CN111172278B

CN111172278B - Grading model for detecting benign and malignant degree of prostate tumor and application thereof

Info

Publication number: CN111172278B
Application number: CN201811340820.XA
Authority: CN
Inventors: 成彤; 周宁
Original assignee: Lisen Imprinting Diagnosis Technology Wuxi Co ltd
Current assignee: Lisen Imprinting Diagnosis Technology Wuxi Co ltd
Priority date: 2018-11-12
Filing date: 2018-11-12
Publication date: 2024-02-09
Anticipated expiration: 2038-11-12
Also published as: CN111172278A; WO2020098604A1

Abstract

The invention relates to a grading model for detecting prostate tumor and application thereof, wherein the model grades the change of a imprinting gene in the prostate tumor by calculating the deletion expression level of the imprinting gene, the abnormal copy number expression level of the imprinting gene and the total expression level of the imprinting gene. The detection model and the device of the invention show the appearance of imprinting missing on the tissue and cell samples of prostate tumor patients in an intuitive way, objectively, intuitively, early and accurately detect the change of imprinting (trace) genes by the method of marking the imprinting genes in situ, and can provide a quantized model, thereby making great contribution to the diagnosis of prostate tumor.

Description

Grading model for detecting benign and malignant degree of prostate tumor and application thereof

Technical Field

The invention relates to the field of biotechnology, relates to the field of gene diagnosis, relates to a grading model and application thereof, relates to a grading model for detecting benign and malignant degrees of prostate tumors and application thereof, and in particular relates to a grading model of a group of imprinting genes in detecting benign and malignant degrees of prostate tumors and a device formed by the grading model.

Background

Prostate cancer is a major health threat for elderly men, with lower morbidity before the age of 55 and with increasing morbidity with age after the age of 55. The number of new prostate cancer patients in the world is 109.5 ten thousand and the death rate is 30.7 ten thousand each year, and the new prostate cancer patients are mainly concentrated in developed countries in Europe and America, and the American prostate cancer is the first incidence rate of male cancers. 6.0 ten thousand patients and 2.7 ten thousand deaths are increased in China every year, which is far lower than European and American deaths, but the trend of the deaths is gradually growing in recent years. The survival time of the prostate cancer patients is closely related to the cancer stage in diagnosis, the five-year survival rate of early prostate cancer reaches 99 percent after treatment, the ten-year survival rate can reach 95 percent, and the five-year survival rate of late prostate cancer is only 28 percent, so that the early diagnosis and early treatment of the prostate cancer have very important significance. At present, early diagnosis means of prostate cancer mainly comprise digital rectal diagnosis and serum PSA detection. The rectal finger diagnosis can diagnose the larger prostate cancer, but the smaller prostate cancer is easy to leak diagnosis, and the accuracy is about 50-75%; serum PSA tests diagnose prostate cancer by the amount of Prostate Specific Antigen (PSA) in the serum, but the accuracy is not very high since the increase in PSA levels in the serum is not significant in 30% of prostate cancer patients, and also in some cases in prostatitis. The only method for diagnosing the prostate cancer is prostate puncture cytology at present, but small prostate cancer is difficult to accurately puncture a cancer focus, and in order to increase the accuracy, the current diagnosis standard usually requires 10-12 punctures to be performed on each patient, so that great pain is caused. Therefore, there is an urgent need to develop more sensitive and accurate early detection means for prostate cancer.

Traditional pathology makes a judgment on the benign or malignant diagnosis of cells based on the relationship of cell size, morphology, invasiveness and surrounding cell tissue. It has a great limitation in the discovery of early changes in cells (cancers), and thus a method for diagnosing cancer at the cellular molecular level has become a research hotspot at one time. With the continuous intensive research in the field of molecular biology, more and more molecular detection techniques are being applied to cancer diagnosis.

Cancer is the generation of uncontrolled cell growth/division caused by epigenetic changes and genetic variations that accumulate over time. Traditional pathological diagnosis makes a judgment on benign and malignant prostate tumors based on the size, morphology and structural variation of cells and tissues. With the development and penetration of molecular biology, more and more molecular detection techniques are applied to the detection of prostate cancer. From the analysis of the progression of cancer, changes in molecular level (epigenetic and genetic) are far ahead of variations in cell morphology and tissue structure. Molecular biological assays are therefore more sensitive to early detection of cancer.

Genomic imprinting is one way of gene regulation in epigenetics. It is characterized in that by methylating alleles from specific parents, one gene is expressed while the other is put into a gene silencing state. This kind of gene is called a blot (marker) gene. A print deletion is an epigenetic change in which the print gene demethylation causes the activation of the silent state allele and the initiation of gene expression. Numerous studies have shown that this phenomenon (print loss) is common to all types of cancer and occurs earlier in time than changes in cell and tissue morphology. Meanwhile, in healthy cells, the proportion of imprinting missing is extremely low, in sharp contrast to cancer cells. Therefore, the methylation state of the imprinted gene can be used as a pathological marker, and the abnormal state of the cells can be analyzed by a specific molecular detection technology.

For the above reasons, new detection systems and detection models are needed for the current diagnosis of prostate cancer, which resolve the molecular marker changes present at the cellular level of prostate cancer based on patient biopsy samples, thereby providing more accurate prognosis and diagnosis information.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention provides a grading model for detecting benign and malignant degrees of prostate tumors and application thereof, wherein the detection device and the model are used for intuitively observing the change of marking genes of the prostate tumors at early stage under single cell and tissue level so as to judge the benign and malignant degrees of the prostate tumors.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a imprinted gene classification model for prostate tumor, which classifies the expression state of imprinted genes by calculating the total expression amount of imprinted genes, the deletion expression amount of imprinted genes and the variation of the copy number abnormal expression amount of imprinted genes in prostate cancer;

wherein the imprinting gene is any one or the combination of at least two of Z1, Z8, Z11 or Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z8 is Dcn, the imprinting gene Z11 is Grb10, and the imprinting gene Z16 is Snrpn/Snurf.

In the present invention, the inventors found that the diagnostic sensitivity to prostate cancer can be up to 70.0% or more by calculating the total expression amount of the imprinting gene, the deletion expression amount of the imprinting gene, and the abnormal expression amount of the copy number of the imprinting gene in prostate tumor of any one of Z1, Z8, Z11, and Z16.

According to the present invention, if only one imprinted gene is detected by preliminary detection, any one of Z1, Z8, Z11 and Z16, preferably any one of Z1, Z11 or Z16, and more preferably Z1 or Z16, can be detected.

