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WO2019041689A1 - 用于结直肠肿瘤的印记基因分级模型及其组成的系统 - Google Patents

用于结直肠肿瘤的印记基因分级模型及其组成的系统 Download PDF

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WO2019041689A1
WO2019041689A1 PCT/CN2017/118956 CN2017118956W WO2019041689A1 WO 2019041689 A1 WO2019041689 A1 WO 2019041689A1 CN 2017118956 W CN2017118956 W CN 2017118956W WO 2019041689 A1 WO2019041689 A1 WO 2019041689A1
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imprinted
imprinted gene
genes
expression amount
gene
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成彤
周宁
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Lisen Imprinting Diagnostics Wuxi Co Ltd
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Lisen Imprinting Diagnostics Wuxi Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • the present application relates to the field of biotechnology, and relates to the field of gene diagnosis, and in particular to a system for imprinting a gene-grading model for colorectal tumors and a composition thereof.
  • Colorectal cancer is a common malignant tumor in the digestive tract. The incidence rate is the third in all malignant tumors, and the mortality rate ranks fourth. About 1.2 million patients worldwide are diagnosed with colorectal cancer each year, and more than 600,000 patients die directly or indirectly from colorectal cancer. According to the World Health Organization (WHO), there were 774,000 cases of global colorectal cancer deaths in 2015. The incidence of colorectal cancer is lower in the age group under 45 years old, but it will increase with age. In recent years, the age of onset is younger.
  • WHO World Health Organization
  • stage I the 5-year survival rates of colorectal cancer patients with different stages in the United States were 90.1% (stage I), 69.2% (stage II and III), and 11.7% (stage IV), respectively, so colorectal cancer Early diagnosis and early treatment are very important. However, the early diagnosis rate of colorectal cancer is very low. The diagnosis rate of early intestinal cancer in the United States is about 25%, while the early diagnosis rate in China is only 10%. It is urgent to develop more sensitive and accurate detection methods.
  • Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by methylation of alleles from a particular parent, such that one gene has only one allele and the other is in a state of gene silencing. This type of gene is called a blot (remember) gene. Deletion of the blot is an epigenetic change in which the allelic gene of the imprinted gene results in a silenced allele being activated and beginning to express the gene.
  • the methylation status of the imprinted gene can be used as a pathological marker to analyze the abnormal state of the cell by a specific molecular detection technique.
  • the analysis of molecular marker changes in colorectal cancer at the cellular level by imprinted gene detection technology can provide more accurate pre-diagnosis and diagnostic information, and has important guiding significance for the determination of the distance adjacent to the cancer during surgery.
  • the present application provides a system for imprinting gene grading models for colorectal tumors and a composition thereof for early visual observation of colorectal tissues at the cellular and tissue levels.
  • the change of the imprinted (marked) gene of cancer determines the degree of benign and malignant and malignant degree of colorectal cancer.
  • the present application provides an imprinted gene grading model for colorectal tumors by calculating the expression status of imprinted genes by changing the amount of imprinted gene expression and the abnormal expression level of imprinted gene copy number in colorectal cancer. Grading
  • the imprinting gene is any one or a combination of at least two of Z1, Z2, Z3 or Z4, the imprinting gene Z1 is Gnas, the imprinting gene Z2 is Igf2r, and the imprinting gene Z3 is Mest.
  • the imprinted gene is Plagl in Z4.
  • any one of Z1 - Z4 can be calculated, preferably Z1 and/or Z3, further preferably Z1.
  • the inventors found that the detection of a Z1 imprinted gene alone can achieve a sensitivity of 93.8% for colorectal cancer, and a Z2 imprinted gene alone can be diagnosed with a sensitivity of 47.9% for colorectal cancer.
  • a Z3 imprinted gene, the sensitivity of diagnosis of colorectal cancer can reach 83.3%, a Z4 imprinted gene alone, the sensitivity of diagnosis of colorectal cancer can reach 58.3%.
  • the combination may be a combination of Z1 and Z2, a combination of Z1 and Z3, a combination of Z1 and Z4, Z2 In combination with Z3, a combination of Z2 and Z4 or a combination of Z3 and Z4, preferably a combination of Z1 and Z3.
  • the inventors have found that by calculating the amount of the imprinted gene deletion expression of the two or more imprinted genes and the abnormal expression level of the imprinted gene copy number, the sensitivity can be further improved, and the combination of the two imprinted genes of the imprinted gene is detected.
  • the diagnostic sensitivity of colorectal cancer can reach more than 60%.
  • the sensitivity of diagnosis to colorectal cancer can reach 100%.
  • the combination of Z1 and Z4 can be sensitive to the diagnosis of colorectal cancer. More than 97.9%, the combination of Z1 and Z2, the sensitivity of diagnosis of colorectal cancer can reach more than 95.8%.
  • the imprinted gene grading model is most preferably a combination of detection Z1-Z4 genes.
  • the imprinted gene is deleted, after the cells are subjected to hematoxylin staining, there are two brown markers in the nucleus, and the abnormal copy number of the imprinted gene is that after the cells are subjected to hematoxylin staining, there are more than two brown markers in the nucleus.
  • the copy number abnormality is caused by abnormal gene replication of cancer cells, resulting in the expression of this gene as triploid or even higher polyploid.
  • the hematoxylin-stained label is selected from, but not limited to, brown, and staining with other colors can also be used for calculation of imprinted gene expression amount, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount.
  • the imprinted gene and the imprinted gene are simultaneously a concept, indicating the same meaning, and can be replaced.
  • the formula for calculating the expression level of the imprinted gene deletion and the abnormal expression amount of the imprinted gene copy is as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed in the cell after hematoxylin staining; and b is a brown mark in the nucleus after imprinting the cell with hematoxylin, and the cell nucleus in which the imprinted gene exists;
  • the c is a hematoxylin-stained cell, and two brown markers are present in the nucleus, and the nucleus of the imprinted gene is deleted;
  • the d is a hematoxylin staining of the cell, and there are two or more brown markers in the nucleus, and the imprinting gene copy number is abnormal.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount are divided into five different grades: the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the four imprinted genes for Z1-Z4 are respectively performed. Five different levels of division.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z1 and Z3 are:
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes Z1 and Z3 have an imprinted gene copy number abnormal expression amount of less than 0.8%;
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z1 and Z3 have an abnormal expression amount of the imprinted gene copy number of 0.8-2.5%;
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of 20-30% and/or the imprinted genes Z1 and Z3 have an imprinted gene copy number abnormal expression amount of 2.5-3.5%;
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of 30-35% and/or the imprinted genes Z1 and Z3 have an imprinted gene copy number abnormal expression amount of 3.5-5%;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z3 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z3 is greater than 5%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z3 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z2 and Z4 are:
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes Z2 and Z4 have an imprinted gene copy number abnormal expression amount of less than 0.8%;
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z2 and Z4 have an abnormal expression amount of the imprinted gene copy number of 0.8-1.5%;
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes Z2 and Z4 have an imprinted gene copy number abnormal expression amount of 1.5-3%;
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of 25-30% and/or the imprinted genes Z2 and Z4 have an imprinted gene copy number abnormal expression amount of 3-5%;
  • the imprinted gene deletion expression amount of the imprinted genes Z2 and Z4 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z2 and Z4 is greater than 5%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z2 and Z4 are independent of each other.
