WO2020098604A1

WO2020098604A1 - Grading model for determining benign and malignant degree of prostate tumors and use thereof

Info

Publication number: WO2020098604A1
Application number: PCT/CN2019/117159
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: 2018-11-12
Filing date: 2019-11-11
Publication date: 2020-05-22
Also published as: CN111172278A; CN111172278B

Abstract

The present invention provides a grading model for detecting prostate tumors and use thereof. Said model performs grading for the changes of imprinted genes in prostate tumors by calculating an imprinted gene deletion expression amount, an imprinted gene copy number abnormality expression amount, and a total imprinted gene expression amount.

Description

[Name of invention formulated by ISA according to Rule 37.2] Grading model for detecting the degree of benign and malignant prostate tumors and its application

Technical field

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

Background technique

Prostate cancer is a major health threat to elderly men. The incidence rate is low before 55 years old, and the incidence rate gradually increases with age after 55 years old. There are 1.095 million new prostate cancer patients and 307,000 deaths every year in the world, mainly in developed countries in Europe and the United States. Prostate cancer in the United States ranks first in male cancer incidence. In China, there are 60,000 new patients and 27,000 deaths each year, far lower than those in Europe and the United States, but in recent years there has been a gradual increase. The survival time of patients with prostate cancer is closely related to the cancer stage at diagnosis. The five-year survival rate of early prostate cancer after treatment reaches 99%, and the ten-year survival rate can also reach 95%, while the five-year survival rate of advanced prostate cancer is only 28 %, So early diagnosis and early treatment of prostate cancer is of great significance. At present, the early diagnosis methods of prostate cancer mainly include digital rectal examination and serum PSA detection. Digital rectal examination can diagnose larger prostate cancer, but smaller prostate cancer is easily missed, with an accuracy rate of about 50-75%; serum PSA detection is diagnosed by the amount of prostate-specific antigen (PSA) in the serum Prostate cancer, but because the PSA level in the serum of 30% of prostate cancer patients is not obvious, and the PSA level of some prostatitis patients will also rise significantly, so the accuracy rate is not very high. At present, the only way to confirm the diagnosis of prostate cancer is prostate puncture cytology, but for small prostate cancer it is difficult to accurately puncture to the cancer focus. In order to increase the accuracy, the current diagnostic standard usually requires 10-12 punctures per patient, resulting in Very painful. Therefore, there is an urgent need to develop more sensitive and accurate methods for early detection of prostate cancer.

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

The occurrence of cancer is the uncontrolled cell growth / division caused by the accumulation of epigenetic changes and genetic mutations over time. Traditional pathological diagnosis is based on the changes in the size, morphology and structure of cells and tissues to make benign and malignant judgments of prostate tumors. With the development and deepening of molecular biology, more and more molecular detection techniques are applied to the detection of prostate 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 diagnosis of prostate cancer 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 prostate cancer are analyzed to provide more accurate pre-diagnosis and diagnosis information.

Summary of the invention

In view of the deficiencies of the prior art and actual needs, the present disclosure provides a grading model for detecting the degree of benign and malignant prostate tumors and its application.

To achieve the above purpose, the present disclosure adopts the following technical solutions:

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

The imprinting gene is any one or a combination of at least two of Z1, Z8, Z11, or Z16. The imprinting gene Z1 is Gnas, the imprinting gene Z8 is Dcn, and the imprinting gene Z11 is Grb10. 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 the 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 proportion of deletions in imprinting is very low. The imprinted gene and imprinted gene are both a concept, meaning the same meaning, and can be replaced.

The inventors found that by calculating the total expression of imprinted genes, the expression of imprinted gene deletion and the abnormal expression of imprinted gene copy number of any imprinted gene in Z1, Z8, Z11 and Z16 in prostate tumors, the sensitivity of diagnosis to prostate cancer can be It reached more than 70.0%.

In an embodiment of this document, if the initial detection detects only one imprinting gene, any imprinting gene of Z1, Z8, Z11, and Z16 may be detected.

In an embodiment of this document, only one imprinting gene is detected, preferably any imprinting gene of Z1, Z11 or Z16 is detected.

In an embodiment herein, only one imprinted gene is detected, most preferably Z1 or Z16.

The inventors found that if a Z1 imprinting gene is detected alone, the diagnostic sensitivity for prostate cancer can reach 82.9%, if a Z8 imprinting gene is detected alone, the diagnostic sensitivity for prostate cancer can reach 70.0%, if a Z11 imprint is detected separately Gene, the diagnostic sensitivity of prostate cancer can reach 78.0%, if a Z16 imprinting gene is detected alone, the diagnostic sensitivity of prostate cancer can reach 95.1%.

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, Z8, Z11, and Z16, preferably Z1 Combination with Z8, combination with Z1 and Z16, combination with Z8 and Z16 or combination with Z11 and Z16.

The inventors 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 detection of imprinted genes Z1, Z8, Z11 and Z16 The combination of any two imprinted genes can achieve a diagnosis sensitivity of more than 82.5% for prostate cancer. When detecting the combination of Z1 and Z8, the diagnosis sensitivity for prostate cancer can reach 92.5%. The combination of Z1 and Z16, Z8 and When the combination of Z16, Z11 and Z16 is combined, the diagnostic sensitivity for prostate 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 Z3, Z4, Z5, Z6, Z10, or Z13; wherein, the imprinting gene Z3 is Peg10, and the imprinting gene Z4 It is Igf2r, the imprinting gene Z5 is Mest, the imprinting gene Z6 is Plagl1, the imprinting gene Z10 is Gatm, and the imprinting gene Z13 is Sgce.

The inventor found that adding Z3, Z4, Z5, Z6, Z10, and Z13 genes for combined diagnosis based on the detection of Z1, Z8, Z11, and Z16 genes not only helps to increase the accuracy of detection, but also adds other probes Assisted diagnosis can further avoid the occurrence of false positives, can further improve the detection accuracy, and thus can achieve accurate classification and judgment of all prostate tumor samples.

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

The deletion of the imprinted gene is that there are two red / brown marks in the nucleus of the cell after hematoxylin staining. The abnormal number of imprinted genes is that there are more than two red / brown marks 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 level (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 Nucleus; the c is the hematoxylin staining of the cell, there are two red / brown markers in the nucleus, imprinting the gene-deficient nuclei; the d is the hematoxylin staining of the cell, there are more than two red / brown markers , Imprinting the nucleus with abnormal gene copy number.