In the present invention, the inventors found that the diagnostic sensitivity to prostate cancer can be up to 82.9% if one Z1 imprinting gene is detected alone, up to 70.0% if one Z8 imprinting gene is detected alone, up to 78.0% if one Z11 imprinting gene is detected alone, and up to 95.1% if one Z16 imprinting gene is detected alone.

According to the invention, the method for calculating the imprinting gene by the model comprises the following steps: if a combination of two imprinted genes of the imprinted genes is detected, the combination may be any two of Z1, Z8, Z11 and Z16, preferably a combination of Z1 and Z8, a combination of Z1 and Z16, a combination of Z8 and Z16 or a combination of Z11 and Z16.

In the present invention, the inventors found that sensitivity can be further improved by calculating the total expression amount of two or more imprinted genes, the amount of expression of deletion of imprinted genes and the amount of abnormal expression of copy numbers of imprinted genes, that the diagnostic sensitivity to prostate cancer can be 82.5% or more by detecting the combination of any two imprinted genes among imprinted genes Z1, Z8, Z11 and Z16, that the diagnostic sensitivity to prostate cancer can be 92.5% or more by detecting the combination of Z1 and Z8, that the diagnostic sensitivity to prostate cancer can be 99.0% or more by detecting the combination of Z1 and Z16, the combination of Z8 and Z16, and the combination of Z11 and Z16.

According to the invention, the imprinted gene further comprises any one or a combination of at least two of Z3, Z4, Z5, Z6, Z10 or Z13; wherein the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z5 is test, the imprinting gene Z6 is Plagl1, the imprinting gene Z10 is Gatm, and the imprinting gene Z13 is Sgce.

In the invention, the inventor finds that on the basis of using the Z1, Z8, Z11 and Z16 genes for detection, the Z3, Z4, Z5, Z6, Z10 and Z13 genes are added for joint diagnosis, so that not only is the detection accuracy improved, but also false positives can be further avoided by adding other probes for auxiliary diagnosis, the detection accuracy can be further improved, and therefore, the accurate classification and judgment of all prostate tumor samples can be realized.

According to the invention, the method for calculating the imprinting gene by the model comprises the following steps: combinations of the imprinted genes were calculated, and combinations of the Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 genes were calculated.

In the invention, the imprinted gene is deleted, two red/brown marks exist in the nucleus after the cells are subjected to hematoxylin staining, the imprinted gene copy number abnormality is that more than two red/brown marks exist in the nucleus after the cells are subjected to hematoxylin staining, and the copy number abnormality is caused by abnormal gene replication of cancer cells, so that the gene appears as triploid or even higher polyploid during expression.

In the present invention, the imprinting gene and the imprinting gene are the same concept, and the same meaning is expressed, and substitution is possible.

Preferably, the formulas for calculating the total expression amount of the imprinted gene, the deletion expression amount of the imprinted gene, and the copy number abnormal expression amount of the imprinted gene are as follows:

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

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

imprinted gene deleted gene expression amount (LOI) =c/(b+c+d) ×100%;

gene expression level (CNV) of the imprinted gene copy number abnormality=d/(b+c+d) ×100%;

Wherein a is a cell nucleus in which no marker exists in the cell nucleus and the imprinted gene is not expressed after the cell is subjected to hematoxylin staining; b is a cell nucleus with a red/brown mark in the cell nucleus and a marking gene after the cell is subjected to hematoxylin staining; c is a cell nucleus with two red/brown marks in the cell nucleus and marking the gene deletion after the cell is subjected to hematoxylin staining; and d is a cell nucleus with more than two red/brown marks in the cell nucleus and abnormal imprinted gene copy number after the cell is subjected to hematoxylin staining.

In the invention, the hematoxylin-stained marker is selected from red or brown, and the stained marker with other colors can be used for calculating the expression level of the imprinting gene, the deletion expression level of the imprinting gene and the abnormal expression level of the copy number of the imprinting gene.

In the invention, the probe is used for judging whether the imprinting gene exists, the imprinting gene is missing or the copy number is abnormal in each cell nucleus through in-situ hybridization and a Hemotoxy (hematoxylin) cell nucleus staining amplification signal under a 40-multiplied or 60-multiplied microscope, and the tumor benign and malignant degree of the sample is judged through calculating the gene expression quantity of the imprinting gene missing gene and the gene expression quantity of the imprinting gene copy number abnormality. Since the sections were only 10 μm, approximately 20% of the nuclei seen under the microscope were incomplete nuclei, i.e. there was a possibility of partial false negatives.

According to the present invention, the total expression level of the imprinted gene, the deletion expression level of the imprinted gene, and the copy number abnormal expression level of the imprinted gene are classified into five different levels, and at least 1200 cells are counted in the most positive region of the sample expression by each probe, and the deletion expression level of the imprinted gene, the copy number abnormal expression level of the imprinted gene, and the total expression level of the imprinted gene are respectively classified into five different levels for the ten imprinted genes of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16.

According to the present invention, the five different grades of the deletion expression level of the imprinting gene, the abnormal expression level of the copy number of the imprinting gene and the total expression level for Z1 are:

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

stage I: the imprinting gene Z1 has a imprinting gene deletion expression level of 10-15%, the imprinting gene copy number abnormal expression level of 1-2% of the imprinting gene Z1 or the total expression level of 20-30% of the imprinting gene Z1, or a combination of at least two of them;

Stage II: the imprinting gene Z1 has a imprinting gene deletion expression level of 15-20%, the imprinting gene copy number abnormal expression level of 2-3% of the imprinting gene Z1 or the total expression level of 30-40% of the imprinting gene Z1, or a combination of at least two of them;

class III: the imprinting gene Z1 has a imprinting gene deletion expression level of 20-25%, the imprinting gene Z1 has an imprinting gene copy number abnormal expression level of 3-5%, or the imprinting gene Z1 has a total expression level of 40-50%, or a combination of at least two thereof;

grade IV: any one or a combination of at least two of the above described imprinted gene Z1, the imprinted gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 5% of the imprinted gene Z1 or the total expression level of more than 50% of the imprinted gene Z1.

According to the present invention, the five different grades of the deletion expression level of the imprinting gene, the abnormal expression level of the copy number of the imprinting gene and the total expression level for Z8 are:

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

Stage I: the imprinting gene Z8 has a imprinting gene deletion expression level of 10-15%, the imprinting gene copy number abnormal expression level of 1-2% of the imprinting gene Z8 or the total expression level of 15-20% of the imprinting gene Z8, or a combination of at least two of them;

stage II: the imprinting gene Z8 has a imprinting gene deletion expression level of 15-20%, the imprinting gene copy number abnormal expression level of 2-5% or the imprinting gene Z8 has a total expression level of 20-30% or a combination of at least two of them;

class III: the imprinting gene Z8 has a imprinting gene deletion expression level of 20-25%, the imprinting gene copy number abnormal expression level of 5-8% or the imprinting gene Z8 has a total expression level of 30-40% or a combination of at least two of them;

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z8 being more than 25%, the imprinting gene copy number abnormal expression level of the imprinting gene Z8 being more than 8%, or the total expression level of the imprinting gene Z8 being more than 40%.

