WO2018214249A1 - 一种印记基因分级模型及其组成的系统和应用 - Google Patents

一种印记基因分级模型及其组成的系统和应用 Download PDF

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WO2018214249A1
WO2018214249A1 PCT/CN2017/092364 CN2017092364W WO2018214249A1 WO 2018214249 A1 WO2018214249 A1 WO 2018214249A1 CN 2017092364 W CN2017092364 W CN 2017092364W WO 2018214249 A1 WO2018214249 A1 WO 2018214249A1
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imprinted
imprinted gene
expression amount
gene
copy number
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French (fr)
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成彤
周宁
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立森印迹诊断技术（无锡）有限公司
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    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • 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
    • 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
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • 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
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20—Supervised data analysis
    • 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
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10056—Microscopic image
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30096—Tumor; Lesion
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems

Definitions

  • the present application relates to the field of biotechnology, and relates to the field of genetic diagnosis, and in particular to a system and application for imprinting gene grading models and their compositions.
  • Cancer is caused by complex genetic and epigenetic changes that accumulate over time at the cellular level, ultimately leading to uncontrolled cell division.
  • the pathological diagnosis of benign and malignant cells by traditional pathology is based on the relationship between cell size, morphology, invasiveness and peripheral cellular tissues. It has great limitations on the discovery of early changes in cells (cancer), so the method of cancer diagnosis at the cellular level has once become a research hotspot. With the continuous research in the field of molecular biology, more and more molecular detection techniques are applied to cancer diagnosis.
  • the benign and malignant lesions and the judgment of cancer typing or cancer stage are largely determined by the morphology of the cells observed on the histopathological sections stained with hematoxylin and eosin.
  • this method has its inherent limitations.
  • the method cannot observe changes in the molecular level of tumors, and molecular changes can provide a more accurate basis for pre-diagnosis and diagnosis.
  • cell morphology diagnosis is subjective judgment, which itself can cause inaccurate diagnosis. The errors caused by such subjective judgments have a great impact on the diagnosis of early cancer, especially the sensitivity and accuracy of early diagnosis of cancer is life-critical for patients.
  • some malignant tumor cells are morphologically very different from benign tumor cells.
  • the research on imprinted genes is mainly based on methylation detection.
  • the detection techniques mainly include restriction enzyme digestion, enrichment and sulfite conversion.
  • the methylation sites of different segments of the target gene are transcriptionally active. The effects are quite different, and even if there is an accurate methylation detection method, the tumor cannot be accurately diagnosed.
  • Genomic imprinting is a way of epigenetic gene regulation, expressed as that for a particular gene, only alleles from a particular parent can be expressed, while another allele appears as a gene silence.
  • imprint (trace) deletion means that the alleles in the imprinted (spot) gene that were originally in a silent state are activated (demethylated), which is the most common and early epigenetic episode in cancer. Change, and this feature can be used as a pathological marker. In contrast, in healthy cell assays, the proportion of blot deletions is very low.
  • the current clinical disease diagnosis requires a new detection system and detection model, based on the patient biopsy sample, to analyze the molecular marker changes present at the cell level of the cancer, thereby providing more accurate pre-diagnosis and diagnostic information.
  • the present application provides a system and application of an imprinted gene grading model and a composition thereof for early and intuitive observation of various types of tumors at the single cell and tissue levels.
  • the change in the imprint (spot) gene determines the degree of benign and malignant tumors.
  • the present application provides an imprinted gene grading model for calculating changes in the amount of imprinted gene expression, imprinted gene deletion expression, and imprinted gene copy number abnormal expression in a tumor.
  • the grading of the imprinted genes is a first aspect, the present application provides an imprinted gene grading model for calculating changes in the amount of imprinted gene expression, imprinted gene deletion expression, and imprinted gene copy number abnormal expression in a tumor. The grading of the imprinted genes.
  • the imprinted gene is deleted, after the cells are subjected to hematoxylin staining, there are two red/brown marks 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 red in the nucleus/ Brown marker, 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, red or 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 imprinted gene is T1-T6, the imprinted gene T1 is Gnas, the imprinted gene T2 is Igf2, the imprinted gene T3 is Peg10, the imprinted gene T4 is Igf2r, and the imprinted gene T5 is Mest, the imprinted gene T6 is Plagl.
  • the formula for calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number are as follows:
  • Imprinted gene deletion gene expression amount c / (b + c + d) ⁇ 100%;
  • the gene expression level of the imprinted gene copy number abnormality d / (b + c + d) ⁇ 100%;
  • a is a hematoxylin staining of cells, and there is no label in the nucleus, and the imprinted gene is not expressed; wherein b is a hematoxylin staining, a red/brown mark is present in the nucleus, and the imprinted gene is present; c is the hematoxylin staining of cells, there are two red/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 red/brown marks and the imprinted gene copy number is abnormal.
  • the imprinted gene expression amount, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount are divided into five different grades, which are imprinted gene deletion expression amount and imprinted gene copy number of six imprinted genes for T1-T6.
  • the abnormal expression levels are divided into five different levels.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T1 are:
  • the imprinted gene T1 has an imprinted gene deletion expression amount of less than 15% and/or the imprinted gene T1 imprinted gene copy number abnormal expression amount is less than 0.5%;
  • the imprinted gene of the imprinted gene T1 has a deletion amount of 15-20% and/or the imprinted gene T1 has an abnormal expression amount of the imprinted gene of 0.5-1.5%;
  • the imprinted gene deletion expression amount of the imprinted gene T1 is 20-25% and/or the imprinted gene copy number abnormal expression amount of the imprinted gene T1 is 1.5-2.5%;
  • the imprinted gene of the imprinted gene T1 has a deletion amount of 25-35% and/or the imprinted gene T1 has an abnormal expression amount of the imprinted gene of 2.5-3.5%;
  • Grade IV The imprinted gene deletion expression amount of the imprinted gene T1 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted gene T1 is greater than 3.5%.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T2, T3 and T4 are:
  • the imprinted gene T2, T3 and T4 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes T2, T3 and T4 have an imprinted gene copy number abnormal expression amount of less than 1%;
  • the imprinted gene T2, T3 and T4 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted gene T2, T3 and T4 imprinted gene copy number abnormal expression amount is 1-2%;
  • Grade II the imprinted genes of the imprinted genes T2, T3 and T4 are expressed in an amount of 20-25% and/or The abnormal expression level of the imprinted gene copy number of the imprinted genes T2, T3 and T4 is 2-3%;
  • the imprinted gene T2, T3 and T4 have an imprinted gene deletion expression amount of 25-35% and/or the imprinted genes T2, T3 and T4 have an imprinted gene copy number abnormal expression amount of 3-4%;
  • the imprinted gene deletion expression amount of the imprinted genes T2, T3 and T4 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T2, T3 and T4 is greater than 4%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T2, T3 and T4 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 T5 and T6 are:
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is less than 0.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 10-15% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 0.5-1.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 1.5-2.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 20-30% and/or the imprinted genes T5 and T6 have an imprinted gene copy number abnormal expression amount of 2.5-3.5%;
  • Grade IV The imprinted gene deletion expression amount of the imprinted genes T5 and T6 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T5 and T6 is greater than 3.5%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T5 and T6 are independent of each other.