  • the present application provides a system for detecting the degree of benign and malignant colorectal tumors, comprising the following elements:
  • sampling unit obtaining a sample to be tested
  • Probe design unit design specific primers according to the imprinted gene sequence
  • the analysis unit determines the level of the imprinted gene deletion expression and the imprinted gene copy number abnormal expression level by calculating the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount by using the model described in the first aspect; The degree of benign and malignant colorectal tumors.
  • the imprinted gene is deleted, after the cells are subjected to hematoxylin staining, there are two brown-labeled nuclei in the nucleus, and the imprinting gene copy number abnormality is that after the cells are subjected to hematoxylin staining, there are two or more brown marks in the nucleus.
  • the copy number abnormality is caused by abnormal gene replication of cancer cells, resulting in the expression of this gene as triploid or even higher polyploid.
  • the hematoxylin-stained label is selected from, but not limited to, brown, and staining with other colors can also be used for calculation of imprinted gene expression amount, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount.
  • the detection system described in the present application is for early and intuitive observation of changes in the imprinted (spot) genes of colorectal tumors at the cellular and tissue levels to determine the benign and malignant degree of colorectal tumors, and to provide the most favorable for patients with early colorectal cancer. Treatment opportunities.
  • the sample to be tested described in the step (1) is derived 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 select according to the needs, and the sample to be tested includes the paraffin section of the tissue. And/or endoscopic screening of the sample, preferably endoscopic screening of the sample.
  • the specific operation procedure of the paraffin section of the tissue is to obtain a human tumor tissue sample, which is fixed in 10% neutral formalin in time, embedded in paraffin, cut into 10 ⁇ m thick, and made into a tissue film with a positively charged slide; Only 10 ⁇ m thick, so some of the microscopically seen nuclei are incomplete, so some false negative gene deletions will occur.
  • the specific operation procedure of the endoscope screening sample is to obtain suspicious tissue under the colorectal microscope, embedded in paraffin, cut into 10 ⁇ m thick, and obtained by using a positively charged slide to form a film.
  • the sampling process is simple, and the colorectal biopsy can be positioned compared to the blood circulation characteristics, and the colorectal biopsy has an experimental sample.
  • the imprinted gene is Z1-Z4, the imprinted gene Z1 is Gnas, the imprinted gene Z2 is Igf2r, the imprinted gene Z3 is Mest, and the imprinted gene is Plagl in Z4.
  • the imprinted genes Z1 (Gnas), Z2 (Igf2r), Z3 (Mest), and Z4 (Plagl1) are expressed in different degrees in normal tumor cell tissues, and expression levels and imprinting states are present when malignant lesions occur. There will be significant changes.
  • the designed probe is designed according to the imprinting genes Z1-Z4, namely Gnas, Igf2r, Mest and Plagl, and specifically selects a sequence as a probe in the endo-spin of each gene, and specifically selects the gene sequence.
  • the imprinting genes Z1-Z4 namely Gnas, Igf2r, Mest and Plagl, and specifically selects a sequence as a probe in the endo-spin of each gene, and specifically selects the gene sequence.
  • the location of specific genes are as follows:
  • the in situ hybridization employs an RNAscope in situ hybridization method.
  • the RNAscope in situ hybridization method uses a single or multi-channel colorimetric kit or a single or multi-channel fluorescent kit, preferably a single channel brown color kit or a multi-channel fluorescent kit.
  • the multi-channel coloring kit or the multi-channel fluorescent kit comprises two or more channels of coloring kits or fluorescent kits, and the two-channel coloring kit or multi-channel fluorescent reagent
  • the cassette can use two imprinted gene probes or a combination of imprinted genes and other genes to express even multiple imprinted genes and non-imprinted genes.
  • the formula for calculating the amount of expression of the imprinted gene and the amount of abnormal expression of the imprinted gene in the model is as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed in the cell after hematoxylin staining; and b is a brown mark in the nucleus after imprinting the cell with hematoxylin, and the cell nucleus in which the imprinted gene exists;
  • the c is a hematoxylin-stained cell, and two brown markers are present in the nucleus, and the nucleus of the imprinted gene is deleted;
  • the d is a hematoxylin staining of the cell, and there are two or more brown markers in the nucleus, and the imprinting gene copy number is abnormal.
  • the hematoxylin-stained label is selected from, but not limited to, brown, and staining with other colors can also be used for calculation of imprinted gene expression amount, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount.
  • the probe is amplified by in situ hybridization and Hemotoxy (hematoxylin) nuclear staining, and the presence of imprinted genes, imprinted gene deletions or copy number abnormalities in each nucleus is determined under a 40 ⁇ or 60 ⁇ microscope.
  • the degree of benign and malignant tumors of the sample is determined by calculating the gene expression amount of the imprinted gene deletion gene and the imprinted gene copy number abnormality. Since the section is only 10 microns, about 20% of the nuclei seen under the microscope are incomplete nuclei, which means that there is a possibility of partial false negatives.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount are divided into five different grades.
  • the five different grades are the division of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the four imprinted genes for Z1-Z4, respectively.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z1 and Z3 are:
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes Z1 and Z3 have an imprinted gene copy number abnormal expression amount of less than 0.8%;
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z1 and Z3 have an abnormal expression amount of the imprinted gene copy number of 0.8-2.5%;
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of 20-30% and/or the imprinted genes Z1 and Z3 have an imprinted gene copy number abnormal expression amount of 2.5-3.5%;
  • the imprinted gene Z1 and Z3 have an imprinted gene deletion expression amount of 30-35% and/or the imprinted genes Z1 and Z3 have an imprinted gene copy number abnormal expression amount of 3.5-5%;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z3 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z3 is greater than 5%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z3 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z2 and Z4 are:
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes Z2 and Z4 have an imprinted gene copy number abnormal expression amount of less than 0.8%;
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z2 and Z4 have an abnormal expression amount of the imprinted gene copy number of 0.8-1.5%;
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes Z2 and Z4 have an imprinted gene copy number abnormal expression amount of 1.5-3%;
  • the imprinted gene Z2 and Z4 have an imprinted gene deletion expression amount of 25-30% and/or the imprinted genes Z2 and Z4 have an imprinted gene copy number abnormal expression amount of 3-5%;
  • the imprinted gene deletion expression amount of the imprinted genes Z2 and Z4 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z2 and Z4 is greater than 5%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z2 and Z4 are independent of each other.
  • the degree of benign and malignant colorectal tumors to be judged is classified into benign colorectal tumor, colorectal cancer potential, early colorectal cancer, mid-stage colorectal cancer, and advanced colorectal cancer.
  • the result of determining the degree of benign and malignant colorectal tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 are both smaller than the I grade, the imprinted gene Z1, and the imprinted gene Z1.
  • the imprinted gene deletion expression level of no more than one imprinted gene in Z2, Z3 and Z4 is grade I and the imprinted gene copy number of the imprinted genes Z1, Z2, Z3 and Z4 is not more than one imprinted gene.
  • the imprinted gene deletion expression level of the two imprinted genes of the grade or imprinted genes Z1, Z2, Z3 and Z4 is I grade and the abnormal expression amount of the imprinted gene copy number of the two imprinted genes is any of 0 grades In the case, it is a benign tumor;
  • the result of determining the degree of benign and malignant color of the colorectal tumor is that the imprinted gene of the two imprinted genes of the imprinted genes Z1, Z2, Z3 and Z4 has an imprinted gene whose expression level is one grade and the imprinted gene of one of the two imprinted genes
  • the copy number abnormal expression level is I
  • at least two imprinted genes of the imprinted genes Z1, Z2, Z3 and Z4 have at least two imprinted genes with imprinted gene expression level I and imprinted genes Z1, Z2, Z3 and Z4.
