WO2020024911A1 - 一种用于检测甲状腺肿瘤良恶性程度的分级模型及其应用 - Google Patents

一种用于检测甲状腺肿瘤良恶性程度的分级模型及其应用 Download PDF

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WO2020024911A1
WO2020024911A1 PCT/CN2019/098209 CN2019098209W WO2020024911A1 WO 2020024911 A1 WO2020024911 A1 WO 2020024911A1 CN 2019098209 W CN2019098209 W CN 2019098209W WO 2020024911 A1 WO2020024911 A1 WO 2020024911A1
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imprinted
gene
genes
expression
copy number
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成彤
周宁
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立森印迹诊断技术有限公司
李星
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Priority to EP19843453.2A priority Critical patent/EP3831961A4/en
Priority to CA3111713A priority patent/CA3111713A1/en
Priority to JP2021529506A priority patent/JP2021531833A/ja
Priority to US17/264,520 priority patent/US20210292852A1/en
Priority to AU2019312636A priority patent/AU2019312636A1/en
Publication of WO2020024911A1 publication Critical patent/WO2020024911A1/zh

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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/10—Ploidy or copy number detection

Definitions

  • the present disclosure relates to the field of biotechnology, such as the field of genetic diagnosis, such as a hierarchical model and its applications, such as a hierarchical model for detecting the benign and malignant degree of thyroid tumors, and its applications, such as a set of imprinted genes in detecting benign and malignant thyroid tumors.
  • a hierarchical model of degree and its constituent devices such as the field of genetic diagnosis, such as a hierarchical model and its applications, such as a hierarchical model for detecting the benign and malignant degree of thyroid tumors, and its applications, such as a set of imprinted genes in detecting benign and malignant thyroid tumors.
  • thyroid cancer According to World Health Organization statistics, in 2012, there were 298,100 new cases of thyroid cancer worldwide, approximately 90,000 newly diagnosed cases in China, and approximately 25,520 newly diagnosed cases in the United States. Most thyroid cancers are highly differentiated and have excellent prognosis. The 5-year survival rate is higher than 95%, but some thyroid cancers metastasize at a very early stage. In addition, less differentiated thyroid and thyroid cancer can be fatal, with a median survival time ranging from 4 months to 5 years. Difficulties in pathological diagnosis of thyroid cancer include the differentiation between benign follicular thyroid tumor (FTA) and malignant follicular thyroid cancer (FTC), and the benign and malignant judgment of thyroid eosinophilic tumor (HCT).
  • FFA benign follicular thyroid tumor
  • FTC malignant follicular thyroid cancer
  • HCT thyroid eosinophilic tumor
  • Some BRAF gene tests can only be used to detect papillary thyroid carcinoma, and have no effect on the diagnosis of FTC and HCT.
  • the incidence of thyroid cancer in China has increased by 4.6 times, and it is still growing.
  • the follicular thyroid gland is difficult to diagnose.
  • Cancer and eosinophilic thyroid tumors account for about 10%, plus 3-4 times the number of suspected cases, it is estimated that about 40,000 to 50,000 cases need to be diagnosed each year, plus the number of patients worldwide, this number will increase to 10 More than 10,000, so it is urgent to develop a detection technology with higher accuracy.
  • Cancer is caused by uncontrolled cell growth / division resulting from epigenetic changes and genetic mutations that accumulate over time.
  • Traditional pathological diagnosis makes benign and malignant thyroid tumors based on cell, tissue size, morphological, and structural variations.
  • molecular detection technologies With the development and deepening of molecular biology, more and more molecular detection technologies are being applied to the detection of thyroid cancer. From the analysis of the development of cancer, the changes at the molecular level (epigenetics and genetics) predate the changes in cell morphology and tissue structure. So molecular biology tests are more sensitive to early detection of cancer.
  • the current diagnosis of thyroid cancer requires a new detection system and detection model. Based on patient biopsy samples, analysis of changes in molecular markers at the cell level of thyroid cancer is provided to provide more accurate pre-diagnosis and diagnostic information.
  • the present disclosure provides an imprinted gene grading model and diagnostic method for detecting the benign and malignant degree of thyroid tumors, and applications thereof.
  • the present disclosure provides a imprinted gene grading model for thyroid tumors.
  • the model measures imprinted genes by calculating the total imprinted gene expression, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount in thyroid cancer. Classification of expression status;
  • the imprint gene is any one or a combination of at least two of Z1, Z11 or Z16, the imprint gene Z1 is Gnas, the imprint gene Z11 is Grb10, and the imprint gene Z16 is Snrpn / Snurf.