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

In an embodiment of this document, 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 gene expression level of the imprinted gene deletion gene and the gene expression level of the imprinted gene copy number abnormality. Since the slice is only 10 μm, about 20% of the nuclei seen under the microscope are incomplete nuclei, 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. Cells were counted, the expression level of the imprinted gene deletion, the abnormal expression of the imprinted gene copy number and the total expression of the imprinted genes for the ten imprinted genes of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 Five different levels are divided.

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

Level 0: The expression level of the imprinting gene deletion of the imprinting gene Z1 is less than 10%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is less than 1%, or the total expression of the imprinting gene Z1 is less than 20% One or a combination of at least two;

Level I: The expression level of the imprinting gene deletion of the imprinting gene Z1 is 10-15%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is 1-2%, or the total expression level of the imprinting gene Z1 is 20 -30% of any one or a combination of at least two;

Level II: The expression level of the imprinting gene deletion of the imprinting gene Z1 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is 2-3%, or the total expression level of the imprinting gene Z1 is 30 -40% of any one or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting gene Z1 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is 3-5%, or the total expression level of the imprinting gene Z1 is 40 -Any one of 50% or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting gene Z1 is greater than 25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is greater than 5%, or the total expression of the imprinting gene Z1 is greater than 50% One or a combination of at least two.

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 Z8 are:

Level 0: The expression level of the imprinting gene deletion of the imprinting gene Z8 is less than 10%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is less than 1%, or the total expression of the imprinting gene Z8 is less than 15% One or a combination of at least two;

Level I: The expression level of the imprinting gene deletion of the imprinting gene Z8 is 10-15%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is 1-2%, or the total expression level of the imprinting gene Z8 is 15 Any one or a combination of at least two of -20%;

Level II: The expression level of the imprinting gene deletion of the imprinting gene Z8 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is 2-5%, or the total expression level of the imprinting gene Z8 is 20 -30% of any one or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting gene Z8 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is 5-8%, or the total expression level of the imprinting gene Z8 is 30 -40% of any one or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting gene Z8 is greater than 25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is greater than 8%, or the total expression of the imprinting gene Z8 is greater than 40% One or a combination of at least two.

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 Z16 are:

Level 0: The expression level of the imprinting gene deletion of the imprinting gene Z16 is less than 15%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z16 is less than 1%, or the total expression of the imprinting gene Z16 is less than 30% One or a combination of at least two;

Level I: The expression of the imprinted gene deletion of the imprinted gene Z16 is 15-20%, the abnormal expression of the imprinted gene copy number of the imprinted gene Z16 is 1-3%, or the total expression of the imprinted gene Z16 is 30 -40% of any one or a combination of at least two;

Level II: The expression of the imprinted gene deletion of the imprinted gene Z16 is 20-25%, the abnormal expression of the imprinted gene copy number of the imprinted gene Z16 is 3-5%, or the total expression of the imprinted gene Z16 is 40 -Any one of 50% or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting gene Z16 is 25-30%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z16 is 5-8%, or the total expression level of the imprinting gene Z16 is 50 -60% of any one or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting gene Z16 is greater than 30%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z16 is greater than 8%, or the total expression of the imprinting gene Z16 is greater than 60% One or a combination of at least two.

In an embodiment of this document, the five different levels of the expression level of the deletion of the imprinted genes, the abnormal expression of the copy number of the imprinted genes and the total expression for Z3, Z11 and Z13 are:

Level 0: the expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is less than 10%, the abnormal expression of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is less than 1% or the imprinting genes Z3 and Z11 The total expression level with Z13 is less than 15% of any one or a combination of at least two;

Level I: The expression level of the deletion of the imprinted genes of the imprinted genes Z3, Z11 and Z13 is 10-15%, and the abnormal expression of the imprinted gene copy number of the imprinted genes Z3, Z11 and Z13 is 1-1.5% or the imprinted The total expression level of genes Z3, Z11 and Z13 is any one of 15-20% or a combination of at least two;

Level II: the expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is 1.5-2.5% or the imprinting The total expression of genes Z3, Z11 and Z13 is any one of 20-30% or a combination of at least two;

Level III: the expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is 2.5-4% or the imprinting The total expression level of genes Z3, Z11 and Z13 is any one of 30-40% or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is greater than 25%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is greater than 4% or the imprinting genes Z3 and Z11 The total expression level with Z13 is greater than 40% of any one or a combination of at least two;

The imprinted gene deletion expression levels, imprinted gene copy number abnormal expression levels and total expression levels of the imprinted genes Z3, Z11 and Z13 are independent of each other.

In an example herein, 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 Z4, Z5, Z6, and Z10 are:

Level 0: The expression level of the deletion of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is less than 10%, the abnormal expression of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is less than 0.5% or the imprinted The total expression of genes Z4, Z5, Z6 and Z10 is less than 15% of any one or a combination of at least two;

Level I: The expression level of the deletion of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is 10-15%, and the abnormal expression amount of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is 0.5-1.5% Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 is any one of 15-20% or a combination of at least two;

Level II: The expression level of the imprinting gene deletion of the imprinting genes Z4, Z5, Z6 and Z10 is 15-20%, and the abnormal expression level of the imprinting gene copy number of the imprinting genes Z4, Z5, Z6 and Z10 is 1.5-2.5% Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 is any one of 20-30% or a combination of at least two;

Level III: the expression level of the imprinting gene deletion of the imprinting genes Z4, Z5, Z6 and Z10 is 20-25%, and the abnormal expression level of the imprinting gene copy number of the imprinting genes Z4, Z5, Z6 and Z10 is 2.5-4% Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 is any one or a combination of at least two of 30-40%;

Level IV: The expression level of the imprinting gene deletion of the imprinting genes Z4, Z5, Z6 and Z10 is greater than 25%, and the abnormal expression of the imprinting gene copy number of the imprinting genes Z4, Z5, Z6 and Z10 is greater than 4% or the imprinting The total expression of genes Z4, Z5, Z6 and Z10 is greater than 40% of any one or a combination of at least two;

The imprinted gene deletion expression levels, imprinted gene copy number abnormal expression levels, and total expression levels of the imprinted genes Z4, Z5, Z6, and Z10 are independent of each other.

In an embodiment of this document, the present disclosure provides a device for detecting the degree of benign and malignant prostate tumors. The imprinted gene grading model of the prostate tumors 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 level of the imprinted gene copy number, through the imprinted gene grading model of the prostate tumor, and thus by the imprinted gene deletion expression and imprinted gene copy The number of abnormal expression levels and total expression levels can be used to judge the degree of benign and malignant prostate tumors.

In an embodiment of this document, the present disclosure provides a method for detecting the degree of benign and malignant prostate tumors, wherein the imprinted gene grading model of the prostate tumors 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 expression situation is calculated by calculating the expression level of the imprinted gene deletion, the abnormal expression of the imprinted gene copy number and the total expression amount, and by the imprinted gene grading model of the prostate tumor, the expression level of the imprinted gene deletion and the imprinted gene copy number are abnormal The level of expression and total expression are used to diagnose the degree of benign and malignant prostate tumors.