  • the present application provides a system for detecting the degree of benign and malignant tumors, comprising the following units:
  • sampling unit obtaining a sample to be tested
  • Probe design unit design specific primers according to the imprinted gene sequence
  • the analysis unit calculates the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number, and the abnormal expression of the imprinted gene and the imprinted gene copy number are expressed by the model described in the first aspect.
  • the level of the amount is used to determine the degree of benign and malignant tumors.
  • the imprinted gene is deleted, after the cells are subjected to hematoxylin staining, there are two red/brown marks 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 red in the nucleus/ Brown marker, 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, red or 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 various types of tumors at the single cell and tissue level to determine the benign and malignant degree of the tumor, and to provide the most favorable treatment opportunity for patients with early cancer. .
  • 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. Any one or a combination of at least two of a needle biopsy cell pellet or an endoscopic screening sample.
  • the specific operation procedure of the paraffin section of the tissue is to obtain a sample of human tumor tissue, and 10% is used in time.
  • the specific operation step of the puncture biopsy cell tablet is to obtain human cells, and the 10% neutral formalin can be fixed in time.
  • the sampling process is simple, and the puncture cells can be positioned compared to the circulating characteristics of the blood, and the puncture of cells as a test sample has its special advantages.
  • the sample to be tested is a biopsy puncture cell.
  • the imprinted gene is T1-T6, the imprinted gene T1 is Gnas, the imprinted gene T2 is Igf2, the imprinted gene T3 is Peg10, the imprinted gene T4 is Igf2r, and the imprinted gene T5 is Mest, the imprinted gene T6 is Plagl.
  • the imprinted genes T1 (Gnas), T2 (Igf2), T3 (Peg10), T4 (Igf2r), T5 (Mest), T6 (Plagl1) have different degrees of expression in normal tumor cell tissues, When a malignant lesion occurs, both the expression level and the imprinted state change significantly.
  • the designed probe is designed according to the imprinting genes T1-T6, namely Gnas, Igf2, Peg10, Igf2r, Mest and Plagl, and specifically selects a sequence as a probe in the inner loop of each gene, specifically
  • the selected gene sequence and the location of the specific gene are as follows:
  • T4 Hs-IGF2R: chr6: 160059099-160060546;
  • the in situ hybridization employs an RNAscope in situ hybridization method.
  • the RNAscope in situ hybridization method uses a single-channel or multi-channel colorimetric kit or a single-channel or multi-channel fluorescent kit, preferably a single-channel red/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 expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy in the model are as follows:
  • Imprinted gene deletion gene expression amount c / (b + c + d) ⁇ 100%;
  • the gene expression level of the imprinted gene copy number abnormality d / (b + c + d) ⁇ 100%;
  • a is a hematoxylin staining of cells, and there is no label in the nucleus, and the imprinted gene is not expressed; wherein b is a hematoxylin staining, a red/brown mark is present in the nucleus, and the imprinted gene is present; c is the hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the imprinted gene is deleted; the d is the hematoxylin staining of the cells, there are more than two red/brown marks in the nucleus, and the imprinting gene copy number is abnormal. .
  • the hematoxylin-stained label is selected from, but not limited to, red or 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 hybridized by in situ hybridization and Hemotoxy (hematoxylin) nuclear staining amplification Signal, under the 40 ⁇ or 60 ⁇ microscope, judge the presence of imprinted genes in each nucleus, imprinted gene deletion or copy number abnormality, and calculate gene expression of imprinted gene expression, imprinted gene deletion gene expression and imprinted gene copy number abnormality.
  • the amount is determined to determine the degree of benign and malignant tumors of the sample. 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 respectively divided into an imprinted gene deletion expression amount and an imprinted gene copy number abnormal expression amount for the six imprinted genes of T1-T6.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T1 are:
  • the imprinted gene T1 has an imprinted gene deletion expression amount of less than 15% and/or the imprinted gene T1 imprinted gene copy number abnormal expression amount is less than 0.5%;
  • the imprinted gene of the imprinted gene T1 has a deletion amount of 15-20% and/or the imprinted gene T1 has an abnormal expression amount of the imprinted gene of 0.5-1.5%;
  • the imprinted gene deletion expression amount of the imprinted gene T1 is 20-25% and/or the imprinted gene copy number abnormal expression amount of the imprinted gene T1 is 1.5-2.5%;
  • the imprinted gene of the imprinted gene T1 has a deletion amount of 25-35% and/or the imprinted gene T1 has an abnormal expression amount of the imprinted gene of 2.5-3.5%;
  • Grade IV The imprinted gene deletion expression amount of the imprinted gene T1 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted gene T1 is greater than 3.5%.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T2, T3 and T4 are:
  • the imprinted gene T2, T3 and T4 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes T2, T3 and T4 have an imprinted gene copy number abnormal expression amount of less than 1%;
  • the imprinted gene T2, T3 and T4 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted gene T2, T3 and T4 imprinted gene copy number abnormal expression amount is 1-2%;
  • the imprinted gene T2, T3 and T4 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes T2, T3 and T4 have an imprinted gene copy number abnormal expression amount of 2-3%;
  • the imprinted gene T2, T3 and T4 have an imprinted gene deletion expression amount of 25-35% and/or the imprinted genes T2, T3 and T4 have an imprinted gene copy number abnormal expression amount of 3-4%;
  • the imprinted gene deletion expression amount of the imprinted genes T2, T3 and T4 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T2, T3 and T4 is greater than 4%.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T5 and T6 are:
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is less than 0.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 10-15% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 0.5-1.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 1.5-2.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 20-30% and/or the imprinted genes T5 and T6 have an imprinted gene copy number abnormal expression amount of 2.5-3.5%;
  • Grade IV The imprinted gene deletion expression amount of the imprinted genes T5 and T6 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T5 and T6 is greater than 3.5%.