  • the absence of the imprint (trace) means that the allele that was originally in the silent state in the imprint (trace) gene is activated (demethylated), which is the most common and earliest epigenetic change in cancer, and This feature can be used as a pathological marker. Relatively speaking, in the detection of healthy cells, the proportion of imprinting deletion is very low.
  • the imprinted gene and imprinted gene have the same concept at the same time, which means the same meaning and can be replaced.
  • the inventors have found that by calculating the amount of imprinted gene deletion expression and the amount of imprinted gene copy number abnormal expression of any of the imprinted genes in Z1, Z11, and Z16 in thyroid tumors, the diagnostic sensitivity to thyroid cancer can reach more than 64.3%.
  • any one of the imprinted genes in Z1, Z11, and Z16 can be detected.
  • any one of the imprinted genes in Z1 or Z11 can be detected.
  • Z1 can be detected if only one imprinted gene is detected in the preliminary detection.
  • the method for calculating the imprint gene by the model is: if a combination of two imprint genes of the imprint gene is detected, the combination may be any two of Z1, Z11, and Z16, and preferably Z1 and Z11 The combination of Z1 and Z16.
  • the combination of the imprinted genes can reach a diagnosis sensitivity of more than 84.6% for thyroid cancer.
  • the diagnosis sensitivity for thyroid cancer can reach 92.9%.
  • the combination of Z1 and Z11 is detected, the sensitivity for thyroid cancer can be reached.
  • the diagnostic sensitivity can reach more than 97.5%.
  • the imprint gene further includes any one or a combination of at least two of Z3, Z4, Z5, Z6, Z8, Z10 or Z13; wherein the imprint gene Z3 is Peg10, and the imprint The gene Z4 is Igf2r, the imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z8 is Dcn, the imprinted gene Z10 is Gatm, and the imprinted gene Z13 is Sgce.
  • the method for calculating the imprint gene by the model is: calculating the imprint gene combination, and calculating the Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 gene combinations.
  • the deletion of the imprinted gene is the presence of two red / brown markers in the nucleus of the cell after hematoxylin staining
  • the abnormal copy number of the imprinted gene is the presence of more than two red / brown markers in the nucleus of the cell after hematoxylin staining.
  • the abnormal copy number is due to the abnormal gene replication of cancer cells, which results in the expression of this gene as a triploid or even higher polyploid.
  • the formula for calculating the expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression amount of the imprinted gene copy number are as follows:
  • a means that there is no marker in the nucleus of the cell after hematoxylin staining, and that the imprinted gene has no expression; and b means that there is a red / brown marker in the nucleus of the cell where the imprinted gene exists after hematoxylin staining.
  • Cell nucleus; c is hematoxylin staining of the cell, there are two red / brown markers in the nucleus of the cell, and the nucleus of the gene deletion is imprinted;
  • d is hematoxylin staining of the cell, there are more than two red / brown markers in the nucleus of the cell , Imprinted nuclei with abnormal gene copy number.
  • the marker after hematoxylin staining is selected from, but not limited to, red or brown, and staining markers with other colors can also be used to imprint gene expression amount, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount. Calculation.
  • the probe is amplified by in situ hybridization and Hemotoxy (hematoxylin) cell nuclear staining to amplify the signal.
  • Hemotoxy hematoxylin
  • the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression level of the imprinted gene copy number are divided into five different levels. At least 1200 are expressed by each probe in the most positive region of the sample. The number of cells was counted, and the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total imprinted gene expression amount of ten imprinted genes for Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 There are five different levels of division.
  • five different levels of the imprinted gene deletion expression, imprinted gene copy number abnormal expression, and total expression for Z1 and Z16 are:
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is less than 12%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is less than 1% or the total expression amount of the imprinted genes Z1 and Z16 Less than 25% of any one or a combination of at least two;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is 12-20%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is 1-2% or the imprinted genes Z1 and Z16 The total expression of any one or a combination of at least two of 25-35%;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is 2-3% or the imprinted genes Z1 and Z16 The total expression of any one or a combination of at least two of 35-45%;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is 3-5% or the imprinted genes Z1 and Z16 The total expression of any one or a combination of at least two of 45-60%;
  • the imprinted gene deletion expression level of the imprinted genes Z1 and Z16 is greater than 30%, the imprinted gene copy number abnormal expression level of the imprinted genes Z1 and Z16 is greater than 5%, or the total expression level of the imprinted genes Z1 and Z16 Greater than 60% of any one or a combination of at least two;
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount of the imprinted genes Z1 and Z16 are independent of each other.