The deletion of the imprinted gene means that there are two red / brown-marked nuclei in the nucleus of the cell after hematoxylin staining. The abnormal copy number of the imprinted gene is that there are more than two red / brown-marked in the nucleus of the cell after hematoxylin staining 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 level of imprinted gene copy number.

The detection device of the present disclosure is used to visually observe the changes of the imprint (trace) genes of prostate 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 prostate 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 processed in a timely manner, 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 disclosure includes paraffin sections of tissues and biopsy samples Or any one or a combination of at least two of the urine exfoliated cell samples.

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 a tissue slide is made with a positively charged glass slide; because It is only 10μm thick, so part of the nucleus seen under the microscope is an incomplete cell nucleus, so some false negative gene deletions will occur.

The specific operation steps of the puncture biopsy sample are to obtain human cells through puncture and fix them with 10% neutral formalin in time.

The specific operation step of the urine exfoliated cell sample is to obtain the patient's urine after massaging the prostate, collect the cells by centrifugation, 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.

Urine exfoliated cells are harmless to patients, the sampling process is simple, and they have special advantages as experimental samples.

In an embodiment of the present invention, the sample to be tested is a biopsy sample or a urine exfoliated cell sample.

In an example herein, the imprinting genes are Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16, the imprinting gene Z1 is Gnas, and the imprinting gene Z3 is Peg10, the 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 imprinting gene Z10 is Gatm, the imprinting gene Z11 is Grb10, the imprinting gene Z13 is Sgce, and the imprinting gene Z16 is Snrpn / Snurf.

The imprinting genes Z1 (Gnas), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce), Z16 (Snrpn / Snurf) has different levels of expression in normal tumor cell tissues. When malignant lesions occur, the expression level and imprinting status will change significantly.

The designed probes are based on the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16, namely Gnas, Peg10, Igf2r, Mest, Plagl1, Dcn, Gatm, Grb10, Sgce and Snrpn / Snurf designed it. Specifically, a sequence was selected as the probe in the inner vortex of each gene. The specific probe was designed by Advanced Cell Diagnostics.

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

In an 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.

In an embodiment of the present invention, the multi-channel color rendering kit or multi-channel fluorescent kit includes two or more channel color rendering kits or fluorescent kits, and the two-channel color rendering kit or multi-channel The fluorescent kit can use two imprinted gene probes 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 level (LOI) = c / (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 Nucleus; the c is the hematoxylin staining of the cell, there are two red / brown markers in the nucleus, imprinting the gene-deficient nuclei; the d is the hematoxylin staining of the cell, there are more than two red / brown markers in the nucleus , Imprinting the nucleus with abnormal gene copy number.

The hematoxylin-stained markers are 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.

In an embodiment of this document, 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 total expression level of the imprinted gene, the expression level of the imprinted gene deletion gene and the gene expression level of the imprinted gene copy number. Because the section 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 at least 1200 cells counted in the most positively expressed area of each probe in the sample, for ten of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 The imprinted gene deletion expression level, imprinted gene copy number abnormal expression level, and total expression level of the imprinted gene are divided 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 are:

The five different levels of the expression level of the deletion of the imprinted gene for Z8, the abnormal expression of the copy number of the imprinted gene and the total expression are:

The five different levels of the expression level of the imprinting gene deletion, the abnormal expression of the imprinting gene copy number and the total expression level for Z16 are:

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 Z3, Z11 and Z13 are:

The five different levels of the expression levels of the deletion of the imprinted genes, the abnormal expression of the copy number of the imprinted genes, and the total expression for Z4, Z5, Z6, and Z10 are:

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

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression levels of the deletion of the imprinted genes and the abnormal expression of the imprinted gene copy number of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are both less than 1. No more than 1 imprinting gene in the level or imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is expressed in level 1 and the imprinting genes Z1, Z3, Z4, Z5 , Z6, Z8, Z10, Z11, Z13, and Z16, if there is no more than one imprinted gene, the imprinted gene copy number abnormal expression level is grade I, which is a benign tumor;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the deletion of the imprinting gene of at least two imprinting genes of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, The imprinted gene copy number of at least 2 imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is level I or the imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of II and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any case in grade II, it is the potential of prostate cancer;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the deletion of the imprinting gene of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade II, The imprinted gene copy number abnormal expression of at least 2 imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II or imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of III and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any of the cases of grade III, which is early prostate cancer;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the imprinting gene deletion of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade III, At least two imprinted genes of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have an imprinted gene copy number abnormal expression level of grade III or imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an expression level of imprinting gene deletion of grade IV and among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any of the grade IV, then it is mid-stage prostate cancer;

The result of the diagnosis of the benign and malignant degree of prostate tumor is that the expression level of the deletion of the imprinting gene of at least 2 imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV or imprinting The abnormal expression of the imprinted gene copy number of at least two imprinted genes in genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade IV, which is advanced prostate cancer.

In an embodiment herein, the present disclosure provides a imprinted gene grading model of the prostate tumor or the device used for prostate tumor detection and / or treatment.

In an embodiment herein, the present disclosure provides a imprinted gene grading model of the prostate or the use of the device for preparing drugs or devices for prostate tumor detection and / or treatment.

In an example herein, the result of diagnosing the degree of benign and malignant prostate tumors is the amount of imprinted gene deletion expression and imprinted gene copy of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The number of abnormal expression levels is less than 1 or the imprinted genes of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 are not more than 1 imprinted gene. Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 of not more than one imprinted gene imprinted gene copy number abnormal expression level is grade I, it is a benign tumor;

In an example herein, the result of diagnosing the degree of benign and malignant prostate tumors is the imprinting gene of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The deletion expression level is level I, and the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is the level I or imprinted gene Z1 No more than 1 imprinting gene in Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of II and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, The abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z11, Z13 and Z16 is any one of grade II, it is the potential of prostate cancer;

In an example herein, the result of diagnosing the degree of benign and malignant prostate tumors is the imprinting gene of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The deletion expression level is grade II, and the abnormal expression level of the imprinted gene copy number of at least 2 imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II or imprinted gene Z1 No more than 1 imprinting gene in Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of III and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, The abnormal expression of the imprinted gene copy number of no more than one imprinted gene in Z11, Z13, and Z16 is any one of grade III, it is early prostate cancer;

In an example herein, the result of diagnosing the degree of benign and malignant prostate tumors is the imprinting gene of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The deletion expression level is grade III, and the imprint gene copy number abnormal expression level of at least two imprint genes of imprint genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade III or imprint gene Z1. No more than 1 imprinting gene in Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of IV and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, The abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z11, Z13 and Z16 is any one of grade IV, it is mid-stage prostate cancer;

In an example herein, the result of diagnosing the degree of benign and malignant prostate tumors is the deletion of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The expression level is grade IV or the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV, it is advanced prostate cancer .