  • the determining the degree of benign and malignant tumor is divided into benign, malignant potential, early malignant tumor, Metaphase malignancies and advanced malignancies.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, T4, T5 and T6 are both level 0, and the imprinted gene T1 is
  • the imprinted gene deletion expression of one of the T2, T3, T4, T5 and T6 imprinted genes is not greater than the imprinted gene copy number of the imprinted gene of the class I and the imprinted genes T1, T2, T3, T4, T5 and T6
  • the amount of abnormal expression is not greater than the level of the imprinted gene of the two imprinted genes of the class I or the imprinted genes T1, T2, T3, T4, T5 and T6, and the amount of the imprinted gene is not greater than the class I and the imprinted gene copy number of the two imprinted genes If the abnormal expression level is any of the 0 grades, it is a benign tumor.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount of the two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 is I level and the two imprinted genes are The imprinting gene whose expression level of the imprinted gene is abnormally expressed at level I, at least three imprinted genes of the imprinted genes T1, T2, T3, T4, T5 and T6 is the level I and the imprinted genes T1, T2, T3,
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of T4, T5 and T6 is the level of imprinted gene deletion expression of no more than one imprinted gene in class I or imprinted genes T1, T2, T3, T4, T5 and T6 If the amount of the imprinted gene deletion is in any of the second grade, it is judged to be a malignant potential.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both II.
  • the level of imprinted gene deletion and the amount of imprinted gene deletion in the class or imprinted genes T1, T2, T3, T4, T5 and T6 are both grade III, which is an early malignant tumor.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both III.
  • the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of no more than one imprinted gene in the graded or imprinted genes T1, T2, T3, T4, T5 and T6 are all grade IV, and are metaphase malignant tumors.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both IV Grade, it is an advanced malignant tumor.
  • the tumor is a tumor conventional in the art, and the application is selected from, but not limited to, any one of thyroid tumor, lung tumor or brain tumor or a combination of at least two.
  • the present application provides a model of the first aspect or the system of the second aspect, for use in the preparation of a medicament for tumor detection.
  • the tumor is a tumor conventional in the art, and the application is selected from, but not limited to, any one of thyroid tumor, lung tumor or brain tumor or a combination of at least two.
  • the tumor is detected to determine the type of tumor, including benign, malignant, early malignancy, metaphase malignancy, and advanced malignancy.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, T4, T5 and T6 are both level 0, and the imprinted gene T1 is
  • the imprinted gene deletion expression of one of the T2, T3, T4, T5 and T6 imprinted genes is not greater than the imprinted gene copy number of the imprinted gene of the class I and the imprinted genes T1, T2, T3, T4, T5 and T6
  • the amount of abnormal expression is not greater than the level of the imprinted gene of the two imprinted genes of the class I or the imprinted genes T1, T2, T3, T4, T5 and T6, and the amount of the imprinted gene is not greater than the class I and the imprinted gene copy number of the two imprinted genes If the abnormal expression level is any of the 0 grades, it is judged to be a benign tumor.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount of the two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 is I level and the two imprinted genes are The imprinting gene whose expression level of the imprinted gene is abnormally expressed at level I, at least three imprinted genes of the imprinted genes T1, T2, T3, T4, T5 and T6 is the level I and the imprinted genes T1, T2, T3,
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of T4, T5 and T6 is grade I or imprinted
  • the malignant potential is a condition in which the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of no more than one imprinted gene in T1, T2, T3, T4, T5, and T6 are both in the second order.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both II.
  • the level of imprinted gene deletion and the amount of imprinted gene deletion in the class or imprinted genes T1, T2, T3, T4, T5 and T6 are both grade III, which is an early malignant tumor.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both III.
  • the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of no more than one imprinted gene in the graded or imprinted genes T1, T2, T3, T4, T5 and T6 are all grade IV, and are metaphase malignant tumors.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both IV Grade, it is an advanced malignant tumor.
  • each tumor to different imprinted genes is different, and each index of different tumors may have a top and bottom fluctuation of 20%.
  • the detection model and system described in the present application express the performance of the imprinted deletion on the sample of the tumor patient for the first time in an intuitive manner, and objectively, intuitively, earlyly and accurately detect the in situ labeling of the imprinted gene.
  • Imprinted (trace) gene changes and can provide quantitative models that make a significant contribution to the diagnosis of molecular pathology;
  • This application establishes a detection system that directly diagnoses the absence of imprinting from the patient's biopsy cells, and can determine the degree of benign and malignant tumors before surgery, thereby providing a basis for surgery and precision treatment.
  • This application can accurately determine the type of tumor, fill in the limitations of current tissue morphological diagnosis, and enable early accurate diagnosis to provide assistance for later targeted treatment;
  • This application is the first case to detect the expression of imprinted genes at a single cell and tissue level, and to qualitatively, quantitatively and spatially locate the expression of imprinted genes at the cellular level, indicating the loss of organ tissue imprinting and tumorigenesis. Relationship of stages;
  • the disease-related gene-imprinted deletion site found in the detection method of the present application may be used to guide the subsequent treatment and administration of the targeted drug or technical method for silencing, eliminating, and rearranging the gene.