  • the five different grades of Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 are divided into five different levels: the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount. for:
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have less than 10% expression of the imprinted genes;
  • the abnormal expression level of the imprinted gene copy number of Z13 is less than 0.5% or the total expression amount of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is less than 15% or a combination of at least two ;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression of 10-15%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 imprinted gene copy number abnormal expression amount is 0.5-1% or the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 total expression amount is any one of 15-20% Or a combination of at least two;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression of 15-20%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10,
  • the imprinted gene copy number abnormal expression amount of Z11 and Z13 is 1-2% or the total expression amount of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is 20-30% Or a combination of at least two;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression of 20-25%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 imprinted gene copy number abnormal expression amount is 2-3% or the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 total expression amount is any one of 30-40% Or a combination of at least two;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression level greater than 25%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Abnormal expression of imprinted gene copy number of Z13 is greater than 3% or the total expression of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is greater than 40% or a combination of at least two ;
  • the imprinted genes of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 are independently expressed, and the imprinted gene copy number abnormal expression amount and total expression amount are independent of each other.
  • the present disclosure provides a device for detecting the benign and malignant degree of a thyroid tumor, which adopts the above model and includes the following units:
  • sampling unit obtain the sample to be tested
  • Probe design unit design specific primers based on the imprinted gene sequence
  • Detection unit performing in situ hybridization between the probe of step (2) and the sample to be tested;
  • the analysis unit calculates the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression amount of the imprinted gene copy, and passes the model described in the first aspect, thereby determining the abnormal expression amount of the imprinted gene and the imprinted copy number of the imprinted gene.
  • the level of expression to determine the benign and malignant degree of thyroid tumors.
  • the deletion of the imprinted gene refers to the presence of two red / brown labeled nuclei in the nucleus of the cell after hematoxylin staining, and the abnormal copy number of the imprinted gene indicates the presence of more than two red / brown labeled nuclei in the cell nucleus after the cell is subjected to hematoxylin staining.
  • the abnormal copy number is due to abnormal gene replication by cancer cells, which results in the expression of this gene as a triploid or even higher polyploid.
  • the marker after hematoxylin staining is selected from, but not limited to, red or brown. Staining markers with other colors can also be used for calculation of the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression amount of the imprinted gene copy number.
  • the detection device is used to intuitively observe the changes of the imprinted (trace) genes of thyroid tumors at an early level at the level of cells and tissues to judge the degree of benign and malignant tumors, and provide the most favorable treatment opportunities for patients with early thyroid tumors.
  • the present disclosure provides a method for detecting the benign and malignant degree of a thyroid tumor, using the model or the device, including the following steps:
  • the analysis unit calculates the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount through the model, so as to determine the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount by the model. Diagnosis of benign and malignant thyroid tumors.
  • the test sample described in step (1) is from human tissues and / or cells.
  • the sample to be tested is feasible as long as the RNA is processed in a timely manner. Those skilled in the art can select according to the needs, which is not particularly limited here.
  • the sample to be tested includes paraffin sections of tissues, thyroid puncture cells Any one or a combination of at least two of the smears.
  • the specific operation steps of the paraffin section of the tissue are to obtain a human tumor tissue sample, which is fixed in 10% neutral formalin in time, embedded in paraffin, cut into a thickness of 10 ⁇ m, and a tissue piece is made from a positively charged glass slide; because Only 10 ⁇ m thick, so part of the nucleus seen under the microscope, so some false negative gene deletions will occur.
  • the specific operation steps of the thyroid puncture cell smear are to obtain human cells through puncture, and it can be fixed in time with 10% neutral formalin.
  • the biopsy is less harmful to the patient and the sampling process is simple, compared with the circulation characteristics of the blood, the biopsy can also be positioned.
  • the biopsy has its special advantages as an experimental sample.
  • test sample is a thyroid puncture cell smear.
  • the imprint genes are Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the imprint gene Z1 is Gnas
  • the imprint gene Z3 is Peg10
  • the imprint gene Z4 is Igf2r
  • the imprint gene Z5 is Mest
  • the imprint gene Z6 is Plagl1
  • the imprint gene Z8 is Dcn
  • the imprint gene Z10 is Gatm
  • the imprint gene Z11 is Grb10
  • the imprint gene Z13 is Sgce
  • the imprinted gene Z16 is Snrpn / Snurf.