Compared with the related art, in the examples herein, the detection model and device are used to express the performance of the imprinted genes on the samples of patients with esophageal tumors and / or gastric tumors in an intuitive way. , 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 prostate cancer with hematoxylin-stained nuclei of the present disclosure, wherein the a is after hematoxylin staining of the cell, there is no marker in the nucleus, and the imprinted gene is not expressed; the b is after hematoxylin staining of the cell, There is a red / brown mark in the nucleus, the imprinted gene is present; the c is the two red / brown marks in the nucleus after the cell is stained with hematoxylin, the imprinted gene is missing; the d is the nucleus after the cell is stained with hematoxylin There are more than two red / brown marks in the memory, and the imprinted gene copy number is abnormal;

Fig. 2 (a) is the expression status of 10 genes in pathological sections of grade 0 prostate tumors, Fig. 2 (b) is the expression status of 10 genes in pathological sections of grade I prostate cancer, and Fig. 2 (c) is grade II The expression status of 10 genes in pathological slices of prostate cancer. Figure 2 (d) is the expression status of 10 genes in pathological slices of grade III prostate cancer. Figure 2 (e) is the expression status of 10 genes in pathological slices of grade IV prostate cancer. Gene expression status;

Figure 3 (a) is the intensity of imprinting genes Z1, Z8, Z11 and Z16 for the loss of imprinting of prostate cancer, and Figure 3 (b) is the intensity of imprinting genes Z1, Z8, Z11 and Z16 for the copy number abnormality of prostate cancer. 3 (c) is the intensity of the total expression of imprinted genes Z1, Z8, Z11, and Z16 on prostate cancer, and FIG. 3 (d) is the imprinted deletion of the imprinted genes Z3, Z4, Z5, Z6, Z10, and Z13 on prostate cancer. Intensity, Figure 3 (e) is the intensity of the imprinted genes Z3, Z4, Z5, Z6, Z10 and Z13 for prostate cancer copy number abnormality, and Figure 3 (f) is the imprinted genes Z3, Z4, Z5, Z6, Z10 and Z13 The intensity of the total expression of prostate cancer, 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;

Fig. 4 (a) is the intensity of the imprinting gene Z1 imprinting deletion, copy number abnormality and total expression, Fig. 4 (b) is the imprinting gene Z8 imprinting deletion, copy number abnormality and total expression intensity, and Fig. 4 (c) is Imprinting gene Z11 imprinting deletion, abnormal copy number and intensity of total expression, Figure 4 (d) is imprinting gene Z16 imprinting deletion, copy number abnormality and total expression intensity, and FIG. 4 (e) is imprinting gene Z3 imprinting loss, Abnormal copy number and intensity of total expression, Figure 4 (f) is the imprinting gene Z4 imprint deletion, abnormal copy number and total expression intensity, and Figure 4 (g) is imprinting gene Z5 imprint deletion, copy number abnormal and total expression Figure 4 (h) is the intensity of imprinting gene Z6 imprinting deletion, abnormal copy number and total expression level, Figure 4 (i) is the imprinting gene Z10 imprinting deletion, abnormal copy number and total expression intensity, figure 4 (j) is the intensity of the imprinting gene Z13 imprinting deletion, copy number abnormality and total expression level, where LOI is the expression level of the imprinting gene deletion gene, CNV is the gene expression level of the imprinting gene copy number abnormality, and TE is the total expression level of the imprinting gene ;

Figure 5 (a) is the imprinting gene Z1 applied to 44 cases of prostate cancer pathological sections, the distribution range and grading criteria of imprinting deletion and copy number abnormality, and FIG. 5 (b) is the imprinting gene Z8 applied to 44 cases of prostate cancer pathological sections. , The distribution range and grading standards of imprinting deletion and copy number abnormality, Figure 5 (c) is the imprinting gene Z11 applied to 44 cases of prostate cancer pathological sections, the distribution range and grading standards of imprinting deletion and copy number abnormality, Figure 5 (d ) Is the imprinting gene Z16 applied to 44 cases of prostate cancer pathological sections, the distribution range and grading criteria of imprinting deletion and copy number abnormality. Figure 5 (e) is the imprinting gene Z3 applied to 44 cases of prostate cancer pathological sections. The distribution range and grading standard of copy number abnormality. Figure 5 (f) is the imprinting gene Z4 applied to 44 cases of prostate cancer pathological sections. The distribution range and grading standard of imprinting deletion and copy number abnormality are shown in FIG. 5 (g). Z5 was applied to 44 cases of prostate cancer pathological slices, and the distribution range and grading criteria of imprinting loss and copy number abnormality. Figure 5 (h) is the imprinting gene Z6 applied to 44 cases of prostate cancer pathological slices. Distribution range and grading standard. Figure 5 (i) shows the imprinting gene Z10 applied to 44 cases of prostate cancer pathological sections. The distribution range and grading standard of imprinting deletion and copy number abnormality are shown in Figure 5 (j). Cases of prostate cancer pathological sections, the distribution range and grading standard of imprinting deletion and copy number abnormality, where LOI is the expression level of the imprinting gene deletion gene, CNV is the gene expression level of the imprinting gene copy number abnormality, and TE is the total expression level of imprinting gene ;

Fig. 6 (a) is the expression status of 10 genes in urine samples of benign prostate tumors, and Fig. 6 (b) is the expression status of 10 genes in urine samples of prostate cancer.

detailed description

To further elaborate on the technical measures adopted by the present disclosure and their effects, the technical solutions of the present disclosure will be further described in the following with reference to the drawings and through specific implementations, but the present disclosure is not limited to the scope of the embodiments.

Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by the methylation of alleles from a specific parent, so that only one allele of a gene is expressed, while the other is in a gene silence state. Genes of this kind are called imprinted genes. Imprint deletion is an epigenetic change in which demethylation of an imprinted gene causes the silent allele to be activated and start 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 disclosed in the present disclosure intuitively express the performance of the imprint missing on the samples of patients with prostate 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 prostate tumors;

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

The present disclosure can be used for early screening of prostate cancer through urine. The sample collection method is simple. It is suitable for large-scale medical checkups, as well as prostate cancer postoperative and drug efficacy monitoring. Especially for the follow-up follow-up of suspected relapse patients, you can buy time. Make a significant contribution to saving the lives of patients;

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 drug or technology that causes the gene tumor to be silenced, eliminated, and rearranged by the deletion site of the related imprinting gene of the prostate tumor The method can be used to guide the later treatment and medication.