  • Figure 1 is a diagram showing the relationship between imprinted gene deletion and cancer in an embodiment of the present invention
  • a is a hematoxylin-stained cell, and there is no label in the nucleus, and the imprinted gene is not expressed
  • b is a hematoxylin After staining, there is a red/brown mark in the nucleus, and the imprinted gene is present
  • the c is the hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the imprinted gene is deleted
  • the d is the hematoxylin staining of the cells. After that, there are more than two red/brown marks in the nucleus, and the imprinted gene copy number is abnormal;
  • Fig. 3 shows the expression of six genes in the pathological sections of different malignant degrees of thyroid cancer according to the embodiment of the present invention, wherein Fig. 3(a) shows the expression of six genes in the pathological section of grade 0 thyroid cancer, Fig. 3 (Fig. 3 b) the expression of six genes in the pathological section of grade I thyroid cancer, Figure 3 (c) shows the expression of six genes in the pathological section of grade II thyroid cancer, and Figure 3 (d) shows the grade III thyroid cancer. The expression of six genes in pathological sections, Figure 3 (e) shows the expression of six genes in the pathological sections of grade IV thyroid cancer;
  • Figure 4 is a diagram showing the application of six genes in the pathological section of 68 cases of thyroid cancer according to an embodiment of the present invention.
  • the distribution range and grading criteria of the loss and copy number anomalies wherein Fig. 4(a) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in the pathological section of 68 cases of thyroid cancer with imprinted gene T1, Fig. 4(b) The imprinted gene T2 was applied to the pathological sections of 68 cases of thyroid cancer, and the distribution range and grading standard of imprinting deletion and copy number abnormality.
  • Figure 4(c) shows that imprinted gene T3 was applied to 68 cases of thyroid cancer pathological sections with imprinted deletion and The distribution range and grading standard of copy number abnormality, Fig.
  • FIG. 4(d) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in the pathological section of 68 cases of thyroid cancer
  • the imprinting gene is shown in Fig. 4(e) T5 was applied to the pathological sections of 68 cases of thyroid cancer, the distribution range and grading standard of imprinting deletion and copy number abnormality.
  • Figure 4(f) was used to imprint the gene T6 in 68 cases of thyroid cancer pathological sections with imprinting deletion and copy number abnormality. Distribution range and grading standards.
  • the method for detecting the imprinted gene comprises the following steps:
  • tissue cell sections (10 ⁇ m) of thyroid cancer and placing them in a 10% neutral formalin solution to prevent RNA degradation for 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 T1 (Gnas), T2 (Igf2), T3 (Peg10), T4 (Igf2r), T5 (Mest) and T6 (Plagl1), specifically in the inner loop of each gene. Select a paragraph The sequence serves as a probe, and the specific selected gene sequences and specific gene positions are as follows:
  • T4 Hs-IGF2R: chr6: 160059099-160060546;
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-2.5% for grade II, Imprinted gene deletion expression level is 25-35% and/or imprinted gene copy number abnormal expression level is 2.5-3.5% as grade III, imprinting base The level of expression of the deletion is greater than 35% and/or the abnormal expression level of the imprinted gene copy number is greater than 3.5% as the IV level;
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2-3% for grade II, The amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 3-4% is grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 4%.
  • 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 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2-3% for grade II, The amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 3-4% is grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 4%.
  • 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 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2-3% for grade II, The amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 3-4% is grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 4%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 15-20% and/or imprinted gene copy number abnormal expression level is 1.5-2.5% for grade II, Imprinted gene deletion The expression level is 20-30% and/or the abnormal expression level of the imprinted gene copy number is 2.5-3.5% for the grade III, the imprinted gene deletion expression amount is greater than 30% and/or the imprinted gene copy number abnormal expression amount is greater than 3.5% for the grade IV. ;
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 15-20% and/or imprinted gene copy number abnormal expression level is 1.5-2.5% for grade II,
  • the imprinted gene deletion expression amount is 20-30% and/or the imprinted gene copy number abnormal expression amount is 2.5-3.5%, the imprinting gene deletion expression amount is more than 30%, and/or the imprinted gene copy number abnormal expression amount is more than 3.5%. It is level IV.
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, T4, T5 and T6 are both 0, and the imprinted genes T1, T2, T3
  • the imprinted gene deletion expression level of one of the imprinted genes in T4, T5, and T6 is not greater than the abnormal expression level of the imprinted gene copy number of one of the imprinted genes of class I and imprinted genes T1, T2, T3, T4, T5, and T6.
  • the imprinted gene deletion expression amount of each of the two imprinted genes of the class I or the imprinted genes T1, T2, T3, T4, T5 and T6 is not greater than the class I and the abnormal expression amount of the imprinted gene copy number of the two imprinted genes If it is any of the 0 grades, it is judged to be a benign tumor;
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount of the two imprinted genes in the imprinted genes T1, T2, T3, T4, T5, and T6 is I grade and the imprint of one of the two imprinted genes
  • the gene expression copy number is abnormal, and the imprinted gene deletion level of at least 3 imprinted genes of class I, imprinted genes T1, T2, T3, T4, T5 and T6 is grade I and imprinted genes T1, T2, T3, T4, T5
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of T6 and T6 is the imprinted gene deletion expression amount and the imprinted gene deletion of no more than one imprinted gene in class I or imprinted genes T1, T2, T3, T4, T5 and T6 If the expression level is any of the grade II, it is a malignant potential;
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both grade II or imprinted.
  • the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of no more than one imprinted gene in the genes T1, T2, T3, T4, T5 and T6 are all grade III, and are early malignant tumors;
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are all grade III or imprinted.
  • the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of no more than one imprinted gene in the genes T1, T2, T3, T4, T5 and T6 are all grade IV, and it is a metaphase malignant tumor;
  • the result of determining the degree of benign and malignant tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both IV, then It is an advanced malignant tumor.
  • the puncture biopsy cells use a positively charged adhesive slide, each smear contains more than 50 cells, push the puncture onto the slide, and pressurize with another slide, after 10% Neutral formalin was fixed for 1 h, placed in a 70% ethanol solution for 10 min, and air-dried at room temperature.
  • the other detection methods were the same as those in Example 1, and the results were similar to those in Example 1.
  • the detection model and system described in the present application express the performance of the imprinted defect on the sample of the tumor patient for the first time in an intuitive manner, and the method of in situ labeling of the imprinted gene is objective, intuitive, early, and accurate. Changes in the imprinted (trace) gene are detected and quantitative models can be provided to make a significant contribution to the diagnosis of molecular pathology.