According to the present invention, the five different grades of the deletion expression level of the imprinting gene, the abnormal expression level of the copy number of the imprinting gene and the total expression level for Z16 are:

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

stage I: the imprinting gene Z16 has a imprinting gene deletion expression level of 15-20%, the imprinting gene Z16 has an imprinting gene copy number abnormal expression level of 1-3% or the imprinting gene Z16 has a total expression level of 30-40% or a combination of at least two of them;

stage II: the imprinting gene Z16 has an imprinting gene deletion expression level of 20-25%, an imprinting gene copy number abnormal expression level of 3-5% of the imprinting gene Z16, or a total expression level of 40-50% of the imprinting gene Z16, or a combination of at least two thereof;

class III: the imprinting gene Z16 has an imprinting gene deletion expression level of 25-30%, an imprinting gene copy number abnormal expression level of 5-8% of the imprinting gene Z16, or a total expression level of 50-60% of the imprinting gene Z16, or a combination of at least two thereof;

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z16 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z16 being more than 8%, or the total expression level of the imprinting gene Z16 being more than 60%.

According to the present invention, the five different grades of the deletion expression amount of the imprinting gene, the copy number abnormality expression amount of the imprinting gene, and the total expression amount for Z3, Z11, and Z13 are divided into:

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

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

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

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

Grade IV: any one or a combination of at least two of the imprinting genes Z3, Z11 and Z13 having a imprinting gene deletion expression level of more than 25%, the imprinting genes Z3, Z11 and Z13 having an imprinting gene copy number abnormal expression level of more than 4%, or the imprinting genes Z3, Z11 and Z13 having a total expression level of more than 40%;

in the present invention, the imprinted gene deletion expression level, imprinted gene copy number abnormal expression level and total expression level of the imprinted genes Z3, Z11 and Z13 are independent of each other.

According to the present invention, the five different grades of the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level for Z4, Z5, Z6 and Z10 are divided into:

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

stage I: the imprinted gene deletion expression level of the imprinted genes Z4, Z5, Z6 and Z10 is 10-15%, the imprinted gene copy number abnormal expression level 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 or a combination of at least two of 15-20%;

Stage II: the imprinted gene deletion expression level of the imprinted genes Z4, Z5, Z6 and Z10 is 15-20%, the imprinted gene copy number abnormal expression level of the imprinted 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 or a combination of at least two of 20-30%;

class III: the imprinted gene deletion expression level of the imprinted genes Z4, Z5, Z6 and Z10 is 20-25%, the imprinted gene copy number abnormal expression level of the imprinted 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%;

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

in the present invention, the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 are independent of each other.

In a second aspect, the present invention provides a device for detecting benign and malignant prostate tumor, comprising the following units:

(1) Sampling unit: obtaining a sample to be tested;

(2) Probe design unit: designing a specific primer according to the imprinting gene sequence;

(3) And a detection unit: performing in situ hybridization on the probe in the step (2) and a sample to be detected;

(4) Analysis unit: microscopic imaging analysis of the expression of the imprinted gene;

wherein the analysis unit calculates the total expression level of the imprinted gene, the abnormal expression level of the imprinted gene deletion expression level and the abnormal expression level of the imprinted gene copy number, and the imprinted gene classification model according to the first aspect, so as to judge the benign and malignant degree of the prostate tumor by the grades of the imprinted gene deletion expression level, the abnormal expression level of the imprinted gene copy number and the total expression level.

In the invention, the imprinted gene is deleted, two red/brown marked cell nuclei exist in the cell nuclei after the cells are subjected to hematoxylin staining, the imprinted gene copy number abnormality is the condition that more than two red/brown marked cell nuclei exist in the cell nuclei after the cells are subjected to hematoxylin staining, and the copy number abnormality is caused by abnormal gene replication of cancer cells, so that the gene appears as triploid or even higher polyploid when expressed.

In the invention, the hematoxylin-stained marker is selected from red or brown, and the stained marker with other colors can be used for calculating the total expression quantity of the imprinted gene, the deletion expression quantity of the imprinted gene and the abnormal expression quantity of the imprinted gene copy number.

The detection device is used for intuitively observing the change of the marking (trace) genes of the prostate tumor at the early stage under the cellular and tissue level so as to judge the benign and malignant degree of the tumor, and provides the most favorable treatment opportunity for early-stage prostate tumor patients.

According to the invention, the sample to be tested in step (1) is derived from human tissue and/or cells.

In the invention, the sample to be tested can be selected according to the needs of a person skilled in the art as long as the RNA is subjected to timely fixation, and the sample to be tested comprises any one or a combination of at least two of paraffin sections of tissues, puncture biopsy samples or urine exfoliated cell samples.

The paraffin section of the tissue comprises the specific operation steps of obtaining a human tumor tissue sample, fixing the tissue sample by 10% neutral formalin in time, embedding the tissue sample by paraffin, cutting the tissue sample into 10 mu m thick, and preparing a tissue sheet by using a positively charged slide; since only 10 μm thick, a part of the nuclei are incomplete under the microscope, so that a partially false negative gene deletion occurs.

The specific operation steps of the biopsy sample are that human cells are obtained through puncturing, and the biopsy sample is fixed by 10% neutral formalin in time.

The specific operation steps of the urine exfoliated cell sample are that urine of a patient is obtained after prostate is massaged, cells are collected through centrifugation, and the urine exfoliated cell sample is timely fixed by 10% neutral formalin.

In the invention, because the puncture biopsy has little harm to a patient, the sampling process is simple, compared with the blood circulation characteristic, the puncture biopsy can be positioned, and the puncture biopsy has special advantages as an experimental sample.

In the invention, urine shed cells have no harm to patients, the sampling process is simple, and the urine shed cells have special advantages as experimental samples.

Preferably, the sample to be tested is a puncture biopsy sample or a urine shed cell sample.

Preferably, the imprinting genes are Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z5 is test, 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.

In the invention, the imprinting genes Z1 (Gnas), Z3 (Peg 10), Z4 (Igf 2 r), Z5 (test), Z6 (Plagl 1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb 10), Z13 (Sgce), and Z16 (Snrpn/Snurf) are expressed in normal tumor cell tissues to different degrees, and the expression quantity and imprinting state are obviously changed when malignant lesions occur.