Example 1 Imprinted gene analysis of prostate cancer

The method for detecting imprinted genes includes the following steps:

(1) Obtain a tissue cell section of prostate cancer (10 microns), place it in 10% neutral formalin solution for fixation to prevent RNA degradation, fixation time is 24 hours, paraffin embedding (FFPE), the The slides need to be unloaded with positive charges, and the slices are baked in a 40 ° C oven for more than 3 hours;

(2) Dewaxing according to the RNASCope sample processing method, blocking the endogenous peroxidase activity in the sample, enhancing permeability and exposing RNA molecules;

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

The design probe is based on imprinting genes Z1 (Gnas), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce) and Z16 (Snrpn / Snurf) were designed. Specifically, a sequence was selected as the probe in the inner spinner of each gene. The specific probe was 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 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 level (LOI) = c / (b + c + d) × 100%;

Among them, a, b, c, and d are as shown in FIG. 1. The a is a cell nucleus after hematoxylin staining, and there is no marker in the nucleus, and the imprinted gene is not expressed. The b is the cell nucleus after hematoxylin staining. There is a red / brown mark in the nucleus that marks the nucleus where the gene is present; c is the nucleus where two red / brown markers are present in the nucleus after the cell is stained with hematoxylin; the d is the nucleus where the cell is hematoxylin 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 obtained by puncturing somatic cells of a suspiciously diseased human body, fixed with 10% neutral formalin solution for more than 24 hours, and other detection methods are the same as in Example 1.

As can be seen from Figure 3 (a) -Figure 3 (f), each gene of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, Z16 responds to prostate cancer or corresponds to prostate The intensity and state of the missing imprint of cancer expression are different.

The specific sensitivity of each imprinted gene to prostate cancer is shown in Figure 4 (a)-Figure 4 (j). As can be seen from Figure 4 (a)-Figure 4 (d), the imprinting gene Z1 is missing and the copy number is abnormal Rapid rise in malignant potential and early stage of prostate cancer, slowed down in the middle stage of prostate cancer stage, and rapidly increased to a high level in the stage of advanced prostate cancer, the expression of imprinted gene Z1 increased rapidly in malignant potential and early stage of prostate cancer Ascending, the rate of increase in the stage of advanced prostate cancer is slowed down to a higher level; the deletion of the imprinting gene Z8, abnormal copy number and increased expression increase rapidly in the stage of early prostate cancer, and the rate of increase in the stage of advanced prostate cancer is slowed to reach Higher levels; imprinting gene Z11 imprinting loss, copy number abnormality and increased expression increased rapidly in the early stage of prostate cancer, slowed down in the advanced stage of prostate cancer, reaching a very high level; imprinting gene Z16 imprinted in The malignant potential stage began to appear, slowly rising in early prostate cancer, rapidly rising in mid-stage prostate cancer, and slowing up to advanced prostate cancer stage, reaching a very high level, the abnormal copy number of imprinting gene Z16 rapidly increased in malignant potential stage To a very high level, it continues to rise slowly during the development of early to advanced prostate cancer. The increase in the expression of the imprinting gene Z16 increases rapidly in the malignant potential and early prostate cancer stage, and the growth rate slows down in the advanced stage of prostate cancer, reaching a very high s level;

As can be seen from Figure 4 (e) -Figure 4 (j), the deletion of imprinting gene Z3 and the abnormal copy number began to appear in the early stage of prostate cancer, and there was no significant increase in advanced prostate cancer. The expression of imprinting gene Z3 The increase began to appear in the early stage of prostate cancer, slowly increased in the middle and late stage of prostate cancer, and the level was also low; the imprinting gene Z4 imprinting deletion and copy number abnormality increased rapidly in early stage prostate cancer, and slowed down in the middle stage prostate cancer stage , The rate of increase in the stage of advanced prostate cancer has accelerated, but the level is still not high. The increase in the expression of the imprinting gene Z4 rises faster in the early and middle stages of prostate cancer, and the rate of increase in the stage of advanced prostate cancer slows down, and the level is still not high. ; Imprinting gene Z5 imprinting deletion and copy number abnormality rise rapidly in early prostate cancer, the rate of rise slows in the middle stage of prostate cancer stage, and the rate of increase to the stage of advanced prostate cancer has accelerated, but the level is still not high, imprinting gene Z5 The increase in expression increased rapidly in the early and middle stages of prostate cancer, but slowed down in the advanced stage of prostate cancer, and the level was still not high; the deletion of the imprinting gene Z6, abnormal copy number, and increased expression increased rapidly in the early stage of prostate cancer , The rate of increase in the stage of advanced prostate cancer is slowed down and the level is not high; the deletion of the imprinting gene Z10 increases rapidly in the early stage of prostate cancer, and the rate of increase slows down in the advanced stage of prostate cancer, reaching a higher level, a copy of the imprinting gene Z10 Abnormal number and increased expression increased rapidly in the early and middle stages of prostate cancer, and slowed down in the advanced stage of prostate cancer, reaching a higher level; the deletion of the imprinting gene Z13 and abnormal copy number increased rapidly in early prostate cancer. The rate of increase in the stage of prostate cancer in the middle and late stages is slow, and the level is not high. The increase in the expression level of the imprinting gene Z13 increases rapidly in the stage of early prostate cancer, and the rate of increase in the stage of prostate cancer in the middle stage is slow, and there is no significant increase in the stage of advanced prostate cancer not tall.

Example 3 Imprinted gene analysis of 44 prostate tumor samples

The tissues of 44 patients with prostate tumors including biopsy samples were obtained. The detection method was the same as in Example 1.