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Abstract

本申请为基因诊断领域，具体提供了一种印记基因分级模型及其组成的系统和应用，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肿瘤中的变化进行分级。

Description

一种印记基因分级模型及其组成的系统和应用 技术领域
本申请涉及生物技术领域，涉及基因诊断领域，具体涉及一种印记基因分级模型及其组成的系统和应用。
背景技术
根据国际癌症数据统计，全球每年死于癌症的大约八百多万人。所以早期发现，确诊，治疗尤为重要。癌症的产生是由于在细胞层面上，随时间的推移而积累的复杂的基因变异和表观遗传改变，最终导致不受控制的细胞分裂。
传统病理学对细胞的良恶性诊断是基于细胞的大小，形态，侵润性和周边细胞组织的关系来作出判断的。它对细胞(癌症)的早期变化的发现有很大的局限性，因此细胞分子水平的癌症诊断方法，一度成为研究热点。随着人们在分子生物学领域的不断深入研究，越来越多的分子检测技术被运用到癌症诊断中。
目前，病变组织的良恶性和对癌症分型或癌症阶段的判断在很大程度上，都是根据被苏木素和伊红染液染色的病理组织切片上观察到的细胞形态来决定的。但是，这个方法有其固有的局限性。首先，该方法不能观察肿瘤发生时分子层面的改变，而分子的改变能够提供更为精准的预诊和诊断的依据；其次，细胞形态学诊断是主观判断，这本身会造成诊断的不准确。这种主观判断造成的误差对于早期癌症的诊断影响巨大，尤其是癌症早期诊断的敏感性和精确性对病人而言是性命攸关的。最后，部分恶性肿瘤细胞在形态学上与良性肿瘤细胞差异极小。
从遗传学的角度讲，肿瘤的发展是一个多基因变化的过程，表观遗传的修 饰对于肿瘤的发生、诊断和治疗具有重要意义，也逐渐在临床上也得到了应用。已有大量科学研究证实了印记状态基因重新表达与细胞癌变的相关性。
目前临床上对于印记基因的研究主要基于甲基化检测，其检测技术主要包括即限制性酶切法、富集法和亚硫酸盐转化法，然而目的基因不同区段甲基化位点对基因转录活性的影响存在很大的差别，即使存在一个准确的甲基化检测方法，也不能准确地对肿瘤进行诊断。
基因组印记是表观遗传学基因调控的一种方式，表现为对于特定基因，只有来自特定亲代的等位基因可以表达，而另外一个等位基因则表现为基因沉寂。大量的研究证明，印记(迹)缺失是指印记(迹)基因中原先处在沉寂状态的等位基因被激活(去甲基化)，是癌症中最常见和最早期就发生的表观遗传改变，并且这个特性可以用作病理标记。相对而言，在健康细胞检测中，印迹缺失比例很低。
基于上述原因，目前的临床疾病诊断需要新的检测系统和检测模型，基于患者活检样本，解析癌症在细胞层面上存在的分子标记物变化，以此提供更精确的预诊和诊断信息。
发明内容
针对现有技术的不足及实际的需求，本申请提供了一种印记基因分级模型及其组成的系统和应用，该检测系统和模型是用于单细胞和组织水平下早期直观地观察各类型肿瘤的印记(迹)基因的变化从而判断肿瘤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，本申请提供了一种印记基因分级模型，所述模型通过计算印迹基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量在肿瘤中的变 化对印记基因进行分级。
本申请中，所述印记基因缺失为将细胞进行苏木素染色后，细胞核内存在两个红色/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红色/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红色或棕色，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，所述印记基因为T1-T6，所述印记基因T1为Gnas，所述印记基因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1。
优选地，所述计算印记基因表达量、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；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/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，细胞核内存在两个红色/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 存在两个以上红色/棕色标记，印记基因拷贝数异常。
优选地，所述印记基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级为针对T1-T6的六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T1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T1的印记基因缺失表达量小于15％和/或所述印记基因T1的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T1的印记基因缺失表达量为15-20％和/或所述印记基因T1的印记基因拷贝数异常表达量为0.5-1.5％；
II级：所述印记基因T1的印记基因缺失表达量为20-25％和/或所述印记基因T1的印记基因拷贝数异常表达量为1.5-2.5％；
III级：所述印记基因T1的印记基因缺失表达量为25-35％和/或所述印记基因T1的印记基因拷贝数异常表达量为2.5-3.5％；
IV级：所述印记基因T1的印记基因缺失表达量大于35％和/或所述印记基因T1的印记基因拷贝数异常表达量大于3.5％。
优选地，所述针对T2、T3和T4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T2、T3和T4的印记基因缺失表达量小于15％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量小于1％；
I级：所述印记基因T2、T3和T4的印记基因缺失表达量为15-20％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为1-2％；
II级：所述印记基因T2、T3和T4的印记基因缺失表达量为20-25％和/或 所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为2-3％；
III级：所述印记基因T2、T3和T4的印记基因缺失表达量为25-35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为3-4％；
IV级：所述印记基因T2、T3和T4的印记基因缺失表达量大于35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量大于4％。
本申请中，所述印记基因T2、T3和T4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T5和T6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T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T5和T6的印记基因缺失表达量为10-15％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.5％；
II级：所述印记基因T5和T6的印记基因缺失表达量为15-20％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.5-2.5％；
III级：所述印记基因T5和T6的印记基因缺失表达量为20-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-3.5％；
IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于3.5％。
本申请中，所述印记基因T5和T6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情况；
其中，所述分析单元通过计算印记基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量，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，细胞核内存在两个红色/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红色/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红色或棕色，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单细胞和组织水平下早期直观地观察各类型肿瘤的印记(迹)基因的变化从而判断肿瘤的良恶性及恶性程度，为早期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、穿刺活检细胞压片或内窥镜筛查样本中的任意一种或至少两种的组合。
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。
所述穿刺活检细胞压片具体操作步骤为获取人体细胞，及时用10％中性福尔马林固定即可。
本申请中，由于穿刺取细胞对病人伤害小，取样过程简单，相较于血液的循环特性，穿刺细胞还能定位，穿刺细胞作为实验样本有其特殊的优势。
优选地，所述待测样本为活检穿刺细胞。
优选地，所述印记基因为T1-T6，所述印记基因T1为Gnas，所述印记基因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1。
本申请中，所述印记基因T1(Gnas)，T2(Igf2)，T3(Peg10)，T4(Igf2r)，T5(Mest)，T6(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T1-T6，即Gnas，Igf2，Peg10，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：
T1(Hs-GNAS)：chr6：143968979-143985134；
T2(Hs-IGF2)：chr11：2133666-2135366；
T3(Hs-PEG10)：chr7：94656592-94663333；
T4(Hs-IGF2R)：chr6：160059099-160060546；
T5(Hs-Mest)：chr7：130492340-130495367；
T6(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，优选为单通道红色/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；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/棕色标记，印记基因拷贝数异常。
本申请中，所述苏木素染色后的标记选自但不限于红色或棕色，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，通过计算印记基因表达量、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T1-T6的六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T1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T1的印记基因缺失表达量小于15％和/或所述印记基因T1的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T1的印记基因缺失表达量为15-20％和/或所述印记基因T1的印记基因拷贝数异常表达量为0.5-1.5％；
II级：所述印记基因T1的印记基因缺失表达量为20-25％和/或所述印记基因T1的印记基因拷贝数异常表达量为1.5-2.5％；
