic cancer, slowly rises in the middle stage of pancreatic cancer, and quickly rises to a higher level in the advanced stage of pancreatic cancer; the imprinting of the imprinting gene Z15 begins to appear in the early stage of pancreatic cancer. In the middle and late stages of pancreatic cancer, it gradually rises to a higher level, and the copy number abnormality of imprinting gene Z15 begins to appear in the stage of malignant potential, and gradually rises to a higher level during the development of early to advanced pancreatic cancer. The expression of imprinting gene Z15 Increased amounts begin to appear in the early stage of pancreatic cancer, in the mid-stage pancreas A stage of rapid rise to a high level, to maintain the stability in the late stages of pancreatic cancer.

Example 3 Imprinting gene analysis of 45 pancreatic tumor samples

The tissues of 45 patients with pancreatic cancer including biopsy samples were obtained, and the detection method was the same as in Example 1.

As can be seen from Fig. 5 (a) -Fig. 5 (o), the ratios of the missing probes and copy number abnormalities of 15 probes in 45 pancreatic tumor tissue samples showed a distribution from low to high, according to the distribution of different probes Trend, we calculated the grading standard shown by the dotted line in the figure, which can divide the missing imprint and the abnormal copy number of each probe into 5 levels from low to high.

The specific ratings are as follows:

As can be seen from FIG. 5 (a), for the imprinting gene Z1, the expression of the imprinting gene deletion is less than 15%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 25%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 15-20%, the abnormal expression of the imprinted gene copy number is 1.5-3%, or the total expression of the imprinted gene is 25-35% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 20-25%, the abnormal expression of imprinted gene copy number is 3-5%, or the total expression of imprinted gene is 35-45%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 25-30%, the abnormal expression of the imprinted gene copy number is 5-8%, or the total expression of the imprinted gene is 45-55%, or at least two The combination of species is grade III, and any one or a combination of at least two of the imprinted gene deletion expression is greater than 30%, the imprinted gene copy number abnormal expression is greater than 8%, or the total imprinted gene expression is greater than 55%.

As can be seen from FIG. 5 (b), for the imprinting gene Z5, the expression of the imprinting gene deletion is less than 15%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 25%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 15-20%, the abnormal expression of the imprinted gene copy number is 1.5-3%, or the total expression of the imprinted gene is 25-35% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 20-25%, the abnormal expression of imprinted gene copy number is 3-5%, or the total expression of imprinted gene is 35-45%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 25-30%, the abnormal expression of the imprinted gene copy number is 5-8%, or the total expression of the imprinted gene is 45-55%, or at least two The combination of species is grade III, and any one or a combination of at least two of the imprinted gene deletion expression is greater than 30%, the imprinted gene copy number abnormal expression is greater than 8%, or the total imprinted gene expression is greater than 55%.

As can be seen from FIG. 5 (c), for the imprinting gene Z10, the expression level of the imprinting gene deletion is less than 15%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 25%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 15-20%, the abnormal expression of the imprinted gene copy number is 1.5-3%, or the total expression of the imprinted gene is 25-35% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 20-25%, the abnormal expression of imprinted gene copy number is 3-5%, or the total expression of imprinted gene is 35-45%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 25-30%, the abnormal expression of the imprinted gene copy number is 5-8%, or the total expression of the imprinted gene is 45-55%, or at least two The combination of species is grade III, and any one or a combination of at least two of the imprinted gene deletion expression is greater than 30%, the imprinted gene copy number abnormal expression is greater than 8%, or the total imprinted gene expression is greater than 55%.

As can be seen from FIG. 5 (d), for the imprinting gene Z11, the expression level of the imprinting gene deletion is less than 15%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 25%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 15-20%, the abnormal expression of the imprinted gene copy number is 1.5-3%, or the total expression of the imprinted gene is 25-35% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 20-25%, the abnormal expression of imprinted gene copy number is 3-5%, or the total expression of imprinted gene is 35-45%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 25-30%, the abnormal expression of the imprinted gene copy number is 5-8%, or the total expression of the imprinted gene is 45-55%, or at least two The combination of species is grade III, and any one or a combination of at least two of the imprinted gene deletion expression is greater than 30%, the imprinted gene copy number abnormal expression is greater than 8%, or the total imprinted gene expression is greater than 55%.

As can be seen from FIG. 5 (e), for the imprinting gene Z16, the expression of the imprinting gene deletion is less than 15%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 25%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 15-20%, the abnormal expression of the imprinted gene copy number is 1.5-3%, or the total expression of the imprinted gene is 25-35% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 20-25%, the abnormal expression of imprinted gene copy number is 3-5%, or the total expression of imprinted gene is 35-45%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 25-30%, the abnormal expression of the imprinted gene copy number is 5-8%, or the total expression of the imprinted gene is 45-55%, or at least two The combination of species is grade III, and any one or a combination of at least two of the imprinted gene deletion expression is greater than 30%, the imprinted gene copy number abnormal expression is greater than 8%, or the total imprinted gene expression is greater than 55%.