As can be seen from Fig. 5 (a) -Fig. 5 (j), the ratios of the loss of imprinting and copy number abnormality of 10 probes in 44 prostate 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 be divided into 5 grades for the missing imprint and abnormal copy number of each probe 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 level of the imprinting gene deletion is less than 10%, the imprinting gene copy number abnormal expression is less than 1%, or the total imprinting gene expression is less than 20%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 10-15%, the expression level of the imprinted gene copy number is 1-2% or the total expression of the 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 2-3% 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 3-5%, 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 5%, or the total imprinted gene expression is greater than 50%; the combination is at least grade IV;

As can be seen from FIG. 5 (b), for the imprinting gene Z8, any of the imprinting gene deletion expression is less than 10%, the imprinting gene copy number abnormal expression is less than 1%, or the total imprinting gene expression is less than 15%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 10-15%, the imprinted gene copy number abnormal expression is 1-2%, or the total expression of the imprinted gene is 15-20% 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%, or the total expression of imprinted gene is 20-30%, 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 5-8% or the total expression of the imprinted gene is 30-40%, or at least two The combination of species is grade III, 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 8%, or the imprinted gene total expression is greater than 40%; the combination is at least grade IV;

As can be seen from FIG. 5 (c), for the imprinting gene Z11, the expression of the imprinting gene deletion is less than 10%, the imprinting gene copy number abnormal expression is less than 1%, or the total imprinting gene expression is less than 15%. Or a combination of at least two is grade 0, the expression level of the imprinted gene deletion is 10-15%, the abnormal expression of the imprinted gene copy number is 1-1.5% or the total expression of the imprinted gene is 15-20% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the imprinted gene copy number abnormal expression is 2.5-4%, or the total expression of the imprinted gene is 30-40%, 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 4%, or the imprinted gene total expression is greater than 40%; the combination is at least grade IV;

As can be seen from FIG. 5 (d), for the imprinted gene Z16, the expression of the imprinted gene deletion is less than 15%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 30%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 15-20%, the expression level of the imprinted gene copy number is 1-3% or the total expression of the imprinted gene is 30-40% or The combination of at least two is grade I, the expression level of the imprinted gene deletion is 20-25%, the abnormal expression of the imprinted gene copy number is 3-5%, or the total expression of the imprinted gene is 40-50%. 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 50-60%, 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 30%, imprinted gene copy number abnormal expression greater than 8%, or imprinted gene total expression greater than 60% is combined; grade IV;

As can be seen from FIG. 5 (e), for the imprinted gene Z3, the expression level of the imprinted gene deletion is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 15%. Or a combination of at least two is grade 0, the expression level of the imprinted gene deletion is 10-15%, the abnormal expression of the imprinted gene copy number is 1-1.5% or the total expression of the imprinted gene is 15-20% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the imprinted gene copy number abnormal expression is 2.5-4%, or the total expression of the imprinted gene is 30-40%, 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 4%, or the imprinted gene total expression is greater than 40%; the combination is at least grade IV;

As can be seen from FIG. 5 (f), for the imprinting gene Z4, the expression level of the imprinting gene deletion is less than 10%, the imprinting gene copy number abnormal expression is less than 0.5%, or the total imprinting gene expression is less than 15%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 10-15%, the expression level of the imprinted gene copy number is 0.5-1.5% or the total expression of the imprinted gene is 15-20% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the imprinted gene copy number abnormal expression is 2.5-4%, or the total expression of the imprinted gene is 30-40%, 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 4%, or the imprinted gene total expression is greater than 40%; the combination is at least grade IV;

As can be seen from FIG. 5 (g), for the imprinting gene Z5, the expression level of the imprinting gene deletion is less than 10%, the imprinting gene copy number abnormal expression is less than 0.5%, or the total imprinting gene expression is less than 15%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 10-15%, the expression level of the imprinted gene copy number is 0.5-1.5% or the total expression of the imprinted gene is 15-20% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the imprinted gene copy number abnormal expression is 2.5-4%, or the total expression of the imprinted gene is 30-40%, 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 4%, or the imprinted gene total expression is greater than 40%; the combination is at least grade IV;

As can be seen from FIG. 5 (h), for the imprinting gene Z6, the expression level of the imprinting gene deletion is less than 10%, the imprinting gene copy number abnormal expression is less than 0.5%, or the total imprinting gene expression is less than 15%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 10-15%, the expression level of the imprinted gene copy number is 0.5-1.5% or the total expression of the imprinted gene is 15-20% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the imprinted gene copy number abnormal expression is 2.5-4%, or the total expression of the imprinted gene is 30-40%, 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 4%, or the imprinted gene total expression is greater than 40%; the combination is at least grade IV;

As can be seen from FIG. 5 (i), for the imprinting gene Z10, the expression of the imprinting gene deletion is less than 10%, the imprinting gene copy number abnormal expression is less than 0.5%, or the total imprinting gene expression is less than 15%. Or a combination of at least two of 0, the expression level of the imprinted gene deletion is 10-15%, the expression level of the imprinted gene copy number is 0.5-1.5% or the total expression of the imprinted gene is 15-20% or The combination of at least two is grade I, the expression level of imprinted gene deletion is 15-20%, the abnormal expression of imprinted gene copy number is 1.5-2.5% or the total expression of imprinted gene is 20-30%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the imprinted gene copy number abnormal expression is 2.5-4%, or the total expression of the imprinted gene is 30-40%, 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 4%, or the imprinted gene total expression is greater than 40%; the combination is at least grade IV;

As can be seen from FIG. 5 (j), for the imprinting gene Z13, the expression of the imprinting gene deletion is less than 10%, the imprinting gene copy number abnormal expression is less than 1%, or the total imprinting gene expression is less than 15%. Or a combination of at least two is grade 0, the expression level of the imprinted gene deletion is 10-15%, the abnormal expression of the imprinted gene copy number is 1-1.5% or the total expression of the imprinted gene is 15-20% 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 1.5-2.5% or the total expression of imprinted gene is 20-30%, or at least The combination of the two is grade II, the expression level of the imprinted gene deletion is 20-25%, the imprinted gene copy number abnormal expression is 2.5-4%, or the total expression of the imprinted gene is 30-40%, 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 4%, or the imprinted gene total expression is greater than 40%.

From the comprehensive analysis of these 44 prostate tumor samples, we can draw:

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

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the imprinting gene deletion of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade III, At least two imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have an abnormal expression level of imprinted gene copy number of grade III or imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an expression level of imprinting gene deletion of grade IV and among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any of the grade IV, then it is mid-stage prostate cancer;

Example 4 Imprinting gene analysis of urine exfoliated cell samples

The urine exfoliated cell sample is that the prostate tumor patient collects urine after prostate massage, collects cells by centrifugation, and fixes with 10% neutral formalin solution for more than 24 hours. The other detection methods are the same as in Example 1.

As can be seen from Figure 6 (a) -Figure 6 (b), Figure 6 (a) is a benign prostate tumor, and Figure 6 (b) is a prostate cancer. There is no loss of imprint and abnormal copy number in the urine of the benign prostate tumor. There are a large number of cells with missing imprints and abnormal copy numbers in the urine cells of prostate cancer.

In summary, the detection model and system disclosed in the present disclosure intuitively express the performance of imprinting loss on samples of patients with prostate tumors. By in-situ marking of imprinting genes, the method is objective, intuitive, early and accurate Detecting changes in imprint (trace) genes, and can provide a quantified model, make a great contribution to the diagnosis of prostate tumors.