  • the imprinted gene copy number abnormal expression level is one of the class II or imprinted genes Z1, Z2, Z3 and Z4, and the imprinted gene deletion expression amount and the imprinted gene deletion expression amount are all of the II levels.
  • the situation is the potential of colorectal cancer;
  • the result of determining the degree of benign and malignant colorectal tumor is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is II, and at least at least one of the imprinting genes Z1, Z2, Z3 and Z4
  • the abnormal expression level of the imprinted gene copy number of the two imprinted genes is Grade II or the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted genes of Z1, Z2, Z3 and Z4 are both Grade III, then For early colorectal cancer;
  • the result of determining the degree of benign and malignant colorectal tumor is that the imprinted gene deletion expression level of at least two imprinted genes of the imprinted genes Z1, Z2, Z3 and Z4 is at least grade III, and at least at least one of the imprinted genes Z1, Z2, Z3 and Z4
  • the abnormal expression level of the imprinted gene copy number of the two imprinted genes is Grade III or the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted genes of Z1, Z2, Z3 and Z4 are both Grade IV, then For mid-stage colorectal cancer;
  • the result of determining the degree of benign and malignant colorectal tumor is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is at least grade IV or at least at least one of the imprinted genes Z1, Z2, Z3 and Z4
  • the abnormal expression level of the imprinted gene copy number of the two imprinted genes was grade IV, and it was advanced malignant colorectal cancer.
  • the present application provides the use of the model of the first aspect or the system of the second aspect in the manufacture of a medicament for detecting and/or treating colorectal cancer.
  • the colorectal cancer is detected to determine the degree of benign and malignant colorectal tumors, and the degree of benign and malignant colorectal tumors is divided into benign, colorectal cancer potential, early colorectal cancer, mid-stage colorectal cancer, and advanced colorectal cancer.
  • the result of determining the degree of benign and malignant colorectal tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 are less than grade I, the imprinted gene Z1, and the imprinted gene
  • the imprinted gene deletion expression level of no more than one imprinted gene in Z1, Z2, Z3, and Z4 is Grade I and the imprinted gene copy number abnormal expression amount of no more than one imprinted gene in the imprinted genes Z1, Z2, Z3, and Z4
  • the imprinted gene deletion expression level of the two imprinted genes of the class I or imprinted genes Z1, Z2, Z3 and Z4 is I and the abnormal expression amount of the imprinted gene copy number of the two imprinted genes is any of 0 In one case, it is a benign tumor;
  • the result of determining the degree of benign and malignant color of the colorectal tumor is that the imprinted gene deletion expression amount of the two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is I level and one of the two imprinted genes
  • the abnormal expression level of the imprinted gene copy number is I
  • the imprinted gene deletion expression level of at least 3 imprinted genes of the imprinted genes Z1, Z2, Z3 and Z4 is I grade and at least 2 of the imprinted genes Z1, Z2, Z3 and Z4
  • the imprinted gene copy number abnormal expression level of the imprinted gene is I or the imprinted gene, and the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3 and Z4 are all in the second level. In one case, it is the potential of colorectal cancer;
  • the result of determining the degree of benign and malignant color of the colorectal tumor is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is II, and the imprinting genes Z1, Z2, Z3 and Z4
  • the abnormal expression level of the imprinted gene copy number of at least 2 imprinted genes is Grade II or the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3 and Z4 are both grade III. , is early colorectal cancer;
  • the result of determining the degree of benign and malignant colorectal tumor is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is grade III, and the imprinting genes Z1, Z2, Z3 and Z4
  • the abnormal expression level of the imprinted gene copy number of at least 2 imprinted genes is Grade III or the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3 and Z4 are both Grade IV , is a mid-stage colorectal cancer;
  • the result of determining the degree of benign and malignant color of the colorectal tumor is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is IV or the imprinting genes Z1, Z2, Z3 and Z4
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes is grade IV, and it is advanced malignant colorectal cancer.
  • the detection model and system described in the present application visually express the performance of the imprinted deletion on the sample of the colorectal cancer patient, and objectively, intuitively, earlyly and accurately detect the in situ labeling of the imprinted gene.
  • the imprinting (trace) gene changes and can provide a quantitative model that makes a significant contribution to the diagnosis of colorectal tumors;
  • the detection system of the present application can determine the degree of benign and malignant colorectal tumors before surgery in patients with colorectal cancer, thereby providing a basis for surgery and precision treatment, which is a revolutionary breakthrough in the diagnosis of colorectal tumors in the field of cell molecules. ;
  • This application can accurately determine the type of colorectal tumor.
  • the sensitivity of detection of samples with malignant potential above the level is 93.8%; if combined with detection of Z1 and Z3 probes, above the malignant potential Graded sample detection sensitivity can be increased to 100%, greatly improving the early stage of colorectal cancer, and clear diagnosis, especially for early screening and postoperative follow-up of cancer, especially for follow-up of suspected relapsed patients. Time, making a significant contribution to saving the lives of patients;
  • This application can detect the benign and malignant tissues of adjacent tissues, and can provide guidance for the determination of the distance adjacent to the cancer during surgery to ensure complete resection of the diseased tissue and greatly reduce the recurrence rate of the patient;
  • the detection method of the present application is different from the immunohistochemical method, which reduces false positives and other negative effects, and not only the target of silencing, culling, rearrangement of the gene by the deletion site of the imprinted gene associated with the colorectal tumor found. To the drug or technical method, it can be used to guide the later treatment and medication.
  • 1 is a pathological section of colorectal cancer in a hematoxylin-stained nuclei in an embodiment of the present application, wherein the a is a hematoxylin-stained cell, and there is no label in the nucleus, and the imprinted gene is not expressed; After hematoxylin staining, there is a brown mark in the nucleus, and the imprinted gene exists; the c is the hematoxylin staining of the cells, there are two brown marks in the nucleus, and the imprinted gene is deleted; the d is the cell nucleus after hematoxylin staining There are more than two brown marks in memory, and the imprinted gene copy number is abnormal;
  • Fig. 2(a) shows the expression status of four genes in the pathological section of grade 0 colorectal cancer
  • Fig. 2(b) shows the expression status of four genes in the pathological section of grade I colorectal cancer
  • Fig. 2(c) is The expression status of four genes in the pathological section of grade II colorectal cancer
  • Fig. 2(d) shows the expression status of four genes in the pathological section of grade III colorectal cancer
  • Fig. 2(e) shows the grade IV colorectal cancer.
  • Fig. 3(a) shows the intensity of imprinting deletion of 4 genes for colorectal cancer
  • Fig. 3(b) shows the intensity of copy number abnormality of 4 genes for colorectal cancer
  • LOI is the amount of imprinted gene deletion gene expression
  • CNV is the gene expression amount of abnormal copy number of imprinted gene
  • Figure 4 (a) shows the intensity of the imprinted gene Z1 imprint deletion and copy number abnormality
  • Figure 4 (b) shows the intensity of the imprinted gene Z2 imprint deletion and copy number abnormality
  • Figure 4 (c) shows the imprinted gene Z3 imprint deletion and copy number.
  • the intensity of the abnormality is the intensity of the imprinting gene Z4 imprint deletion and copy number abnormality
  • LOI is the imprinted gene deletion gene expression level
  • CNV is the gene expression amount of the imprinted gene copy number abnormality
  • Fig. 5(a) shows the distribution range and grading standard of imprinted gene and Z1 in 73 cases of colorectal cancer pathological sections
  • Fig. 5(b) shows the imprinted gene Z2 applied to 73 cases of colorectal cancer pathology.