In the present invention, the design probes were designed based on the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16, that is, gnas, peg10, igf2r, test, plagl1, dcn, gatm, grb10, sgce and Snrpn/Snurf, specifically, a sequence was selected as a probe within the intron of each gene, and a specific probe was designed by Advanced CellDiagnostics company.

Preferably, the in situ hybridization employs an RNAscope in situ hybridization method.

Preferably, the RNAscope in situ hybridization method uses a single-channel or multi-channel chromogenic kit or a single-channel or multi-channel fluorescent kit, preferably a single-channel red/brown chromogenic kit or a multi-channel fluorescent kit.

In the invention, the multi-channel coloring kit or the multi-channel fluorescent kit comprises two or more than two channels of coloring kits or fluorescent kits, and the two channels of coloring kits or the multi-channel fluorescent kits can use two imprinting gene probes or the joint expression of imprinting genes and other genes or even the comprehensive expression of a plurality of imprinting genes and non-imprinting genes.

According to the present invention, the formulas for calculating the total expression amount of the imprinted gene, the deletion expression amount of the imprinted gene and the copy number abnormal expression amount of the imprinted gene in the model are as follows:

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

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

imprinted gene deleted gene expression amount (LOI) =c/(b+c+d) ×100%;

In the invention, the probe is used for judging whether the imprinting gene exists, the imprinting gene is missing or the copy number is abnormal in each cell nucleus through in-situ hybridization and a Hemotoxy (hematoxylin) cell nucleus staining amplification signal under a 40-multiplied or 60-multiplied microscope, and the tumor benign and malignant degree of the sample is judged through calculating the total imprinting gene expression quantity, the imprinting gene missing gene expression quantity and the gene expression quantity with abnormal imprinting gene copy number. Since the sections were only 10 microns, approximately 20% of the nuclei seen under the microscope were incomplete nuclei, i.e. there was a possibility of partial false negatives.

According to the present invention, the imprinted gene deletion expression level, imprinted gene copy number abnormal expression level and total expression level are classified into five different levels.

The five different grades are that at least 1200 cells are counted in a region where each probe of the sample expresses most positive, and the imprinted gene deletion expression amounts, imprinted gene copy number abnormality expression amounts and total expression amounts of ten imprinted genes for Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are respectively divided.

The five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity aiming at Z1 are divided into:

The five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity aiming at Z8 are divided into:

The five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity of the imprinting gene aiming at Z16 are divided into the following grades:

The five different grades of the deletion expression quantity of the imprinting genes, the abnormal expression quantity of the copy number of the imprinting genes and the total expression quantity of the imprinting genes aiming at Z3, Z11 and Z13 are divided into:

The five different grades of the deletion expression quantity of the imprinting genes, the copy number abnormal expression quantity of the imprinting genes and the total expression quantity aiming at Z4, Z5, Z6 and Z10 are divided into:

According to the present invention, the judging benign and malignant degree of prostate tumor is classified into benign tumor, prostate cancer potential, early stage prostate cancer, intermediate stage prostate cancer and advanced stage prostate cancer.

Preferably, the result of the determination of the benign and malignant degree of the prostate tumor is that the imprinting gene deletion expression amount and the imprinting gene copy number abnormal expression amount of each of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are smaller than the I-level or that the imprinting gene deletion expression amount of not more than 1 imprinting gene of the imprinting genes Z1, Z3, Z4, Z5, Z8, Z10, Z11, Z13 and Z16 is I-level and that the imprinting gene copy number abnormal expression amount of not more than 1 imprinting gene of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is I-level, and is a benign tumor;

preferably, the prostate cancer is a prostate cancer in which the result of the determination of the benign and malignant extent of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting gene among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II;

Preferably, the prostate cancer is one in which the result of the determination of the benign and malignant degree of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z16 is level II, the imprinting gene copy number abnormality expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z13, and Z16 is level II, or that the imprinting gene deletion expression level of not more than 1 imprinting gene among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is level III, and the imprinting gene copy number abnormality expression level of not more than 1 imprinting gene among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is level III;

preferably, the prostate cancer is a prostate cancer in the middle stage if the result of the determination of the benign or malignant extent of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting gene among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV;

Preferably, the result of the determination of the benign and malignant extent of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV-level or that the imprinting gene copy number abnormality expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV-level, and it is advanced prostate cancer.

In a third aspect, the present invention provides a imprinted gene fractionation model as described in the first aspect or a device as described in the second aspect for use in the preparation of a medicament or kit for prostate tumor detection and/or treatment.

According to the present invention, the benign and malignant degree of prostate tumor is judged to be classified into benign tumor, prostate cancer potential, early-stage prostate cancer, mid-stage prostate cancer and advanced-stage prostate cancer;

according to the present invention, the result of judging the benign and malignant degree of prostate tumor is that the imprinting gene deletion expression amount and the imprinting gene copy number abnormal expression amount of each of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are smaller than the imprinting gene deletion expression amount of the I-class or not more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z5, Z6, Z11, Z13 and Z16 are the I-class and the imprinting gene copy number abnormal expression amount of not more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is the I-class, and is a benign tumor;

According to the present invention, the prostate cancer potential is determined if the result of judging the benign and malignant degree of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, or that the imprinting gene deletion expression level of not more than 1 imprinting gene among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, and the imprinting gene copy number abnormality expression level of not more than 1 imprinting gene among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of level II;

according to the present invention, the prostate cancer is a prostate cancer in which the result of judging the benign and malignant extent of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level III, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting gene among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level III, or in any one of early stages;

According to the present invention, the prostate cancer is a prostate cancer in the middle stage if the result of judging the benign or malignant extent of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV;

according to the present invention, the result of judging the benign and malignant extent of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV-level or that the imprinting gene copy number abnormality expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV-level, and it is advanced prostate cancer.

Compared with the prior art, the invention has the following beneficial effects:

(1) The detection model and the device of the invention show the appearance of imprinting missing on the sample of the prostate tumor patient in an intuitive way, objectively, intuitively, early and accurately detect the change of imprinting (trace) genes by the method of marking the imprinting genes in situ, and can provide a quantized model to make great contribution to the diagnosis of prostate tumor;

(2) The detection device can obtain the judgment of benign and malignant degree of the prostate tumor by puncturing cells or urine shed cells before the operation of the prostate tumor patient, thereby providing basis for the operation and the accurate treatment, and being a revolutionary breakthrough in the field of cell molecules for diagnosing the prostate tumor;

(3) The invention can carry out early screening on the prostate cancer through urine, has simple sample collection method, is suitable for large-scale physical examination and general investigation, and monitoring the postoperative and drug curative effects of the prostate cancer, especially for tracking follow-up visit of suspected recurrent patients, can strive for time, and makes great contribution to saving the lives of the patients;

(4) The detection method is different from an immunohistochemical method, false positive and other negative effects are reduced, and moreover, the detection method can be used for guiding later treatment and medication through a target medicament or a technical method for silencing, removing and rearranging the gene by the found prostate tumor related imprinted gene deletion site.