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

Claims

An imprinted gene grading model for prostate tumors, which 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 prostate tumors;

The imprinting gene is any one or a combination of at least two of Z1, Z8, Z11, or Z16. The imprinting gene Z1 is Gnas, the imprinting gene Z8 is Dcn, and the imprinting gene Z11 is Grb10. The imprinting gene Z16 is Snrpn / Snurf.
The imprinted gene grading model of prostate tumor according to claim 1, wherein the method for calculating imprinted genes by the model is as follows:

Calculate any one of Z1, Z8, Z11 or Z16, preferably any one of Z1, Z11 or Z16.
The imprinted gene grading model of prostate tumor according to claim 1 or 2, wherein the method for calculating imprinted genes by the model is as follows: Z1 or Z16 is calculated.
The imprinted gene grading model of prostate tumor according to any one of claims 1-3, wherein the method for calculating imprinted genes by the model is: any two of Z1, Z8, Z11 or Z16, preferably Z1 and Z8 combination, Z1 and Z16 combination, Z8 and Z16 combination or Z11 and Z16 combination.
The imprinting gene grading model of prostate tumor according to any one of claims 1-4, wherein the imprinting gene further comprises any one of Z3, Z4, Z5, Z6, Z10, or Z13 or a combination of at least two; Wherein, 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 Z10 is Gatm, and the imprinting gene Z13 is Sgce.
The imprinted gene grading model for prostate tumors according to any one of claims 1 to 5, wherein the method for calculating imprinted genes in the model is to calculate the combination of imprinted genes and calculate Z1, Z3, Z4, Z5, Z6, Z8 , Z10, Z11, Z13 and Z16 combination of ten imprinted genes.
The imprinted gene grading model of prostate tumor according to any one of claims 1 to 6, 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 Nucleus; the c is the hematoxylin staining of the cell, there are two red / brown markers in the nucleus, imprinting the gene-deficient nucleus; the d is the hematoxylin staining of the cell, there are more than two red / brown markers in the nucleus , Imprinting the nucleus with abnormal gene copy number.
The imprinted gene grading model for prostate tumors according to any one of claims 1 to 7, wherein the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level, and the imprinted gene total expression level are divided into five different levels.
The imprinted gene grading model of prostate tumor according to any one of claims 1-8, wherein the five different grades are for Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 Among the ten imprinted genes, the expression level of imprinted gene deletion, the abnormal expression of imprinted gene copy number and the total expression of imprinted gene were divided into five different levels.
The imprinted gene grading model of prostate tumor according to any one of claims 1-9, wherein the expression level of the imprinted gene deletion expression of Z1, the imprinted gene copy number abnormal expression amount and the total expression amount are divided into five different The level is:

Level 0: The expression level of the imprinting gene deletion of the imprinting gene Z1 is less than 10%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is less than 1%, or the total expression of the imprinting gene Z1 is less than 20% One or a combination of at least two;

Level I: The expression level of the imprinting gene deletion of the imprinting gene Z1 is 10-15%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is 1-2%, or the total expression level of the imprinting gene Z1 is 20 -30% of any one or a combination of at least two;

Level II: The expression level of the imprinting gene deletion of the imprinting gene Z1 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is 2-3%, or the total expression level of the imprinting gene Z1 is 30 -40% of any one or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting gene Z1 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is 3-5%, or the total expression level of the imprinting gene Z1 is 40 -Any one of 50% or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting gene Z1 is greater than 25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z1 is greater than 5%, or the total expression of the imprinting gene Z1 is greater than 50% One or a combination of at least two;

The five different levels of the expression level of the deletion of the imprinted gene for Z8, the abnormal expression of the copy number of the imprinted gene and the total expression are:

Level 0: The expression level of the imprinting gene deletion of the imprinting gene Z8 is less than 10%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is less than 1%, or the total expression of the imprinting gene Z8 is less than 15% One or a combination of at least two;

Level I: The expression level of the imprinting gene deletion of the imprinting gene Z8 is 10-15%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is 1-2%, or the total expression level of the imprinting gene Z8 is 15 Any one or a combination of at least two of -20%;

Level II: The expression level of the imprinting gene deletion of the imprinting gene Z8 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is 2-5%, or the total expression level of the imprinting gene Z8 is 20 -30% of any one or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting gene Z8 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is 5-8%, or the total expression level of the imprinting gene Z8 is 30 -40% of any one or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting gene Z8 is greater than 25%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z8 is greater than 8%, or the total expression of the imprinting gene Z8 is greater than 40% One or a combination of at least two;

The five different levels of the expression level of the imprinting gene deletion, the abnormal expression of the imprinting gene copy number and the total expression level for Z16 are:

Level 0: The expression level of the imprinting gene deletion of the imprinting gene Z16 is less than 15%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z16 is less than 1%, or the total expression of the imprinting gene Z16 is less than 30% One or a combination of at least two;

Level I: The expression of the imprinted gene deletion of the imprinted gene Z16 is 15-20%, the abnormal expression of the imprinted gene copy number of the imprinted gene Z16 is 1-3%, or the total expression of the imprinted gene Z16 is 30 -40% of any one or a combination of at least two;

Level II: The expression of the imprinted gene deletion of the imprinted gene Z16 is 20-25%, the abnormal expression of the imprinted gene copy number of the imprinted gene Z16 is 3-5%, or the total expression of the imprinted gene Z16 is 40 -Any one of 50% or a combination of at least two;

Level III: The expression level of the imprinting gene deletion of the imprinting gene Z16 is 25-30%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z16 is 5-8%, or the total expression level of the imprinting gene Z16 is 50 -60% of any one or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting gene Z16 is greater than 30%, the abnormal expression level of the imprinting gene copy number of the imprinting gene Z16 is greater than 8%, or the total expression of the imprinting gene Z16 is greater than 60% One or a combination of at least two;

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 Z3, Z11 and Z13 are:

Level 0: the expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is less than 10%, the abnormal expression of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is less than 1% or the imprinting genes Z3 and Z11 The total expression level with Z13 is less than 15% of any one or a combination of at least two;

Level I: The expression level of the deletion of the imprinted genes of the imprinted genes Z3, Z11 and Z13 is 10-15%, and the abnormal expression of the imprinted gene copy number of the imprinted genes Z3, Z11 and Z13 is 1-1.5% or the imprinted The total expression level of genes Z3, Z11 and Z13 is any one of 15-20% or a combination of at least two;

Level II: the expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is 15-20%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is 1.5-2.5% or the imprinting The total expression of genes Z3, Z11 and Z13 is any one of 20-30% or a combination of at least two;