  • the distribution range and grading standard of imprinting deletion and copy number abnormality shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 73 pathological sections of colorectal cancer with imprinting gene Z3.
  • 5(d) is the imprinting gene Z4 applied to 73 cases of colorectal cancer pathological sections, the distribution range and grading standard of imprinting deletion and copy number abnormality
  • LOI is the imprinting gene deletion gene expression level
  • CNV is the imprinting gene copy number abnormal gene The amount of expression.
  • the method for detecting the imprinted gene comprises the following steps:
  • tissue cells of colorectal cancer cut into 10 ⁇ m thick, and fixed in 10% neutral formalin solution to prevent RNA degradation, fixed time is 24 hours, paraffin embedding (FFPE), The slide needs to be loaded with a positive charge, and the slice is baked in an oven at 40 ° C for more than 3 hours;
  • FFPE paraffin embedding
  • Design probe design specific primers according to the imprinted gene sequence
  • the designed probe is designed according to the imprinting genes Z1 (Gnas), Z2 (Igf2r), Z3 (Mest) and Z4 (Plagl1), and specifically selects a sequence as a probe in the inner loop of each gene, and specifically selects
  • Z1 Gas
  • Z2 Igf2r
  • Z3 Mest
  • Z4 Plagl1
  • the formula for calculating the amount of expression of the imprinted gene and the amount of abnormal expression of the imprinted gene in the model is as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a, b, c, and d are as shown in FIG. 1 , wherein a is a cell nucleus in which no hemoglobin is stained in the nucleus and the imprinted gene is not expressed; and b is a hematoxylin staining of the cell.
  • a is a cell nucleus in which no hemoglobin is stained in the nucleus and the imprinted gene is not expressed; and b is a hematoxylin staining of the cell.
  • a brown mark in the nucleus to imprint the nucleus of the gene There is a brown mark in the nucleus to imprint the nucleus of the gene
  • the c is the hematoxylin staining of the cell, there are two brown marks in the nucleus, and the nucleus of the imprinted gene is deleted
  • the d is the cell nucleus after hematoxylin staining
  • the nuclei with abnormal copy number of the imprinted gene are imprinted.
  • the colorectal biopsy sample was obtained by taking a suspicious lesion under a colorectal microscope, fixed in a 10% neutral formalin solution for 24 hours, embedded in paraffin (FFPE), and cut into 10 micron thick sections.
  • FFPE paraffin
  • Fig. 4(a)-Fig. 4(d) The sensitivity of each imprinted gene to colorectal cancer is shown in Fig. 4(a)-Fig. 4(d).
  • the deletion and copy number abnormality of imprinted gene Z1 appear at the earliest stage of colorectal cancer lesions, in malignant potential and early stage The stage of rectal cancer was significantly increased; the imprinted gene Z2 was less sensitive in the early stage of colorectal cancer lesions, and some copy number abnormalities occurred in the malignant potential stage, but did not continue to rise in the early and middle stages of colorectal cancer, and the imprinted deletion occurred in the late stage.
  • Tissues from 73 patients with colorectal cancer including colorectal biopsy samples (10 micron), were obtained in the same manner as in Example 1.
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 0.8%, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 0.8-2.5% for grade I, imprinted gene deletion expression level is 20-30% and/or imprinted gene copy number abnormal expression level is 2.5-3.5% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 3.5-5% for the grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 5%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 0.8%, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 0.8-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3% for grade II,
  • the amount of imprinted gene deletion is 25-30% and/or the abnormal expression of imprinted gene copy number is 3-5% to grade III, the imprinted gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed by more than 5%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 0.8%, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 0.8-2.5% for grade I, imprinted gene deletion expression level is 20-30% and/or imprinted gene copy number abnormal expression level is 2.5-3.5% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 3.5-5% for the grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 5%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 0.8%, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 0.8-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3% for grade II,
  • the amount of imprinted gene deletion is 25-30% and/or the abnormal expression of imprinted gene copy number is 3-5% to grade III, the imprinted gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed by more than 5%. It is level IV.
  • the result of judging the degree of benign and malignant colorectal tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 are less than grade I, and the imprinted genes Z1, Z2, Z3 and Z4
  • the imprinted gene deletion expression level of no more than one imprinted gene is grade I and the imprinted gene copy number of no more than one imprinted gene in the imprinted genes Z1, Z2, Z3 and Z4 is abnormally expressed in grade I or imprinted gene Z1
  • the imprinting gene deletion expression level of the two imprinted genes in Z2, Z3, and Z4 is Grade I and the abnormal expression level of the imprinted gene copy number of the two imprinted genes is in any of the 0 grades, and is benign Tumor
  • the result of determining the degree of benign and malignant color of the colorectal tumor is that the imprinted gene of the two imprinted genes of the imprinted genes Z1, Z2, Z3 and Z4 has a deletion gene expression level of I and an imprinted gene copy of one of the two imprinted genes
  • the number of abnormal expression levels is I, and the imprinting gene of at least 3 imprinting genes in the imprinting genes Z1, Z2, Z3, and Z4 is deleted at a level I and the imprinting genes Z1, Z2, Z3, and Z4 are at least 2 imprinted genes.
  • the abnormal expression level of the imprinted gene copy number is one of the grade II or the imprinted gene of the imprinted genes Z1, Z2, Z3 and Z4, and the imprinted gene deletion expression amount and the imprinted gene deletion expression amount are all in the second level. Then judge the potential of colorectal cancer;
  • the result of determining the degree of benign and malignant colorectal tumors is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is II, and at least 2 of the imprinted genes Z1, Z2, Z3 and Z4
  • the imprinted gene copy number abnormal expression level of the imprinted gene is Grade II or the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted gene of Z1, Z2, Z3 and Z4 are both Grade III, then Early colorectal cancer;
  • the result of determining the degree of benign and malignant color of the colorectal tumor is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is III, and at least 2 of the imprinted genes Z1, Z2, Z3 and Z4
  • the imprinted gene copy number abnormal expression level of the imprinted gene is III or the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted gene Z1, Z2, Z3 and Z4 are both IV, then Mid-stage colorectal cancer;
  • the result of determining the degree of benign and malignant colorectal tumors is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z2, Z3 and Z4 is IV or at least 2 of the imprinted genes Z1, Z2, Z3 and Z4
  • the imprinted gene copy number abnormal expression level of the imprinted gene is grade IV, which is advanced colorectal cancer.
  • the detection model and system described in the present application visually express the performance of the imprinted deletion on the sample of the colorectal cancer patient, and the method of in situ labeling of the imprinted gene is objective, intuitive, early, and accurate.
  • the change in the imprinted (trace) gene is detected and a quantitative model can be provided to make a significant contribution to the diagnosis of colorectal tumors.