Drawings

FIG. 1 is a pathological section of prostate cancer of hematoxylin stained nuclei according to the present invention, wherein a is that after the cells are hematoxylin stained, no marker exists in the nuclei and the imprinted gene is not expressed; the step b is that after the cells are subjected to hematoxylin staining, a red/brown mark exists in the nuclei of the cells, and a mark gene exists; the step c is that after the cells are subjected to hematoxylin staining, two red/brown marks exist in the nuclei of the cells, and the imprinted genes are deleted; the step d is that after the cells are subjected to hematoxylin staining, more than two red/brown marks exist in the nuclei of the cells, and the number of copies of the imprinted genes is abnormal;

fig. 2 (a) is the expression state of 10 genes in the pathological section of the grade 0 prostate tumor, fig. 2 (b) is the expression state of 10 genes in the pathological section of the grade I prostate cancer, fig. 2 (c) is the expression state of 10 genes in the pathological section of the grade II prostate cancer, fig. 2 (d) is the expression state of 10 genes in the pathological section of the grade III prostate cancer, and fig. 2 (e) is the expression state of 10 genes in the pathological section of the grade IV prostate cancer;

fig. 3 (a) shows the intensities of the imprinting deletions of the imprinting genes Z1, Z8, Z11 and Z16 on the prostate cancer, fig. 3 (b) shows the intensities of the imprinting genes Z1, Z8, Z11 and Z16 on the copy number abnormality of the prostate cancer, fig. 3 (c) shows the intensities of the total expression amounts of the imprinting genes Z1, Z8, Z11 and Z16 on the prostate cancer, fig. 3 (d) shows the intensities of the imprinting deletions of the imprinting genes Z3, Z4, Z5, Z6, Z10 and Z13 on the prostate cancer, fig. 3 (e) shows the intensities of the copy number abnormality of the imprinting genes Z3, Z4, Z6, Z10 and Z13 on the prostate cancer, fig. 3 (f) shows the total expression amounts of the imprinting genes Z3, Z4, Z5, Z6, Z10 and Z13 on the prostate cancer, where LOI is the gene expression amount of the imprinting gene copy number abnormality, and CNV is the total expression amount of the imprinting gene TE;

FIG. 4 (a) is the intensity of the imprinting gene Z1 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (b) is the intensity of the imprinting gene Z8 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (c) is the intensity of the imprinting gene Z11 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (d) is the intensity of the imprinting gene Z16 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (e) is the intensity of the imprinting gene Z3 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (f) is the intensity of the imprinting gene Z4 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (g) is the intensity of the imprinting gene Z5 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (h) is the intensity of the imprinting gene Z6 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (i) is the intensity of the imprinting gene Z13 imprinting, the copy number abnormality and the total amount of expression, wherein the gene I is the gene expression of the imprinting gene is the imprinting gene CNTE imprinting;

FIG. 5 (a) shows the distribution ranges and classification criteria of the imprinting gene Z1 for 44 cases of prostate cancer pathological sections, FIG. 5 (b) shows the distribution ranges and classification criteria of the imprinting gene Z8 for 44 cases of prostate cancer pathological sections, FIG. 5 (c) shows the distribution ranges and classification criteria of the imprinting gene Z11 for 44 cases of prostate cancer pathological sections, FIG. 5 (d) shows the distribution ranges and classification criteria of the imprinting gene Z16 for 44 cases of prostate cancer pathological sections, the distribution ranges and classification criteria of the imprinting gene Z3 for 44 cases of prostate cancer pathological sections, FIG. 5 (f) shows the distribution ranges and classification criteria of the imprinting gene Z4 for 44 cases of prostate cancer pathological sections, FIG. 5 (g) shows the distribution ranges and classification criteria of the imprinting gene Z5 for 44 cases of prostate cancer pathological sections, FIG. 5 (h) shows the distribution ranges and classification criteria of the imprinting gene Z6 for 44 cases of prostate cancer pathological sections, FIG. 5 (i) shows the distribution ranges and classification criteria of the imprinting gene Z10 for 44 cases of prostate cancer pathological sections, the distribution ranges and classification criteria of the imprinting gene Z13 for 44 cases of prostate cancer pathological sections, and FIG. 5 (j) shows the distribution ranges and classification criteria of the imprinting gene Z13 for 44 cases of prostate cancer pathological sections, LOI is the gene expression quantity of the imprinting gene deletion, CNV is the gene expression quantity of the imprinting gene with abnormal copy number, and TE is the total expression quantity of the imprinting gene;

FIG. 6 (a) shows the expression state of 10 genes in a urine shed cell sample of benign prostate tumor, and FIG. 6 (b) shows the expression state of 10 genes in a urine shed cell sample of prostate cancer.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below by the specific embodiments in combination with the accompanying drawings, but the invention is not limited to the examples.

EXAMPLE 1 imprinted Gene analysis of prostate cancer

The detection method of the imprinting gene comprises the following steps:

(1) Obtaining tissue cell sections (10 microns) of the prostate cancer, placing the tissue cell sections into 10% neutral formalin solution for fixation to prevent RNA degradation, wherein the fixation time is 24 hours, paraffin embedding (FFPE), the slide glass needs positive charges for removing the slide glass, and the sections are baked for more than 3 hours in a 40 ℃ oven;

(2) Dewaxing according to a sample treatment method of RNASCope, sealing the endogenous peroxidase activity in the sample, enhancing the permeability and exposing RNA molecules;

(3) Designing a probe: designing a specific primer according to the imprinting gene sequence;

the design probes were designed based on the imprinted genes Z1 (Gnas), Z3 (Peg 10), Z4 (Igf 2 r), Z5 (test), Z6 (Plagl 1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb 10), Z13 (Sgce) and Z16 (Snrpn/Snuf), with a specific sequence selected as a probe within the introns of each gene, and the specific probes were designed by Advanced CellDiagnostics company.