III级：所述印记基因T1的印记基因缺失表达量为25-35％和/或所述印记基因T1的印记基因拷贝数异常表达量为2.5-3.5％；
IV级：所述印记基因T1的印记基因缺失表达量大于35％和/或所述印记基因T1的印记基因拷贝数异常表达量大于3.5％。
优选地，所述针对T2、T3和T4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T2、T3和T4的印记基因缺失表达量小于15％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量小于1％；
I级：所述印记基因T2、T3和T4的印记基因缺失表达量为15-20％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为1-2％；
II级：所述印记基因T2、T3和T4的印记基因缺失表达量为20-25％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为2-3％；
III级：所述印记基因T2、T3和T4的印记基因缺失表达量为25-35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为3-4％；
IV级：所述印记基因T2、T3和T4的印记基因缺失表达量大于35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量大于4％。
优选地，所述针对T5和T6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T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T5和T6的印记基因缺失表达量为10-15％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.5％；
II级：所述印记基因T5和T6的印记基因缺失表达量为15-20％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.5-2.5％；
III级：所述印记基因T5和T6的印记基因缺失表达量为20-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-3.5％；
IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于3.5％。
优选地，所述判断肿瘤的良恶性程度分为良性、恶性潜能、早期恶性肿瘤、 中期恶性肿瘤和晚期恶性肿瘤。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量均为0级、印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因缺失表达量不大于I级和印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因拷贝数异常表达量均不大于I级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量不大于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，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6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，则判断为恶性潜能。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为II级或印记基因T1、T2、T3、T4、T5和T6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，则为早期恶性肿瘤。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为III级或印记基因T1、T2、T3、T4、T5和T6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，则为中期恶性肿瘤。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为IV级，则为晚期恶性肿瘤。
优选地，所述肿瘤为本领域常规的肿瘤都是可行的，本申请选自但不限于甲状腺肿瘤、肺肿瘤或脑肿瘤中的任意一种或至少两种的组合。
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用于制备肿瘤检测的药物。
优选地，所述肿瘤为本领域常规的肿瘤都是可行的，本申请选自但不限于甲状腺肿瘤、肺肿瘤或脑肿瘤中的任意一种或至少两种的组合。
优选地，所述肿瘤检测为判断肿瘤的类型，所述肿瘤类型包括良性、恶性潜能、早期恶性肿瘤、中期恶性肿瘤和晚期恶性肿瘤。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量均为0级、印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因缺失表达量不大于I级和印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因拷贝数异常表达量均不大于I级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量不大于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，则判断为良性肿瘤。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6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，则为恶性潜能。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为II级或印记基因T1、T2、T3、T4、T5和T6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，则为早期恶性肿瘤。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为III级或印记基因T1、T2、T3、T4、T5和T6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，则为中期恶性肿瘤。
优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为IV级，则为晚期恶性肿瘤。
本申请中，针对不同的肿瘤对各个印记基因的敏感性不同，不同肿瘤的各个指标会有上下20％的浮动。
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，首次以直观的方法表现了印记缺失在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，为分子病理学的诊断做出巨大贡献；
(2)本申请创立了一种直接从病人穿刺活检细胞中诊断印记缺失的检测方系统，可以在肿瘤病人手术前得出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肿瘤的革命性突破；
(3)本申请可以精确的判断肿瘤的类型，填补了目前组织细胞形态学诊断的局限性，能够做到早期的精确诊断，为后期的靶向性治疗提供帮助；
(4)本申请是首例可以在单个细胞和组织水平下检测印记基因表达情况，并可以对印记基因在细胞水平的表达进行定性、定量研究和空间定位，指出器官组织印记缺失与肿瘤的发生阶段的关系；
(5)本申请区别于免疫组化方法，减少了假阳性和其他负面作用；
(6)本申请检测方法中发现的疾病相关基因印记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是本发明实施例的印记基因缺失和癌症的关系图；
图2是本发明实施例的苏木素染色细胞核的甲状腺癌的病理切片，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，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/棕色标记，印记基因拷贝数异常；
图3本发明实施例的6个基因在甲状腺癌不同恶性程度的病理切片中的表达情况，其中，图3(a)为0级甲状腺癌的病理切片中6个基因的表达情况，图3(b)为I级甲状腺癌的病理切片中6个基因的表达情况，图3(c)为II级甲状腺癌的病理切片中6个基因的表达情况，图3(d)为III级甲状腺癌的病理切片中6个基因的表达情况，图3(e)为IV级甲状腺癌的病理切片中6个基因的表达情况；
图4是本发明实施例的6个基因应用于68例甲状腺癌病理切片中，印记缺 失和拷贝数异常的分布范围和分级标准，其中，图4(a)为印记基因T1应用于68例甲状腺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图4(b)为印记基因T2应用于68例甲状腺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图4(c)为印记基因T3应用于68例甲状腺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图4(d)为印记基因T4应用于68例甲状腺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图4(e)为印记基因T5应用于68例甲状腺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图4(f)为印记基因T6应用于68例甲状腺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准。
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 甲状腺癌的印记基因分析
所述的印记基因的检测方法，包括如下步骤：
(1)获取甲状腺癌的组织细胞切片(10微米)，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T1(Gnas)、T2(Igf2)、T3(Peg10)、T4(Igf2r)、T5(Mest)和T6(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：
T1(Hs-GNAS)：chr6：143968979-143985134；
T2(Hs-IGF2)：chr11：2133666-2135366；
T3(Hs-PEG10)：chr7：94656592-94663333；
T4(Hs-IGF2R)：chr6：160059099-160060546；
T5(Hs-Mest)：chr7：130492340-130495367；
T6(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情况，结果如图3-5所示。
从图3(a)-图3(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。
从图4(a)-图4(f)可以看出，68例甲状腺肿瘤组织样本中6个探针的印记缺失和拷贝数异常的比例呈现从低到高的分布，根据不同探针的分布趋势，我们计算得到了图中虚线所示的分级标准，可以将每个探针的印记缺失和拷贝数异常分别从低到高分成5个等级。
具体等级分型如下：
从图4(a)可以看出，对于所述印记基因T1，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0.5％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0.5-1.5％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1.5-2.5％为II级，印记基因缺失表达量为25-35％和/或印记基因拷贝数异常表达量为2.5-3.5％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3.5％为IV级；