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Abstract

本申请提供了用于结直肠肿瘤的印记基因分级模型及其组成的系统，所述模型通过计算印记基因缺失表达量和印记基因拷贝数异常表达量对印记基因在结直肠肿瘤中的变化进行分级，其中，所述印记基因为Z1、Z2、Z3或Z4中的任意一个或至少两个的组合，所述印记基因Z1为Gnas，所述印记基因Z2为Igf2r，所述印记基因Z3为Mest，所述印记基因Z4为Plagl1。

Description

用于结直肠肿瘤的印记基因分级模型及其组成的系统 技术领域
本申请涉及生物技术领域，涉及基因诊断领域，具体涉及用于结直肠肿瘤的印记基因分级模型及其组成的系统。
背景技术
结直肠癌是消化道中常见的恶性肿瘤，发病率位居所有恶性肿瘤的第三位，死亡率位居第四位。每年全球有约120万名患者被确诊为结直肠癌，有超过60万名患者直接或间接死于结直肠癌。据世界卫生组织(WHO)统计，2015年全球结直肠癌死亡774000例。结直肠癌发病率在45岁以下年龄段较低，但会随着年龄的增大而增加，近年来发病年龄有年轻化趋势。
2001至2007年问，美国不同分期的结直肠癌患者的5年生存率分别为90.1％(I期)，69.2％(II期和III期)和11.7％(IV期)，因此结直肠癌的早期诊断和早期治疗具有非常重要的意义。但是结直肠癌的早期诊断率非常低，在美国早期肠癌诊断率约为25％，而在中国早期诊断率仅为10％，急需开发更灵敏、更准确的检测手段。
此外，结直肠癌病人手术时为保证病变组织完全切除会连带切除距病变区域一定距离的癌旁组织，因为病变组织是否切除完全直接关系到复发率和复发时间，因此对于癌旁距离的选择十分关键。但是基于细胞和组织形态结构的传统病理学难以区分癌旁组织的良恶性。
从癌症的发展过程分析，分子层面的改变(表观遗传学和基因学)远早于细胞形态和组织结构的变异，所以分子生物学检测对癌症早期的检测更敏感。基因组印记是表观遗传学中基因调控的一种方式。其特点是，通过甲基化来自 特定亲代的等位基因，使某个基因只有一个等位基因表达，而另一个则陷入基因沉默状态。该种类的基因，被称为印迹(记)基因。印迹缺失是印迹基因去甲基化导致沉默状态的等位基因被激活并且开始基因表达的一种表观遗传改变。大量研究表明，该现象(印迹缺失)普遍存在于各类癌症并且发生时间早于细胞和组织形态改变。与此同时，在健康细胞中，印迹缺失比例极低，与癌细胞成鲜明对比。所以，印迹基因的甲基化状态可以作为病理标记，通过特定分子检测技术，对细胞异常状态进行分析。
基于上述原因，通过印迹基因检测技术解析结直肠癌在细胞层面上存在的分子标记物变化，可以提供更精确的预诊和诊断信息，并对手术时癌旁距离的确定具有重要的指导意义。
发明内容
针对现有技术的不足及实际的需求，本申请提供了用于结直肠肿瘤的印记基因分级模型及其组成的系统，该检测系统和模型是用于细胞和组织水平下早期直观地观察结直肠癌的印记(迹)基因的变化从而判断结直肠癌的良恶性及恶性程度。
为达到上述目的，本申请采用以下技术方案：
第一方面，本申请提供了用于结直肠肿瘤的印记基因分级模型，所述模型通过计算印记基因缺失表达量和印记基因拷贝数异常表达量在结直肠癌中的变化对印记基因的表达状态进行分级；
其中，所述印记基因为Z1、Z2、Z3或Z4中的任意一个或至少两个的组合，所述印记基因Z1为Gnas，所述印记基因Z2为Igf2r，所述印记基因Z3为Mest，所述印记基因在Z4为Plagl1。
根据本申请，若只检测一个印记基因，可以计算Z1-Z4中的任意一个，优 选为Z1和/或Z3，进一步优选为Z1。
本申请中，发明人发现，单独检测一个Z1印记基因，对结直肠癌的诊断敏感度可以达到93.8％，单独检测一个Z2印记基因，对结直肠癌的诊断敏感度可以达到47.9％，单独检测一个Z3印记基因，对结直肠癌的诊断敏感度可以达到83.3％，单独检测一个Z4印记基因，对结直肠癌的诊断敏感度可以达到58.3％。
根据本申请，若检测印记基因的两个印记基因的组合，计算Z1-Z4中的任意两个，所述组合可以是Z1和Z2的组合，Z1和Z3的组合，Z1和Z4的组合，Z2和Z3的组合，Z2和Z4的组或Z3和Z4的组合，优选为Z1和Z3的组合。
本申请中，发明人发现通过计算两个或两个以上的印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量可以进一步提高敏感度，检测印记基因的两个印记基因的组合，对结直肠癌的诊断敏感度可以达到60％以上，检测Z1和Z3的组合时，对结直肠癌的诊断敏感度可以达到100％，Z1和Z4的组合时，对结直肠癌的诊断敏感度可以达到97.9％以上，Z1和Z2的组合时，对结直肠癌的诊断敏感度可以达到95.8％以上。
根据本申请，所述印记基因分级模型最优选为检测Z1-Z4基因的组合。
本申请中，所述印记基因缺失为将细胞进行苏木素染色后，细胞核内存在两个棕色标记，所述印记基因拷贝数异常为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记，所述拷贝数异常是由于癌细胞异常地进行基因复制，导致这个基因表达时呈现为三倍体甚至更高的多倍体的情况。
本申请中，所述苏木素染色后的标记选自但不限于棕色，用其他颜色进行染色标记也可用于印迹基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。
本申请中，所述印记基因与印迹基因同时一个概念，表示同一个意思，可 以进行替换。
优选地，所述计算印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常印记基因表达量＝b/(b+c+d)×100％；
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；
其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记，印记基因拷贝数异常的细胞核。
优选地，所述印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级为针对Z1-Z4的四个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分的五个不同的等级。
优选地，所述针对Z1和Z3的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因Z1和Z3的印记基因缺失表达量小于15％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量小于0.8％；
I级：所述印记基因Z1和Z3的印记基因缺失表达量为15-20％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为0.8-2.5％；
II级：所述印记基因Z1和Z3的印记基因缺失表达量为20-30％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为2.5-3.5％；
III级：所述印记基因Z1和Z3的印记基因缺失表达量为30-35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为3.5-5％；
IV级：所述印记基因Z1和Z3的印记基因缺失表达量大于35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量大于5％。
本申请中，所述印记基因Z1和Z3的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
优选地，所述针对Z2和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因Z2和Z4的印记基因缺失表达量小于15％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量小于0.8％；
I级：所述印记基因Z2和Z4的印记基因缺失表达量为15-20％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为0.8-1.5％；
II级：所述印记基因Z2和Z4的印记基因缺失表达量为20-25％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为1.5-3％；
III级：所述印记基因Z2和Z4的印记基因缺失表达量为25-30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为3-5％；
IV级：所述印记基因Z2和Z4的印记基因缺失表达量大于30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量大于5％。
本申请中，所述印记基因Z2和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
第二方面，本申请提供了一种用于检测结直肠肿瘤良恶性程度的系统，包括如下单元：
(1)取样单元：获取待测样本；
(2)探针设计单元：根据印记基因序列设计特异性引物；
(3)检测单元：将步骤(2)的探针与待测样本进行原位杂交；
(4)分析单元：显微镜成像分析印记基因的表达状态；
其中，所述分析单元通过计算印记基因缺失表达量和印记基因拷贝数异常表达量，通过第一方面所述的模型，从而通过印记基因缺失表达量和印记基因拷贝数异常表达量的等级来判断结直肠肿瘤的良恶性程度。
本申请中，所述印记基因缺失为将细胞进行苏木素染色后，细胞核内存在两个棕色标记的细胞核，所述印记基因拷贝数异常为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记的细胞核，所述拷贝数异常是由于癌细胞异常地进行基因复制，导致这个基因表达时呈现为三倍体甚至更高的多倍体的情况。
本申请中，所述苏木素染色后的标记选自但不限于棕色，用其他颜色进行染色标记也可用于印迹基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。
本申请所述检测系统是用于细胞和组织水平下早期直观地观察结直肠肿瘤的印记(迹)基因的变化从而判断结直肠肿瘤的良恶性及恶性程度，为早期结直肠肿瘤患者提供最有利的治疗机会。
根据本申请，步骤(1)所述的待测样本来自于人的组织和/或细胞。
本申请中，所述待测样本只要RNA经过及时固定的处理都是可行的，本领域技术人员可以根据需要进行选择，在此不做特殊限定，本申请所述待测样本包括组织的石蜡切片和/或内窥镜筛查样本，优选为内窥镜筛查样本。
所述组织的石蜡切片具体操作步骤为获取人体肿瘤组织样本，及时用10％中性福尔马林固定，石蜡包埋，切成10μm厚，用带正电荷的玻片制成组织片子； 因为只有10μm厚，因此显微镜下看见的有一部分为不完整的细胞核，所以会出现部分假阴性的基因缺失。
所述内窥镜筛查样本具体操作步骤为在结直肠镜下获取可疑组织，石蜡包埋，切成10μm厚，用带正电荷的玻片制成片子即得。