As can be seen from FIG. 5 (f), for the imprinted gene Z2, the expression of the imprinted gene deletion is less than 11%, the imprinted gene copy number abnormal expression is less than 1.5%, or the total imprinted gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (g), for the imprinting gene Z3, the expression of the imprinting gene deletion is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (h), for the imprinting gene Z4, the expression level of the imprinting gene deletion is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (i), for the imprinting gene Z6, the expression of the imprinting gene deletion is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (j), for the imprinting gene Z8, the expression level of the imprinting gene deletion is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (k), for the imprinting gene Z9, the expression of the imprinting gene deletion is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (l), for the imprinting gene Z12, the imprinting gene deletion expression is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the imprinting gene total expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (m), for the imprinting gene Z13, the expression of the imprinting gene deletion is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (n), for the imprinting gene Z14, the expression of the imprinting gene deletion is less than 11%, the imprinting gene copy number abnormal expression is less than 1.5%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, any one or a combination of at least two of imprinted gene deletion expression greater than 25%, imprinted gene copy number abnormal expression greater than 6%, or imprinted gene total expression greater than 50% is combined; grade IV;

As can be seen from FIG. 5 (o), for the imprinted gene Z15, the expression of the imprinted gene deletion is less than 11%, the imprinted gene copy number abnormal expression is less than 1.5%, or the total imprinted gene expression is less than 20%. Or a combination of at least two of 0, the expression level of imprinted gene deletion is 11-15%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30% or The combination of at least two is level I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 2.5-4% or the total expression of imprinted gene is 30-40%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 4-6% or the total expression of the imprinted gene is 40-50%, or at least two The combination of species is grade III, and any one or a combination of at least two of the imprinted gene deletion expression is greater than 25%, the imprinted gene copy number abnormal expression is greater than 6%, or the total imprinted gene expression is greater than 50%.

From the comprehensive analysis of these 45 cases of pancreatic cancer tumors, we can draw:

The diagnosis of benign and malignant pancreatic tumors is divided into benign tumors, pancreatic cancer potential, early pancreatic cancer, mid-stage pancreatic cancer and advanced pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is the expression level of the imprinting gene deletion of the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Abnormal expression of imprint gene copy number is less than level 1 or imprint genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 no more than 1 imprint The imprinting gene deletion expression level of the gene is class I and no more than 1 imprinting gene among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the imprinted gene copy number is grade I, it is a benign tumor;

In summary, the detection model and system of the present invention intuitively express the performance of imprinting loss on samples of patients with pancreatic tumors. The method of in situ labeling of imprinted genes is objective, intuitive, early and accurate Detecting changes in imprint (trace) genes, and can provide a quantified model, make a huge contribution to the diagnosis of pancreatic tumors.

The applicant declares that the present invention illustrates the detailed method of the present invention through the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, choice of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

An imprinted gene grading model for pancreatic tumors. The model grades the expression state of imprinted genes by calculating the changes in the total expression of imprinted genes, the expression of imprinted genes, and the abnormal expression of imprinted gene copy number in pancreatic tumors. ;

Wherein, the imprinting gene is any one or a combination of at least two of Z1, Z5, Z10, Z11, or Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z5 is Mest, and the imprinting gene Z10 is Gatm The imprinting gene Z11 is Grb10, and the imprinting gene Z16 is Snrpn / Snurf.
The imprinting gene grading model according to claim 1, wherein the method for calculating imprinting genes by the model is as follows:

Calculate any one of Z1, Z5, Z10, Z11 or Z16.
The imprinted gene grading model according to claim 1 or 2, wherein the method for calculating imprinted genes by the model is as follows: any one of Z1, Z11 or Z16 is calculated, preferably Z11 or Z16.
The imprinting gene grading model according to any one of claims 1 to 3, wherein the method for calculating imprinting genes by the model is: calculating a combination of any two imprinting genes of Z1, Z5, Z10, Z11 or Z16.
The imprinting gene grading model according to any one of claims 1 to 4, wherein the method for calculating imprinting genes by the model is: calculating the combination of Z1 and Z10, the combination of Z5 and Z11, the combination of Z10 and Z16 or Z11 And Z16 combination.
The imprinting gene grading model according to any one of claims 1-5, wherein the imprinting gene further comprises any one of Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14, or Z15 or A combination of at least two; wherein the imprinting gene Z2 is Igf2, the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z6 is Plagl1, the imprinting gene Z8 is Dcn, the The imprinting gene Z9 is Dlk1, the imprinting gene Z12 is Peg3, the imprinting gene Z13 is Sgce, the imprinting gene Z14 is Slc38a4, and the imprinting gene Z15 is Diras3.
The imprinting gene grading model according to any one of claims 1-6, wherein the method for calculating imprinting genes by the model is: calculating the combination of imprinting genes, calculating Z1, Z2, Z3, Z4, Z5, Z6, Z8 , Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 combinations of fifteen imprinted genes.
The imprinted gene grading model according to any one of claims 1 to 7, wherein the formula for calculating the total expression amount of imprinted genes, the expression amount of imprinted gene deletion, and the abnormal expression amount of imprinted gene copy number is as follows:

Total expression level = (b + c + d) / (a + b + c + d) × 100%;

Normal imprinted gene expression = b / (b + c + d) × 100%;

Imprinted gene deletion gene expression = c / (b + c + d) × 100%;

Imprinted gene copy number abnormal gene expression = d / (b + c + d) × 100%;

Wherein, a is a cell nucleus after hematoxylin staining, there is no marker in the nucleus, and the imprinted gene is not expressed; b is a red / brown mark in the nucleus after the cell is hematoxylin stained, and the imprinting gene is present Cell nuclei; c is two red / brown markers in the nucleus of the cell after hematoxylin staining, marking the nuclei where the gene is missing; d is more than two red / brown markers in the nucleus of the cell after hematoxylin staining , Imprinting the nucleus with abnormal gene copy number.
The imprinted gene grading model according to any one of claims 1 to 8, wherein the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level, and the total imprinted gene total expression level are divided into five different levels.
The imprinted gene grading model according to claim 9, wherein the five different levels are for Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 Five different grades of Z16's fifteen imprinted genes, the expression of imprinted gene deletion, the abnormal expression of imprinted gene copy number, and the total expression of imprinted genes, respectively;

The five different levels of the expression level of the imprinted gene deletion, the abnormal expression of the imprinted gene copy number, and the total expression level for Z1 and Z11 are:

Level 0: The expression level of the imprinted gene deletion of the imprinted genes Z1 and Z11 is less than 15%, the abnormal expression of the imprinted gene copy number of the imprinted genes Z1 and Z11 is less than 1.5%, or the total expression of the imprinted genes Z1 and Z11 Less than 25% of any one or a combination of at least two;

Level I: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is 1.5-3% or the imprinting genes Z1 and Z11 The total expression level is 25-35% of any one or a combination of at least two;

Level II: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is 3-5%, or the imprinting genes Z1 and Z11 The total expression level is 35-45% of any one or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is 25-30%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is 5-8% or the imprinting genes Z1 and Z11 The total expression level of 45-55% is any one or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting genes Z1 and Z11 is greater than 30%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z1 and Z11 is greater than 8%, or the total expression level of the imprinting genes Z1 and Z11 Any one or a combination of at least two of more than 55%;

The expression levels of the missing expression of imprinted genes, abnormal expression of copy number of imprinted genes and total expression of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 The different levels are:

Level 0: the imprinting genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15, and Z16 are less than 11% of the expression level of the imprinting genes, the imprinting genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression is less than 1.5% or the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9 , Z10, Z12, Z13, Z14, Z15 and Z16 total expression is less than 20% of any one or a combination of at least two;

Level I: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are 11-15%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression level is 1.5-2.5% or the imprinted genes Z2, Z3, Z4, Z5, Z6 , Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, the total expression is 20-30% of any one or a combination of at least two;

Level II: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are 15-20%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression level is 2.5-4% or the imprinted genes Z2, Z3, Z4, Z5, Z6 , Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, the total expression level is any one or a combination of at least two of 30-40%;

Level III: The expression levels of the deletion of the imprinted genes of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are 20-25%, and the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression level is 4-6% or the imprinted genes Z2, Z3, Z4, Z5, Z6 , Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, the total expression level is any one of 40-50% or a combination of at least two;

Level IV: The imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 25% of the expression level of the imprinted gene deletion, the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 imprinted gene copy number abnormal expression is greater than 6% or the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9 , Z10, Z12, Z13, Z14, Z15 and Z16 have a total expression greater than 50% of any one or a combination of at least two.
A device for detecting the degree of pancreatic tumor benign and malignant, which uses the imprinted gene grading model according to any one of claims 1-10, including the following units:

(1) Sampling unit: Obtain the sample to be tested;

(2) Probe design unit: design specific primers based on the imprinted gene sequence;

(3) Detection unit: in situ hybridization of the probe of step (2) and the sample to be tested;

(4) Analysis unit: Microscope imaging analyzes the expression of imprinted genes;