Level III: the expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is 20-25%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is 2.5-4% or the imprinting The total expression level of genes Z3, Z11 and Z13 is any one of 30-40% or a combination of at least two;

Level IV: The expression level of the imprinting gene deletion of the imprinting genes Z3, Z11 and Z13 is greater than 25%, the abnormal expression level of the imprinting gene copy number of the imprinting genes Z3, Z11 and Z13 is greater than 4% or the imprinting genes Z3 and Z11 The total expression level with Z13 is greater than 40% of any one or a combination of at least two;

The five different levels of the expression levels of the deletion of the imprinted genes, the abnormal expression of the copy number of the imprinted genes, and the total expression for Z4, Z5, Z6, and Z10 are:

Level 0: The expression level of the deletion of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is less than 10%, the abnormal expression of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is less than 0.5% or the imprinted The total expression of genes Z4, Z5, Z6 and Z10 is less than 15% of any one or a combination of at least two;

Level I: The expression level of the deletion of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is 10-15%, and the abnormal expression amount of the imprinted genes of the imprinted genes Z4, Z5, Z6 and Z10 is 0.5-1.5% Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 is any one of 15-20% or a combination of at least two;

Level II: The expression level of the imprinting gene deletion of the imprinting genes Z4, Z5, Z6 and Z10 is 15-20%, and the abnormal expression level of the imprinting gene copy number of the imprinting genes Z4, Z5, Z6 and Z10 is 1.5-2.5% Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 is any one of 20-30% or a combination of at least two;

Level III: the expression level of the imprinting gene deletion of the imprinting genes Z4, Z5, Z6 and Z10 is 20-25%, and the abnormal expression level of the imprinting gene copy number of the imprinting genes Z4, Z5, Z6 and Z10 is 2.5-4% Or the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 is any one or a combination of at least two of 30-40%;

Level IV: The expression level of the imprinting gene deletion of the imprinting genes Z4, Z5, Z6 and Z10 is greater than 25%, and the abnormal expression of the imprinting gene copy number of the imprinting genes Z4, Z5, Z6 and Z10 is greater than 4% or the imprinting The total expression levels of genes Z4, Z5, Z6 and Z10 are greater than 40% of any one or a combination of at least two.
A device for detecting the degree of benign and malignant prostate tumors, wherein the imprinted gene grading model for prostate tumors according to any one of claims 1-10 is used, 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 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 amount, and by 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 degree of benign and malignant prostate tumors.
A method for detecting the degree of benign and malignant prostate tumors, wherein the imprinted gene grading model for prostate tumors according to any one of claims 1-10 is used, 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 expression is determined by calculating the expression level of the imprinted gene deletion, the abnormal expression of the imprinted gene copy number, and the total expression level, and by the imprinted gene grading model according to any one of claims 1-10, thereby expressing by the imprinted gene deletion The amount, imprinted gene copy number abnormal expression level and total expression level are used to diagnose the degree of benign and malignant prostate 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 a tissue, a biopsy sample or a urine exfoliated cell 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 coloring kit or a single-channel or multi-channel fluorescent kit, preferably a single-channel Red / brown colored kit or multi-channel fluorescent kit.
The method according to any one of claims 12 to 16, wherein the degree of benign and malignant diagnosis of prostate tumors is divided into benign prostate tumors, prostate cancer potential, early prostate cancer, intermediate prostate cancer, and advanced prostate cancer.
The method according to any one of claims 12-17, wherein the result of diagnosing the degree of benign and malignant prostate tumors is the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The imprinted gene deletion expression and imprinted gene copy number abnormal expression are less than the imprint of not more than 1 imprinted gene in level I or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 The gene deletion expression level is grade I and the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade I, then It is a benign prostate tumor;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the deletion of the imprinting gene of at least two imprinting genes of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, The imprinted gene copy number of at least 2 imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is level I or the imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of II and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any case in grade II, it is the potential of prostate cancer;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the deletion of the imprinting gene of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade II, The imprinted gene copy number abnormal expression of at least 2 imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II or imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of III and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any of the cases of grade III, which is early prostate cancer;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the imprinting gene deletion of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade III, At least two imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have an abnormal expression level of imprinted gene copy number of grade III or imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an expression level of imprinting gene deletion of grade IV and among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any of the grade IV, then it is mid-stage prostate cancer;

The result of the diagnosis of the benign and malignant degree of prostate tumor is that the expression level of the deletion of the imprinting gene of at least 2 imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV or imprinting The abnormal expression of the imprinted gene copy number of at least two imprinted genes in genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade IV, which is advanced prostate cancer.
The imprinted gene grading model of prostate tumor according to any one of claims 1-10 or the use of the device according to claim 11 for prostate tumor detection and / or treatment.
The use of the imprinted gene grading model for prostate tumors according to any one of claims 1-10 or the device according to claim 11 for the preparation of drugs or devices for detection and / or treatment of prostate tumors.
The use according to claim 20, wherein the degree of diagnosis of benign and malignant prostate tumors is divided into benign prostate tumors, prostate cancer potential, early prostate cancer, intermediate prostate cancer, and advanced prostate cancer.
The use according to claim 21, wherein the result of diagnosing the degree of benign and malignant prostate tumors is the lack of expression of the imprinted genes of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 Abnormal expression of copy number of imprinted genes is less than grade I or the imprinted gene deletion expression of not more than one imprinted gene in imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is I The level of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is not classified as a benign prostate tumor.

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the deletion of the imprinting gene of at least two imprinting genes of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, The imprinted gene copy number of at least 2 imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is level I or the imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of II and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any case in grade II, it is the potential of prostate cancer;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the deletion of the imprinting gene of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade II, The imprinted gene copy number abnormal expression of at least 2 imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II or imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an imprinting gene deletion expression level of III and imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any of the cases of grade III, which is early prostate cancer;

The result of the diagnosis of the degree of benign and malignant prostate tumors is that the expression level of the imprinting gene deletion of at least two imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade III, At least two imprinted genes of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have an abnormal expression level of imprinted gene copy number of grade III or imprinted genes Z1, Z3, Z4, Z5, Z6 No more than 1 imprinting gene in Z8, Z10, Z11, Z13, and Z16 has an expression level of imprinting gene deletion of grade IV and among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 Abnormal expression of the imprinted gene copy number of no more than one imprinted gene is any of the grade IV, then it is mid-stage prostate cancer;

The result of the diagnosis of the benign and malignant degree of prostate tumor is that the expression level of the deletion of the imprinting gene of at least 2 imprinting genes of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV or imprinting The abnormal expression of the imprinted gene copy number of at least two imprinted genes in genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade IV, which is advanced prostate cancer.