本申请中，由于内窥镜筛查样本即结直肠镜活检对病人伤害小，取样过程简单，相较于血液的循环特性，结直肠镜活检还能定位，结直肠镜活检作为实验样本有其特殊的优势。
优选地，所述印记基因为Z1-Z4，所述印记基因Z1为Gnas，所述印记基因Z2为Igf2r，所述印记基因Z3为Mest，所述印记基因在Z4为Plagl1。
本申请中，所述印记基因Z1(Gnas)，Z2(Igf2r)，Z3(Mest)，Z4(Plagl1)在正常肿瘤细胞组织内有不同程度的表达，在发生恶性病变时，表达量和印记状态都会发生明显变化。
本申请中，所述设计探针是根据印记基因Z1-Z4，即Gnas，Igf2r，Mest和Plagl1进行设计的，具体在每个基因的内旋子内选择一段序列作为探针，具体选择的基因序列和具体基因的位置如下：
Z1(Hs-GNAS)：chr20：58839681-58911196；
Z2(Hs-IGF2R)：chr6：160059099-160060546；
Z3(Hs-Mest)：chr7：130492340-130495367；
Z4(Hs-PLAGL1)：chr6：143968979-143985134。
优选地，所述原位杂交采用RNAscope原位杂交方法。
优选地，所述RNAscope原位杂交方法使用单通道或多通道的呈色试剂盒或者单通道或多通道的荧光试剂盒，优选为单通道棕色呈色试剂盒或多通道的荧光试剂盒。
本申请中，所述多通道呈色试剂盒或多通道荧光试剂盒包括两通道或两通道以上的呈色试剂盒或荧光试剂盒，所述两通道的呈色试剂盒或多通道的荧光试剂盒可以使用两个印记基因探针或印记基因和其他基因的联合表达甚至多个印记基因和非印记基因的综合表达。
根据本申请，所述模型中的计算印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常印记基因表达量＝b/(b+c+d)×100％；
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；
其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记，印记基因拷贝数异常的细胞核。
本申请中，所述苏木素染色后的标记选自但不限于棕色，用其他颜色进行染色标记也可用于印迹基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。
本申请中，将探针通过原位杂交，和Hemotoxy(苏木精)细胞核染色扩增信号，在40×或60×显微镜下，判断每一个细胞核内印记基因存在、印记基因缺失或拷贝数异常，通过计算印记基因缺失基因表达量和印记基因拷贝数异常的基因表达量来判定该样本的肿瘤良恶性程度。由于切片仅为10微米，所以在显微镜下所见细胞核大约有20％为不完整细胞核，也就是说有部分假阴性的可能 性存在。
优选地，所述印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级。
优选地，所述五个不同的等级为针对Z1-Z4的四个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分。
优选地，所述针对Z1和Z3的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因Z1和Z3的印记基因缺失表达量小于15％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量小于0.8％；
I级：所述印记基因Z1和Z3的印记基因缺失表达量为15-20％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为0.8-2.5％；
II级：所述印记基因Z1和Z3的印记基因缺失表达量为20-30％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为2.5-3.5％；
III级：所述印记基因Z1和Z3的印记基因缺失表达量为30-35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为3.5-5％；
IV级：所述印记基因Z1和Z3的印记基因缺失表达量大于35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量大于5％。
本申请中，所述印记基因Z1和Z3的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
优选地，所述针对Z2和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因Z2和Z4的印记基因缺失表达量小于15％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量小于0.8％；
I级：所述印记基因Z2和Z4的印记基因缺失表达量为15-20％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为0.8-1.5％；
II级：所述印记基因Z2和Z4的印记基因缺失表达量为20-25％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为1.5-3％；
III级：所述印记基因Z2和Z4的印记基因缺失表达量为25-30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为3-5％；
IV级：所述印记基因Z2和Z4的印记基因缺失表达量大于30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量大于5％。
本申请中，所述印记基因Z2和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
优选地，待判断的结直肠肿瘤的良恶性程度分为良性结直肠肿瘤、结直肠癌潜能、早期结直肠癌、中期结直肠癌和晚期结直肠癌。
优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级、印记基因Z1、印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因Z1、Z2、Z3和Z4中的至少3个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4的至少2 个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则为结直肠癌潜能；
优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为II级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期结直肠癌；
优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为III级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期结直肠癌；
优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期恶性结直肠癌。
第三方面，本申请提供一种如第一方面所述的模型或如第二方面所述的系统在制备结直肠癌检测和/或治疗的药物中的用途。
优选地，所述结直肠癌检测为判断结直肠肿瘤的良恶性程度，所述结直肠肿瘤的良恶性程度分为良性、结直肠癌潜能、早期结直肠癌、中期结直肠癌和晚期结直肠癌。
优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3 和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级、印记基因Z1、印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因Z1、Z2、Z3和Z4中的至少3个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则为结直肠癌潜能；
优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为II级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期结直肠癌；
优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为III级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期结直肠癌；
优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期恶性结直肠癌。
与现有技术相比，本申请具有如下有益效果：
(1)本申请所述检测模型和系统，以直观的方法表现了印记缺失在结直肠肿瘤病人的样本上的表现，通过对印记基因原位标记的方法，客观，直观，早期，精确地检测出印记(迹)基因的变化，并可以提供量化的模型，为结直肠肿瘤的诊断做出巨大贡献；
(2)本申请检测方系统，可以在结直肠肿瘤病人手术前得出结直肠肿瘤良恶性程度的判断，从而为手术及精准治疗提供依据，这是细胞分子领域诊断结直肠肿瘤的革命性突破；
(3)本申请可以精确的判断结直肠肿瘤的类型，单独检测Z1探针时，对恶性潜能以上等级的样本检测敏感度为93.8％；若联合检测Z1和Z3探针时，对恶性潜能以上等级的样本检测敏感度可以增加到100％，极大地提高了对结直肠癌的早期，明确诊断，特别是用在早期普查和癌症术后随访，尤其是对于疑似复发病人的跟踪随访，可以争取时间，为挽救病人生命做出重大贡献；
(4)本申请可以检测癌旁组织的良恶性，可以对手术时癌旁距离的确定提供指导，保证病变组织的完全切除，极大降低病人的复发率；
(5)本申请检测方法区别于免疫组化方法，减少了假阳性和其他负面作用，不仅如此，通过发现的结直肠肿瘤相关印记基因缺失位点的致该基因沉默、剔除、重排的靶向药物或技术方法，可用于指导后期的治疗和用药。
附图说明
图1是本申请实施例中苏木素染色细胞核的结直肠癌的病理切片，其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达；所述b为将细胞进行苏木素染色后，细胞核内存在一个棕色标记，印记基因存在；所述c为将细胞进行苏木素染色后，细胞核内存在两个棕色标记，印记基因缺失；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记，印记基因拷贝数异常；
图2(a)为0级结直肠癌的病理切片中4个基因的表达状态，图2(b)为I级结直肠癌的病理切片中4个基因的表达状态，图2(c)为II级结直肠癌的病理切片中4个基因的表达状态，图2(d)为III级结直肠癌的病理切片中4个基因的表达状态，图2(e)为IV级结直肠癌的病理切片中4个基因的表达状态；