Wherein, the analysis unit calculates the expression of imprinted gene deletion by calculating the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level, and by using the imprinted gene grading model according to any one of claims 1-9. The amount, imprinted gene copy number abnormal expression level and total expression level are used to diagnose the benign and malignant degree of pancreatic tumors.
A method for detecting the degree of pancreatic tumor benign and malignant, which uses the imprinted gene grading model according to any one of claims 1-10, comprising the following steps:

(1) Obtain the sample to be tested;

(2) Design specific primers based on the imprinted gene sequence;

(3) In situ hybridization of the probe of step (2) and the sample to be tested;

(4) Microscopic imaging analysis of the expression of imprinted genes;

Wherein, the analysis unit calculates the expression level of the imprinted gene by calculating the expression level of the imprinted gene deletion, the abnormal expression level of the imprinted gene copy number, and the total expression level, and by using the imprinted gene grading model according to any one of claims 1-10 The amount, imprinted gene copy number abnormal expression level and total expression level are used to diagnose the benign and malignant degree of pancreatic tumors.
The method according to claim 12, wherein the sample to be tested in step (1) is derived from human tissues and / or cells.
The method according to claim 12 or 13, wherein the sample to be tested is a paraffin section of the tissue and / or a biopsy sample.
The method according to any one of claims 12 to 14, wherein the in situ hybridization uses an RNAscope in situ hybridization method.
The method according to any one of claims 12-15, wherein the RNAscope in situ hybridization method uses a single-channel or multi-channel color rendering kit or a single-channel or multi-channel fluorescent kit, preferably a single-channel Red / brown colored kit or multi-channel fluorescent kit.
The device according to any one of claims 12-16, wherein the degree of benign and malignant diagnosis of pancreatic tumors is divided into benign tumors, pancreatic cancer potential, early pancreatic cancer, intermediate pancreatic cancer, and advanced pancreatic cancer.
The method according to any one of claims 12-17, wherein the result of diagnosing the degree of benign and malignant pancreatic tumors is the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 , Z12, Z13, Z14, Z15 and Z16 imprinted gene deletion expression and imprinted gene copy number abnormal expression are less than grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 , Z12, Z13, Z14, Z15 and Z16 of not more than one imprinting gene, the expression level of imprinting gene deletion is level I and imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Abnormal expression of the imprinted gene copy number of not more than one imprinted gene in Z12, Z13, Z14, Z15 and Z16 is grade I, it is a benign tumor;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade I, imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinting genes with abnormal copy number expression of grade I or imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 A copy of the imprinting gene with a level II deletion and no more than one imprinting gene among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade II, which is the potential of pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade II, at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted genes with abnormal copy number expression of grade II or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 A copy of the imprinted gene with a level III deletion and no more than one imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade III, which is early pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade III, imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted genes with abnormal copy number expression of grade III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 A copy of the imprinted gene with an expression level of IV and no more than one imprinted gene among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade IV, which is mid-stage pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is at least two imprinted gene copies of imprinted genes of grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The abnormal expression level is grade IV, which is advanced pancreatic cancer.
A model according to any one of claims 1-10 and / or a device according to claim 10 for pancreatic tumor detection.
A use of the model according to any one of claims 1-10 and / or the device according to claim 10 for preparing drugs or devices for pancreatic tumor detection.
The use according to claim 20, wherein the degree of benign and malignant diagnosis of pancreatic tumors is divided into benign tumors, pancreatic cancer potential, early pancreatic cancer, intermediate pancreatic cancer and advanced pancreatic cancer;

The result of diagnosing the benign and malignant degree of pancreatic tumors is the expression level of the imprinting gene deletion of the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 and Abnormal expression of imprint gene copy number is less than level 1 or imprint genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 no more than 1 imprint The imprinting gene deletion expression level of the gene is class I and no more than 1 imprinting gene among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the imprinted gene copy number is grade I, it is a benign tumor;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least two imprinting genes of imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade I, imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinting genes with abnormal copy number expression of grade I or imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 A copy of the imprinting gene with a level II deletion and no more than one imprinting gene among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade II, which is the potential of pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade II, at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted genes with abnormal copy number expression of grade II or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 A copy of the imprinted gene with a level III deletion and no more than one imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade III, which is early pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is at least 2 imprinting genes among imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is grade III, imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 Imprinted genes with a copy number abnormal expression level of III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 A copy of the imprinted gene with an expression level of IV and no more than one imprinted gene among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 The abnormal expression of the number is any of the cases of grade IV, which is mid-stage pancreatic cancer;

The result of diagnosing the degree of benign and malignant pancreatic tumors is the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The imprinted gene deletion expression level is at least two imprinted gene copies of imprinted genes of grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 The abnormal expression level is grade IV, which is advanced pancreatic cancer.