从图4(b)可以看出，对于所述印记基因T2，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1％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1-2％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2-3％为II级，印记基因缺失表达量为25-35％和/或印记基因拷贝数异常表达量为3-4％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4％为IV级；
从图4(c)可以看出，对于所述印记基因T3，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1％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1-2％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2-3％为II级，印记基因缺失表达量为25-35％和/或印记基因拷贝数异常表达量为3-4％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4％为IV级；
从图4(d)可以看出，对于所述印记基因T4，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1％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1-2％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2-3％为II级，印记基因缺失表达量为25-35％和/或印记基因拷贝数异常表达量为3-4％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4％为IV级；
从图4(e)可以看出，对于所述印记基因T5，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-15％和/或印记基因拷贝数异常表达量为0.5-1.5％为I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1.5-2.5％为II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3.5％为IV级；
从图4(f)可以看出，对于所述印记基因T6，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-15％和/或印记基因拷贝数异常表达量为0.5-1.5％为I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1.5-2.5％为II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3.5％为IV级。
从这68个甲状腺肿瘤的样本综合分析可以得出：
所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量均为0级、印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因缺失表达量不大于I级和印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因拷贝数异常表达量均不大于I级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量不大于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，则判断为良性肿瘤；
所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6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，则为恶性潜能；
所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为II级或印记基因T1、T2、T3、T4、T5和T6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，则为早期恶性肿瘤；
所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为III级或印记基因T1、T2、T3、T4、T5和T6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，则为中期恶性肿瘤；
所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为IV级，则为晚期恶性肿瘤。
实施例2
所述穿刺活检细胞是，使用带有正电荷的粘附载玻片，每张涂片含有50个以上细胞，将穿刺物推到玻片上，用另一张玻片加压推送，经10％中性福尔马林固定1h，放入70％乙醇溶液10min，室温风干，其他检测方法同实施例1，结果类似实施例1。
综上所述，本申请所述检测模型和系统，首次以直观的方法表现了印记缺失在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，为分子病理学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 (13)

  1. 一种印记基因分级模型，其通过计算印记基因的表达量、印记基因缺失表达量和印记基因拷贝数异常表达量在肿瘤中的变化对印记基因进行分级。
  2. 根据权利要求1所述的模型，其中，所述印记基因为T1-T6，所述印记基因T1为Gnas，所述印记基因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1。
  3. 根据权利要求1或2所述的模型，其中，所述计算印记基因的表达量、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；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/棕色标记，印记基因拷贝数异常。
  4. 根据权利要求1-3任一项所述的模型，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为针对T1-T6的六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T1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T1的印记基因缺失表达量小于15％和/或所述印记基 因T1的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T1的印记基因缺失表达量为15-20％和/或所述印记基因T1的印记基因拷贝数异常表达量为0.5-1.5％；
    II级：所述印记基因T1的印记基因缺失表达量为20-25％和/或所述印记基因T1的印记基因拷贝数异常表达量为1.5-2.5％；
    III级：所述印记基因T1的印记基因缺失表达量为25-35％和/或所述印记基因T1的印记基因拷贝数异常表达量为2.5-3.5％；
    IV级：所述印记基因T1的印记基因缺失表达量大于35％和/或所述印记基因T1的印记基因拷贝数异常表达量大于3.5％；
    优选地，所述针对T2、T3和T4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T2、T3和T4的印记基因缺失表达量小于15％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量小于1％；
    I级：所述印记基因T2、T3和T4的印记基因缺失表达量为15-20％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为1-2％；
    II级：所述印记基因T2、T3和T4的印记基因缺失表达量为20-25％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为2-3％；
    III级：所述印记基因T2、T3和T4的印记基因缺失表达量为25-35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为3-4％；
    IV级：所述印记基因T2、T3和T4的印记基因缺失表达量大于35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量大于4％；
    优选地，所述针对T5和T6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T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T5和T6的印记基因缺失表达量为10-15％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.5％；
    II级：所述印记基因T5和T6的印记基因缺失表达量为15-20％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.5-2.5％；
    III级：所述印记基因T5和T6的印记基因缺失表达量为20-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-3.5％；
    IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于3.5％。
  5. 一种用于检测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情况；
    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1-4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肿瘤的良恶性程度。
  6. 根据权利要求5所述的系统，其中，步骤(1)所述的待测样本为活检穿刺细胞和/或内窥镜筛查样本。
  7. 根据权利要求5或6所述的系统，其中，所述印记基因为T1-T6，所述印记基因T1为Gnas，所述印记基因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1。
  8. 根据权利要求5-7任一项所述的系统，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，优选为单通道红色/棕色呈色试剂盒或多通道的荧光试剂盒。
  9. 根据权利要求5-8任一项所述的系统，其中，所述模型中的计算印记基因的表达量、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；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/棕色标记，印记基因拷贝数异常；
    优选地，所述印记基因的表达量、印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级；
    优选地，所述五个不同的等级为针对T1-T6的六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T1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T1的印记基因缺失表达量小于15％和/或所述印记基 因T1的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T1的印记基因缺失表达量为15-20％和/或所述印记基因T1的印记基因拷贝数异常表达量为0.5-1.5％；