图3(a)为4个基因对结直肠癌的印记缺失的强度，图3(b)为4个基因对结直肠癌的拷贝数异常的强度，其中，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量；
图4(a)为印记基因Z1印记缺失和拷贝数异常的强度，图4(b)为印记基因Z2印记缺失和拷贝数异常的强度，图4(c)为印记基因Z3印记缺失和拷贝数异常的强度，图4(d)为印记基因Z4印记缺失和拷贝数异常的强度，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量；
图5(a)为印记基因Z1应用于73例结直肠癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(b)为印记基因Z2应用于73例结直肠癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(c)为印记基因Z3应用于73例结直肠癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(d)为印记基因Z4应用于73例结直肠癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，LOI为印记基因缺失基因表达量， CNV为印记基因拷贝数异常的基因表达量。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
实施例1结直肠癌的印记基因分析
所述的印记基因的检测方法，包括如下步骤：
(1)获取结直肠癌的组织细胞，切成10微米厚，放入10％中性福尔马林溶液中进行固定，以防RNA降解，固定时间为24小时，石蜡包埋(FFPE)，所述玻片需要用正电荷脱载玻片，所述切片在40℃烤箱烘烤3h以上；
(2)按照RNASCope的样品处理方法进行脱蜡处理，封闭样本中内源性过氧化物酶活性，增强通透性并暴露出RNA分子；
(3)设计探针：根据印记基因序列设计特异性引物；
所述设计探针是根据印记基因Z1(Gnas)、Z2(Igf2r)、Z3(Mest)和Z4(Plagl1)进行设计的，具体在每个基因的内旋子内选择一段序列作为探针，具体选择的基因序列和具体基因的位置如下：
Z1(Hs-GNAS)：chr20：58839681-58911196；
Z2(Hs-IGF2R)：chr6：160059099-160060546；
Z3(Hs-Mest)：chr7：130492340-130495367；
Z4(Hs-PLAGL1)：chr6：143968979-143985134。
(4)将步骤(3)的探针与待测样本通过试剂盒进行RNA SCope原位杂交；
(5)信号扩增和苏木精染色，用显微镜成像分析印记基因的表达状态；
所述模型中的计算印记基因缺失表达量和印记基因拷贝数异常表达量的公 式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常印记基因表达量＝b/(b+c+d)×100％；
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；
其中，a、b、c、d如图1所示，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记，印记基因拷贝数异常的细胞核。
从图2(a)-图2(e)可以看出，从0级到IV级的样本中，印记缺失(细胞核内有两个信号点)和拷贝数异常(细胞核内有三个或以上信号点)的细胞比例随恶性程度的增加而逐渐增加。
实施例2
所述结直肠镜活检样本是，在结直肠镜下取出可疑病变组织，10％中性福尔马林溶液固定24h，石蜡包埋(FFPE)，切成10微米厚的切片，其他检测方法同实施例1。
从图3(a)-图3(b)可以看出，Z1，Z2，Z3，Z4每个基因对结直肠癌的反应敏感性或者说对应于结直肠癌表达的印记缺失的强度和状态是不同的。
具体每个印记基因对结直肠癌的敏感度如图4(a)-图4(d)，印记基因Z1的缺失和拷贝数异常出现在结直肠癌病变的最早期，在恶性潜能和早期结直肠癌阶段显著升高；印记基因Z2在结直肠癌病变的初期较不敏感，在恶性潜能阶 段出现部分拷贝数异常，但在结直肠癌早中期没有继续升高，到晚期才出现印记缺失和拷贝数异常的显著增加；印记基因Z3的拷贝数异常在恶性潜能阶段显著上升，印记缺失在结直肠癌早期显著增加，但在中晚期病例中印记缺失和拷贝数异常上升的速度减缓；印记基因Z4的印记缺失和拷贝数异常在随结直肠癌恶性程度的升高而逐渐上升，在晚期结直肠癌中上升最为明显。
实施例3
获取73例结直肠癌病人的组织包括结直肠镜活检样本(10微米)，检测方法同实施例1。
具体等级分型如下：
从图5(a)可以看出，对于所述印记基因Z1，印记基因缺失表达量小于15％和/或印记基因拷贝数异常表达量小于0.8％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为0.8-2.5％为I级，印记基因缺失表达量为20-30％和/或印记基因拷贝数异常表达量为2.5-3.5％为II级，印记基因缺失表达量为30-35％和/或印记基因拷贝数异常表达量为3.5-5％为III级，印记基因缺失表达量大于35％和/或印记基因拷贝数异常表达量大于5％为IV级；
从图5(b)可以看出，对于所述印记基因Z2，印记基因缺失表达量小于15％和/或印记基因拷贝数异常表达量小于0.8％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为0.8-1.5％为I级，印记基因缺失表达量为20-25％和/或印记基因拷贝数异常表达量为1.5-3％为II级，印记基因缺失表达量为25-30％和/或印记基因拷贝数异常表达量为3-5％为III级，印记基因缺失表达量大于30％和/或印记基因拷贝数异常表达量大于5％为IV级；
从图5(c)可以看出，对于所述印记基因Z3，印记基因缺失表达量小于15％和/或印记基因拷贝数异常表达量小于0.8％为0级，印记基因缺失表达量为 15-20％和/或印记基因拷贝数异常表达量为0.8-2.5％为I级，印记基因缺失表达量为20-30％和/或印记基因拷贝数异常表达量为2.5-3.5％为II级，印记基因缺失表达量为30-35％和/或印记基因拷贝数异常表达量为3.5-5％为III级，印记基因缺失表达量大于35％和/或印记基因拷贝数异常表达量大于5％为IV级；
从图5(d)可以看出，对于所述印记基因Z4，印记基因缺失表达量小于15％和/或印记基因拷贝数异常表达量小于0.8％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为0.8-1.5％为I级，印记基因缺失表达量为20-25％和/或印记基因拷贝数异常表达量为1.5-3％为II级，印记基因缺失表达量为25-30％和/或印记基因拷贝数异常表达量为3-5％为III级，印记基因缺失表达量大于30％和/或印记基因拷贝数异常表达量大于5％为IV级。
从这73个结直肠肿瘤的样本综合分析可以得出：
所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级，印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因Z1、Z2、Z3和Z4中的至少3个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中不超 过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则判断为结直肠癌潜能；
所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为II级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期结直肠癌；
所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为III级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期结直肠癌；
所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期结直肠癌。
综上所述，本申请所述检测模型和系统，以直观的方法表现了印记缺失在结直肠肿瘤病人的样本上的表现，通过对印记基因原位标记的方法，客观，直观，早期，精确地检测出印记(迹)基因的变化，并可以提供量化的模型，为结直肠肿瘤的诊断做出巨大贡献。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原 料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种用于结直肠肿瘤的印记基因分级模型，其通过计算印记基因缺失表达量和印记基因拷贝数异常表达量在结直肠肿瘤中的变化对印记基因的表达状态进行分级；
    其中，所述印记基因为Z1、Z2、Z3或Z4中的任意一个或至少两个的组合，所述印记基因Z1为Gnas，所述印记基因Z2为Igf2r，所述印记基因Z3为Mest，所述印记基因在Z4为Plagl1。
  2. 根据权利要求1所述的模型，其中，所述模型计算印记基因的方法如下：
    计算Z1-Z4中的任意一个印记基因。
  3. 根据权利要求1所述的模型，其中，所述模型计算印记基因的方法为：计算Z1-Z4中的任意两个印记基因。
  4. 根据权利要求1所述的模型，其中，所述模型计算印记基因的方法为：计算Z1-Z4的四个印记基因的组合。
  5. 根据权利要求1-4任一项所述的模型，其特征在于，所述计算印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
    总表达量＝(b+c+d)/(a+b+c+d)×100％；
    正常印记基因表达量＝b/(b+c+d)×100％；
    印记基因缺失基因表达量＝c/(b+c+d)×100％；
    印记基因拷贝数异常的基因表达量＝d/(b+c+d)×100％；
    其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记，印记基因拷贝数异常的细胞核。
  6. 根据权利要求1-5中任一项所述的模型，其特征在于，所述印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级为针对Z1-Z4的四个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分的五个不同的等级；
    优选地，所述针对Z1和Z3的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
    0级：所述印记基因Z1和Z3的印记基因缺失表达量小于15％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量小于0.8％；