(4) Carrying out RNA SCope in situ hybridization on the probe in the step (3) and a sample to be detected through a kit;

(5) Signal amplification and hematoxylin staining, and analyzing the expression condition of the imprinted gene by using a microscope for imaging;

the formula for calculating the total expression quantity of the imprinting genes, the deletion expression quantity of the imprinting genes and the abnormal expression quantity of the copy number of the imprinting genes in the model is as follows:

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

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

imprinted gene deleted gene expression amount (LOI) =c/(b+c+d) ×100%;

as shown in fig. 1, a, b, c, d, a is a cell nucleus with no marker in the cell nucleus and no expressed imprinted gene after the cell is subjected to hematoxylin staining; b is a cell nucleus with a red/brown mark in the cell nucleus and a marking gene after the cell is subjected to hematoxylin staining; c is a cell nucleus with two red/brown marks in the cell nucleus and marking the gene deletion after the cell is subjected to hematoxylin staining; and d is a cell nucleus with more than two red/brown marks in the cell nucleus and abnormal imprinted gene copy number after the cell is subjected to hematoxylin staining.

From FIGS. 2 (a) -2 (e), it can be seen that the proportion of cells with a loss of imprint (two signal points in the nucleus) and an abnormality in copy number (three or more signal points in the nucleus) gradually increases with the increase in malignancy in the samples from grade 0 to grade IV.

EXAMPLE 2 imprinted Gene analysis of puncture biopsy samples

The puncture biopsy sample is obtained by puncture of somatic cells of a suspected patient, and is fixed in 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 fig. 3 (a) -3 (f), the intensity and status of the sensitivity of each gene Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, Z16 to prostate cancer or the absence of an imprint corresponding to prostate cancer expression are different.

Specifically, the sensitivity of each imprinting gene to prostate cancer is as shown in fig. 4 (a) -4 (j), and as can be seen from fig. 4 (a) -4 (d), the imprinting deletion and copy number abnormality of imprinting gene Z1 rapidly rise in malignant potential and early stage prostate cancer, slow down in the rising speed of middle stage prostate cancer, and rapidly rise to a very high level in late stage prostate cancer, and the expression level of imprinting gene Z1 rapidly rises in malignant potential and early stage prostate cancer, slow down in the rising speed of middle and late stage prostate cancer, and reach a higher level; the imprinting deletion, copy number abnormality and expression amount increase of the imprinting gene Z8 are rapidly increased in the early stage of the prostatic cancer, and the rising speed is slowed down in the middle and late stage of the prostatic cancer, so that the higher level is achieved; the imprinting deletion, copy number abnormality and expression amount increase of the imprinting gene Z11 are rapidly increased in the early stage of the prostatic cancer, and the rising speed is slowed down in the middle and late stage of the prostatic cancer, so that the high level is achieved; the imprinting deletion of the imprinting gene Z16 begins to appear in the malignant potential stage, slowly rises in early-stage prostate cancer, rapidly rises in middle-stage prostate cancer, and slowly rises to reach a very high level in the late-stage prostate cancer stage, the copy number abnormality of the imprinting gene Z16 rapidly rises to reach a very high level in the malignant potential stage, and continuously slowly rises in the development process of early-stage to late-stage prostate cancer, the expression quantity of the imprinting gene Z16 increases rapidly in the malignant potential and early-stage prostate cancer stage, and the rising speed of the imprinting gene Z16 is slowly reduced in the middle-stage and late-stage prostate cancer stage, so that the level is very high;

As can be seen from fig. 4 (e) -4 (j), the imprinting deletion and copy number abnormality of the imprinting gene Z3 began to appear in early stage of prostate cancer, did not significantly rise in middle and late stage of prostate cancer, the increase in the expression level of the imprinting gene Z3 began to appear in early stage of prostate cancer, and slowly rise in middle and late stage of prostate cancer, and the level was also lower; the imprinting deletion and copy number abnormality of the imprinting gene Z4 rapidly increase in early-stage prostate cancer, slow down in mid-stage prostate cancer, and increase to a certain extent in late-stage prostate cancer, but the level is still not high, the increase of the expression level of the imprinting gene Z4 is rapid in early-stage and mid-stage prostate cancer, slow down in late-stage prostate cancer, and the level is still not high; the imprinting deletion and copy number abnormality of the imprinting gene Z5 are rapid in rising in early stage prostate cancer, slow in rising in mid-stage prostate cancer stage, and rise in rising to late stage prostate cancer stage, but the level is still not high, the expression level of the imprinting gene Z5 is increased faster in rising in early and mid-stage prostate cancer stage, slow in rising in late stage prostate cancer stage, and the level is still not high; the imprinting deletion, copy number abnormality and expression amount increase of the imprinting gene Z6 are rapidly increased in the early stage of the prostatic cancer, the rising speed is slowed down in the middle and late stage of the prostatic cancer, and the level is not high; the imprinting deletion of the imprinting gene Z10 is rapidly increased in early stage and slow down in middle and late stage of prostatic cancer, so that the imprinting deletion reaches a higher level, the copy number abnormality and the expression amount increase of the imprinting gene Z10 are rapidly increased in early and middle stage of prostatic cancer, and the imprinting deletion reaches a higher level; the imprinting deletion and copy number abnormality of the imprinting gene Z13 rapidly increased in early stage prostate cancer, decreased in the middle and late stage prostate cancer stage, and the level was not high, and the increase in the expression amount of the imprinting gene Z13 rapidly increased in early stage prostate cancer stage, decreased in the middle stage prostate cancer stage, no significant increase in the late stage prostate cancer stage, and the level was not high.

EXAMPLE 3 imprinting Gene analysis of 44 prostate tumor samples

Obtaining 44 prostate tumor patients' tissues includes puncturing biopsy samples, and the detection method is the same as in example 1.

As can be seen from FIGS. 5 (a) -5 (j), the ratio of the imprinting deletion and the copy number abnormality of 10 probes in 44 samples of prostate tumor tissue is distributed from low to high, and according to the distribution trend of different probes, the classification standard shown by the dotted line in the graph is calculated, so that the imprinting deletion and the copy number abnormality of each probe can be respectively classified into 5 grades from low to high.