    II级：所述印记基因T1的印记基因缺失表达量为20-25％和/或所述印记基因T1的印记基因拷贝数异常表达量为1.5-2.5％；
    III级：所述印记基因T1的印记基因缺失表达量为25-35％和/或所述印记基因T1的印记基因拷贝数异常表达量为2.5-3.5％；
    IV级：所述印记基因T1的印记基因缺失表达量大于35％和/或所述印记基因T1的印记基因拷贝数异常表达量大于3.5％；
    优选地，所述针对T2、T3和T4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T2、T3和T4的印记基因缺失表达量小于15％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量小于1％；
    I级：所述印记基因T2、T3和T4的印记基因缺失表达量为15-20％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为1-2％；
    II级：所述印记基因T2、T3和T4的印记基因缺失表达量为20-25％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为2-3％；
    III级：所述印记基因T2、T3和T4的印记基因缺失表达量为25-35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量为3-4％；
    IV级：所述印记基因T2、T3和T4的印记基因缺失表达量大于35％和/或所述印记基因T2、T3和T4的印记基因拷贝数异常表达量大于4％；
    优选地，所述针对T5和T6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T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T5和T6的印记基因缺失表达量为10-15％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.5％；
    II级：所述印记基因T5和T6的印记基因缺失表达量为15-20％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.5-2.5％；
    III级：所述印记基因T5和T6的印记基因缺失表达量为20-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-3.5％；
    IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于3.5％。
  10. 根据权利要求5-9中任一项所述的系统，其中，待判断的肿瘤的良恶性程度分为良性、恶性潜能、早期恶性肿瘤、中期恶性肿瘤和晚期恶性肿瘤；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量均为0级、印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因缺失表达量不大于I级和印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因拷贝数异常表达量均不大于I级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量不大于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，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、 T4、T5和T6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T1、T2、T3、T4、T5和T6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，则为恶性潜能；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为II级或印记基因T1、T2、T3、T4、T5和T6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，则为早期恶性肿瘤；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为III级或印记基因T1、T2、T3、T4、T5和T6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，则为中期恶性肿瘤；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为IV级，则为晚期恶性肿瘤。
  11. 一种如权利要求1-4中任一项所述的模型或如权利要求5-10中任一项所述的系统在制备肿瘤检测的药物中的用途。
  12. 根据权利要求11所述的用途，其中，所述肿瘤为甲状腺肿瘤、肺肿瘤或脑肿瘤中的任意一种或至少两种的组合。
  13. 根据权利要求12所述的用途，其中，所述肿瘤检测为判断肿瘤的良恶性程度，所述肿瘤的良恶性程度包括良性、恶性潜能、早期恶性肿瘤、中期恶性肿瘤和晚期恶性肿瘤；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量均为0级、印记 基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因缺失表达量不大于I级和印记基因T1、T2、T3、T4、T5和T6中的1个印记基因的印记基因拷贝数异常表达量均不大于I级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量不大于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，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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均为I级、印记基因T1、T2、T3、T4、T5和T6的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6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，则为恶性潜能；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为II级或印记基因T1、T2、T3、T4、T5和T6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，则为早期恶性肿瘤；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为III级或印记基因T1、T2、T3、T4、T5和T6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，则为中期恶性肿瘤；
    优选地，所述判断肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2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均为IV级，则为晚期恶性肿瘤。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024503A1 (en) * 1994-03-07 1995-09-14 Rapaport, Erich Sensitive cancer test
WO2016154330A1 (en) * 2015-03-23 2016-09-29 Whitehead Institute For Biomedical Research Reporter of genomic methylation and uses thereof

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6235474B1 (en) 1996-12-30 2001-05-22 The Johns Hopkins University Methods and kits for diagnosing and determination of the predisposition for diseases
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US7670775B2 (en) * 2006-02-15 2010-03-02 The Ohio State University Research Foundation Method for differentiating malignant from benign thyroid tissue
US9347088B2 (en) * 2008-06-27 2016-05-24 Institut Pasteur Molecular signature of liver tumor grade and use to evaluate prognosis and therapeutic regimen
US8586310B2 (en) * 2008-09-05 2013-11-19 Washington University Method for multiplexed nucleic acid patch polymerase chain reaction
BRPI1006075A2 (pt) * 2009-01-07 2016-04-19 Myriad Genetics Inc biomarcadores de cancer
EP2233590A1 (en) * 2009-01-28 2010-09-29 AIT Austrian Institute of Technology GmbH Methylation assay
WO2012011952A2 (en) 2010-07-20 2012-01-26 Board Of Trustees Of The University Of Arkansas Copy number variant-dependent genes as diagnostic tools, predictive biomarkers and therapeutic targets
WO2012031008A2 (en) * 2010-08-31 2012-03-08 The General Hospital Corporation Cancer-related biological materials in microvesicles
AU2011305696A1 (en) * 2010-09-20 2013-05-02 Advanced Cell Diagnostics, Inc. Biomarkers for differentiating melanoma from benign nevus in the skin
JP6247934B2 (ja) * 2011-02-04 2017-12-13 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 単一細胞において染色体構造および遺伝子発現を同時に検出するための方法
CN103502473B (zh) * 2011-03-01 2016-03-09 耶鲁大学 胃肠胰神经内分泌肿瘤(gep-nen)的预测

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024503A1 (en) * 1994-03-07 1995-09-14 Rapaport, Erich Sensitive cancer test
WO2016154330A1 (en) * 2015-03-23 2016-09-29 Whitehead Institute For Biomedical Research Reporter of genomic methylation and uses thereof

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