    I级：所述印记基因Z1和Z3的印记基因缺失表达量为15-20％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为0.8-2.5％；
    II级：所述印记基因Z1和Z3的印记基因缺失表达量为20-30％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为2.5-3.5％；
    III级：所述印记基因Z1和Z3的印记基因缺失表达量为30-35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为3.5-5％；
    IV级：所述印记基因Z1和Z3的印记基因缺失表达量大于35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量大于5％；
    优选地，所述针对Z2和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
    0级：所述印记基因Z2和Z4的印记基因缺失表达量小于15％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量小于0.8％；
    I级：所述印记基因Z2和Z4的印记基因缺失表达量为15-20％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为0.8-1.5％；
    II级：所述印记基因Z2和Z4的印记基因缺失表达量为20-25％和/或所述 印记基因Z2和Z4的印记基因拷贝数异常表达量为1.5-3％；
    III级：所述印记基因Z2和Z4的印记基因缺失表达量为25-30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为3-5％；
    IV级：所述印记基因Z2和Z4的印记基因缺失表达量大于30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量大于5％。
  7. 一种用于检测结直肠肿瘤良恶性程度的系统，其包括如下单元：
    (1)取样单元：获取待测样本；
    (2)探针设计单元：根据印记基因序列设计特异性引物；
    (3)检测单元：将步骤(2)的探针与待测样本进行原位杂交；
    (4)分析单元：显微镜成像分析印记基因的表达状态；
    其中，所述分析单元通过计算印记基因缺失表达量和印记基因拷贝数异常表达量，通过权利要求1-6中任一项所述的模型，从而通过印记基因缺失表达量和印记基因拷贝数异常表达量的等级来判断结直肠肿瘤的良恶性程度。
  8. 根据权利要求7所述的系统，其中，步骤(1)所述的待测样本为组织的石蜡切片和/或内窥镜筛查样本。
  9. 根据权利要求7所述的系统，其中，所述印记基因为Z1-Z4，所述印记基因Z1为Gnas，所述印记基因Z2为Igf2r，所述印记基因Z3为Mest，所述印记基因在Z4为Plagl1。
  10. 根据权利要求7所述的系统，其中，所述原位杂交采用RNAscope原位杂交方法；
    优选地，所述RNAscope原位杂交方法使用单通道或多通道的呈色试剂盒或者单通道或多通道的荧光试剂盒，优选为单通道棕色呈色试剂盒或多通道的荧光试剂盒。
  11. 根据权利要求7-10任一项所述的系统，其中，所述模型中的计算印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
    总表达量＝(b+c+d)/(a+b+c+d)×100％；
    正常印记基因表达量＝b/(b+c+d)×100％；
    印记基因缺失基因表达量＝c/(b+c+d)×100％；
    印记基因拷贝数异常的基因表达量＝d/(b+c+d)×100％；
    其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上棕色标记，印记基因拷贝数异常的细胞核。
  12. 根据权利要求7-11中任一项所述的系统，其中，所述印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级；
    优选地，所述五个不同的等级为针对Z1-Z4的四个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分；
    优选地，所述针对Z1和Z3的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
    0级：所述印记基因Z1和Z3的印记基因缺失表达量小于15％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量小于0.8％；
    I级：所述印记基因Z1和Z3的印记基因缺失表达量为15-20％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为0.8-2.5％；
    II级：所述印记基因Z1和Z3的印记基因缺失表达量为20-30％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为2.5-3.5％；
    III级：所述印记基因Z1和Z3的印记基因缺失表达量为30-35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量为3.5-5％；
    IV级：所述印记基因Z1和Z3的印记基因缺失表达量大于35％和/或所述印记基因Z1和Z3的印记基因拷贝数异常表达量大于5％；
    优选地，所述针对Z2和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
    0级：所述印记基因Z2和Z4的印记基因缺失表达量小于15％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量小于0.8％；
    I级：所述印记基因Z2和Z4的印记基因缺失表达量为15-20％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为0.8-1.5％；
    II级：所述印记基因Z2和Z4的印记基因缺失表达量为20-25％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为1.5-3％；
    III级：所述印记基因Z2和Z4的印记基因缺失表达量为25-30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量为3-5％；
    IV级：所述印记基因Z2和Z4的印记基因缺失表达量大于30％和/或所述印记基因Z2和Z4的印记基因拷贝数异常表达量大于5％。
  13. 根据权利要求7-12中任一项所述的系统，其中，待判断的结直肠肿瘤的良恶性程度分为良性、结直肠癌潜能、早期结直肠癌、中期结直肠癌和晚期结直肠癌；
    优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级、印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因拷贝数异常表达 量为I级或印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
    优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因Z1、Z2、Z3和Z4中的至少3个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则为结直肠癌潜能；
    优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为II级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期结直肠癌；
    优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为III级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期结直肠癌；
    优选地，判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期恶 性结直肠癌。
  14. 一种如权利要求1-6中任一项所述的模型或如权利要求7-13中任一项所述的系统在制备结直肠肿瘤检测和/或治疗的药物中的用途。
  15. 根据权利要求14所述的用途，其中，所述结直肠肿瘤检测为判断结直肠肿瘤的良恶性程度，所述结直肠肿瘤的良恶性程度包括良性、结直肠癌潜能、早期结直肠癌、中期结直肠癌和晚期结直肠癌；
    优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级、印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
    优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因Z1、Z2、Z3和Z4中的至少3个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z2、Z3和Z4的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z2、Z3和Z4中的不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则为结直肠癌潜能；
    优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为II级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因 Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期结直肠癌；
    优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为III级、印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z2、Z3和Z4中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期结直肠癌；
    优选地，所述判断结直肠肿瘤的良恶性程度的结果为印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z2、Z3和Z4中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期恶性结直肠癌。
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