The specific classification is as follows:

as can be seen from fig. 5 (a), for the imprinted gene Z1, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1%, or the imprinted gene total expression level of less than 20% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 2%, or the imprinted gene total expression level of 20 to 30% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 2 to 3%, or the imprinted gene total expression level of 30 to 40% is level II, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 3 to 5%, or the imprinted gene total expression level of 40 to 50% is level III, the imprinted gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 5%, or the imprinted gene total expression level of more than 50% is level IV;

As can be seen from fig. 5 (b), for the imprinted gene Z8, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1%, or the imprinted gene total expression level of less than 15% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 2%, or the imprinted gene total expression level of 15 to 20% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 2 to 5%, or the imprinted gene total expression level of 20 to 30% is level II, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 5 to 8%, or the imprinted gene total expression level of 30 to 40% is level III, the imprinted gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 8%, or the imprinted gene total expression level of more than 40% is level IV;

as can be seen from fig. 5 (c), for the imprinted gene Z11, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1% or the imprinted gene total expression level of less than 15% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 1.5% or the imprinted gene total expression level of 15 to 20% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 2.5% or the imprinted gene total expression level of 20 to 30% is level II, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 2.5 to 4% or the imprinted gene total expression level of 30 to 40% is level III, the gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 4% or the combination of at least 40% is level IV;

As can be seen from fig. 5 (d), for the imprinted gene Z16, any one or a combination of at least two of the imprinted gene deletion expression level of less than 15%, the imprinted gene copy number abnormal expression level of less than 1%, or the imprinted gene total expression level of less than 30% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1 to 3%, or the imprinted gene total expression level of 30 to 40% is level I, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 3 to 5%, or the imprinted gene total expression level of 40 to 50% is level II, the imprinted gene deletion expression level of 25 to 30%, the imprinted gene copy number abnormal expression level of 5 to 8%, or the combination of at least two of the imprinted gene total expression level of 50 to 60%, the imprinted gene deletion expression level of more than 30%, the gene copy number abnormal expression level of more than 8%, or the combination of at least two of the imprinted gene total expression level of more than 60% is level IV;

as can be seen from fig. 5 (e), for the imprinted gene Z3, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1% or the imprinted gene total expression level of less than 15% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 1.5% or the imprinted gene total expression level of 15 to 20% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 2.5% or the imprinted gene total expression level of 20 to 30% is level II, the imprinted gene copy number abnormal expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 2.5 to 4% or the imprinted gene total expression level of 30 to 40% is level III, the gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 4% or the combination of at least 40% is level IV;

As can be seen from fig. 5 (f), for the imprinted gene Z4, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 0.5% or the imprinted gene total expression level of less than 15% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 0.5 to 1.5% or the imprinted gene total expression level of 15 to 20% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 2.5% or the imprinted gene total expression level of 20 to 30% is level II, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 2.5 to 4% or the imprinted gene total expression level of 30 to 40% is level III, the imprinted gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 25%, or the imprinted gene copy number of more than 40% is level IV;

as can be seen from fig. 5 (g), for the imprinted gene Z5, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 0.5%, or the imprinted gene total expression level of less than 15% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 0.5 to 1.5%, or the imprinted gene total expression level of 15 to 20% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 2.5%, or the imprinted gene total expression level of 20 to 30% is level II, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 2.5 to 4%, or the imprinted gene total expression level of 30 to 40% is level III, the imprinted gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 25%, or the imprinted gene copy number of more than 40% is level IV;

As can be seen from fig. 5 (h), for the imprinted gene Z6, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 0.5% or the imprinted gene total expression level of less than 15% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 0.5 to 1.5% or the imprinted gene total expression level of 15 to 20% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 2.5% or the imprinted gene total expression level of 20 to 30% is level II, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 2.5 to 4% or the imprinted gene total expression level of 30 to 40% is level III, the imprinted gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 25%, or the imprinted gene copy number of more than 40% is level IV;

as can be seen from fig. 5 (I), for the imprinted gene Z10, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 0.5%, or the imprinted gene total expression level of less than 15% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 0.5 to 1.5%, or the imprinted gene total expression level of 15 to 20% is level I, the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 2.5%, or the imprinted gene total expression level of 20 to 30% is level II, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 2.5 to 4%, or the imprinted gene total expression level of 30 to 40% is level III, the imprinted gene deletion expression level of more than 25%, the imprinted gene copy number abnormal expression level of more than 25%, or the imprinted gene copy number of more than 40% is level IV;

As can be seen from FIG. 5 (j), for the above-mentioned imprinted gene Z13, the amount of the imprinted gene deletion expression is less than 10%, the amount of the imprinted gene copy number abnormal expression is less than 1% or the amount of the imprinted gene total expression is less than 15%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 10 to 15%, the amount of the imprinted gene copy number abnormal expression is 1 to 1.5% or the amount of the imprinted gene total expression is 15 to 20%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 15 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 20 to 30%, the amount of any one or combination of at least two of the imprinted gene copy number abnormal expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the amount of the imprinted gene copy number abnormal expression is 30 to 40%, the amount of any one or combination of at least two of the imprinted gene copy number abnormal expression is greater than 25%, the amount of the imprinted gene copy number IV is greater than 40%.

From a comprehensive analysis of these 44 samples of prostate tumors, it can be derived that:

judging benign and malignant degrees of the prostate tumor to be classified into benign tumor, potential of prostate cancer, early-stage prostate cancer, medium-stage prostate cancer and advanced-stage prostate cancer;

The result of the judgment of the benign and malignant degree of the prostate tumor is that the imprinting gene deletion expression amount and the imprinting gene copy number abnormal expression amount of each of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are smaller than the I level or the imprinting gene deletion expression amount of not more than 1 imprinting gene of the imprinting genes Z1, Z3, Z4, Z6, Z8, Z10, Z11, Z13 and Z16 is I level and the imprinting gene copy number abnormal expression amount of not more than 1 imprinting gene of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is I level, and the prostate tumor is benign tumor;

the prostate cancer is determined by determining the benign and malignant extent of the prostate tumor, wherein the result is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z6, Z10, Z8, Z10, Z11, Z13 and Z16 is level I, or the imprinting gene deletion expression level of no more than 1 imprinting genes among the imprinting genes Z1, Z3, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of level II;

The prostate cancer is obtained when the result of the determination of the benign and malignant extent of the prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z6, Z10, Z8, Z11, Z13 and Z16 is level II, or the imprinting gene deletion expression level of not more than 1 imprinting genes among the imprinting genes Z1, Z3, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level III, and the imprinting gene copy number abnormal expression level of not more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of level III;

the result of the determination of the benign and malignant degree of prostate tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III or that the imprinting gene deletion expression level of no more than 1 imprinting gene among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV and the imprinting gene copy number abnormal expression level of no more than 1 imprinting gene among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of class IV;

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

Example 4 imprinted Gene analysis of urine shed cell samples

The urine exfoliated cell sample is obtained by collecting urine after prostate massage of a prostate tumor patient, centrifuging the collected cells, and fixing the cells for more than 24 hours by 10% neutral formalin solution, and other detection methods are the same as in example 1.

As can be seen from fig. 6 (a) -6 (b), fig. 6 (a) is a benign prostate tumor, fig. 6 (b) is a prostate cancer, and cells without imprint loss and copy number abnormality in urine shed cells of benign prostate tumor, and a large number of cells with imprint loss and copy number abnormality exist in urine shed cells of prostate cancer.

In summary, the detection model and the detection system of the invention represent the representation of imprinting missing on the sample of the prostate tumor patient in an intuitive way, detect the change of imprinting (trace) genes objectively, intuitively, early and accurately by the method of marking imprinting genes in situ, and can provide a quantized model to make a great contribution to the diagnosis of prostate tumor.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A device for detecting benign and malignant prostate tumor, comprising the following elements:

(1) Sampling unit: obtaining a sample to be tested;

(2) Probe design unit: designing a specific probe according to the intron sequence of the imprinted gene;

wherein,

the imprinting gene comprises a combination of Z1, Z8, Z11 and Z16, or a combination of Z8 and Z16, or a combination of Z1, Z11 and Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z8 is Dcn, the imprinting gene Z11 is Grb10, the imprinting gene Z16 is Snrpn/Snurf,

The imprinted gene further comprises any one or a combination of at least two of Z3, Z4, Z5, Z6, Z10 or Z13; wherein the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z5 is test, the imprinting gene Z6 is Plagl1, the imprinting gene Z10 is Gatm, the imprinting gene Z13 is Sgce,

the analysis unit judges the benign and malignant degree of the prostate tumor by calculating the imprinting gene deletion expression quantity, the imprinting gene copy number abnormal expression quantity and the total expression quantity and by a imprinting gene grading model of the prostate tumor, thereby judging the benign and malignant degree of the prostate tumor by the grades of the imprinting gene deletion expression quantity, the imprinting gene copy number abnormal expression quantity and the total expression quantity,

the marking gene grading model grades the expression state of marking genes by calculating the total expression quantity of marking genes, the deletion expression quantity of marking genes and the variation of the copy number abnormal expression quantity of marking genes in prostate tumors;

wherein, the marking genes Z1, Z8, Z11, Z16 and any one or at least two of Z3, Z4, Z5, Z6, Z10 or Z13 are detected by in situ hybridization by adopting a sequence selected from introns of each gene as a probe,

The formulas for calculating the total expression quantity of the imprinting genes, the deletion expression quantity of the imprinting genes and the copy number abnormal expression quantity of the imprinting genes are as follows:

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

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

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

gene expression amount of the imprinted gene copy number abnormality=d/(b+c+d) ×100%;

wherein a is a cell nucleus in which no marker exists in the cell nucleus and the imprinted gene is not expressed after the cell is subjected to hematoxylin staining; b is a cell nucleus with a red/brown mark in the cell nucleus and a marking gene after the cell is subjected to hematoxylin staining; c is a cell nucleus with two red/brown marks in the cell nucleus and marking the gene deletion after the cell is subjected to hematoxylin staining; the d is the cell nucleus with more than two red/brown marks in the cell nucleus after the cell is subjected to hematoxylin staining and the marked gene copy number is abnormal,

the deletion expression level of the imprinting gene, the abnormal copy number expression level of the imprinting gene and the total expression level of the imprinting gene are divided into five different levels;

the five different grades are five different grades respectively divided into a imprinted gene deletion expression amount, an imprinted gene copy number abnormal expression amount and an imprinted gene total expression amount for ten imprinted genes of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16;

When detecting prostate tumor, five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity aiming at Z1 are divided into:

Grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z1 being more than 25%, the imprinting gene copy number abnormal expression level of the imprinting gene Z1 being more than 5%, or the total expression level of the imprinting gene Z1 being more than 50%;

when detecting prostate tumor, five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity aiming at Z8 are divided into:

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z8 being more than 25%, the imprinting gene copy number abnormal expression level of the imprinting gene Z8 being more than 8%, or the total expression level of the imprinting gene Z8 being more than 40%;

when detecting prostate tumors, the five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity of the imprinting gene aiming at Z16 are divided into:

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z16 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z16 being more than 8%, or the total expression level of the imprinting gene Z16 being more than 60%;

in the detection of prostate tumors, the five different classes of the deletion expression level of the imprinting genes, the abnormal expression level of the copy number of the imprinting genes and the total expression level of the imprinting genes for Z3, Z11 and Z13 are:

In the detection of prostate tumors, the five different grades of the deletion expression level of the imprinting genes, the abnormal expression level of the copy number of the imprinting genes and the total expression level of the imprinting genes for Z4, Z5, Z6 and Z10 are divided into:

grade IV: the imprinted gene deletion expression level of the imprinted genes Z4, Z5, Z6 and Z10 is more than 25%, the imprinted gene copy number abnormal expression level of the imprinted genes Z4, Z5, Z6 and Z10 is more than 4%, or the total expression level of the imprinted genes Z4, Z5, Z6 and Z10 is more than 40% or a combination of at least two.

2. The device of claim 1, wherein the sample to be tested in step (1) is derived from human tissue and/or cells.

3. The device of claim 2, wherein the sample to be tested is a needle biopsy sample or a urine shed cell sample.

4. The apparatus of claim 1, wherein said in situ hybridization employs an RNAscope in situ hybridization method.

5. The device of claim 4, wherein the RNAscope in situ hybridization method uses a single-channel or multi-channel chromogenic kit or a single-channel or multi-channel fluorogenic kit.

6. The device of claim 4, wherein the RNAscope in situ hybridization method uses a single channel red/brown color kit or a multichannel fluorescent kit.

7. The device of any one of claims 1-6, wherein the judging the benign malignancy of a prostate tumor is classified as benign prostate tumor, prostate cancer potential, early-stage prostate cancer, mid-stage prostate cancer, and advanced-stage prostate cancer;

the result of the judgment of the benign and malignant degree of the prostate tumor is that the imprinting gene deletion expression amount and the imprinting gene copy number abnormal expression amount of each of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are smaller than the I level or the imprinting gene deletion expression amount of not more than 1 imprinting gene of the imprinting genes Z1, Z3, Z4, Z6, Z8, Z10, Z11, Z13 and Z16 is I level and the imprinting gene copy number abnormal expression amount of not more than 1 imprinting gene of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is I level, and the benign prostate tumor is;

8. Use of a device according to any one of claims 1-7 for the preparation of a medicament or kit for prostate tumor detection.

9. The use according to claim 8, wherein the benign and malignant extent of the prostate tumor is classified into benign prostate tumor, prostate cancer potential, early stage prostate cancer, intermediate stage prostate cancer and advanced stage prostate cancer.