  • the imprinted genes Z1 (Gnas), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce), Z16 (Snrpn / Snurf) is expressed to varying degrees in normal tumor cell tissues. When malignant lesions occur, the expression level and imprinting status will change significantly.
  • the design probe is based on the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16, that is, Gnas, Peg10, Igf2r, Mest, Plagl1, Dcn, Gatm, Grb10, Sgce, and Snrpn / Designed by Snurf, a specific sequence was selected as a probe in the inner rotor of each gene, and the specific probe was designed by Advanced Cell Diagnostics.
  • the in situ hybridization adopts the RNAscope in situ hybridization method.
  • the RNAscope in situ hybridization method uses a single-channel or multi-channel color rendering kit or a single-channel or multi-channel fluorescence kit, preferably a single-channel red / brown coloring kit or a multi-channel Fluorescent kit.
  • the multi-channel color rendering kit or multi-channel fluorescence kit includes two channels or more than two channels of color rendering kits or fluorescence kits.
  • the two-channel color rendering kit or multi-channel fluorescence kit can use two A single imprinted gene probe or the combined expression of imprinted genes and other genes or even the combined expression of multiple imprinted and non-imprinted genes.
  • the formula for calculating the expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression level of the imprinted gene copy number in the model is as follows:
  • a means that there is no marker in the nucleus of the cell after hematoxylin staining, and that the imprinted gene has no expression; and b means that there is a red / brown marker in the nucleus of the cell where the imprinted gene exists after hematoxylin staining.
  • Cell nucleus; c is hematoxylin staining of the cell, there are two red / brown marks in the nucleus of the cell, and the nucleus of the gene deletion is imprinted;
  • d is hematoxylin staining of the cell, there are more than two red / brown marks in the nucleus of the cell Imprinted nuclei with abnormal gene copy number.
  • the marker after hematoxylin staining is selected from, but not limited to, red or brown. Staining markers with other colors can also be used to calculate the expression amount of imprinted genes, the expression expression of imprinted genes, and the expression expression of imprinted gene copy numbers.
  • the probe was amplified by in situ hybridization and Hemotoxy (hematoxylin) nucleus staining to amplify the signal. Under a 40 ⁇ or 60 ⁇ microscope, the presence of imprinted genes in each nucleus, the deletion of imprinted genes, or abnormal copy number were determined. The total gene expression level, the expression level of the imprinted gene, and the expression level of the imprinted gene copy number were abnormal to determine the degree of benign and malignant tumors in the sample. Because 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 some false negatives.
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount are divided into five different levels.
  • the five different grades count at least 1,200 cells in the region where the most positive expression of each probe of the sample is counted, and ten for Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount were separately divided.
  • the five different levels of the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount for Z1 and Z16 are:
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is less than 12%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is less than 1% or the total expression amount of the imprinted genes Z1 and Z16 Less than 25% of any one or a combination of at least two;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is 12-20%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is 1-2% or the imprinted genes Z1 and Z16 The total expression of any one or a combination of at least two of 25-35%;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is 2-3% or the imprinted genes Z1 and Z16 The total expression of any one or a combination of at least two of 35-45%;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is 3-5% or the imprinted genes Z1 and Z16 The total expression of any one or a combination of at least two of 45-60%;
  • the imprinted gene deletion expression level of the imprinted genes Z1 and Z16 is greater than 30%, the imprinted gene copy number abnormal expression level of the imprinted genes Z1 and Z16 is greater than 5%, or the total expression level of the imprinted genes Z1 and Z16 Greater than 60% of any one or a combination of at least two;
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount of the imprinted genes Z1 and Z16 are independent of each other.
  • the five different grades for the Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 imprinted gene deletion expression levels, imprinted gene copy number abnormal expression levels, and total expression levels are:
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have less than 10% expression of the imprinted genes, the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and The abnormal expression level of the imprinted gene copy number of Z13 is less than 0.5% or the total expression amount of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is less than 15% or a combination of at least two ;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression of 10-15%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 imprinted gene copy number abnormal expression amount is 0.5-1% or the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 total expression amount is any one of 15-20% Or a combination of at least two;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression of 15-20%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10,
  • the imprinted gene copy number abnormal expression amount of Z11 and Z13 is 1-2% or the total expression amount of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is 20-30% Or a combination of at least two;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression of 20-25%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 imprinted gene copy number abnormal expression amount is 2-3% or the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 total expression amount is any one of 30-40% Or a combination of at least two;
  • the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 have an expression level greater than 25%, and the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Abnormal expression of imprinted gene copy number of Z13 is greater than 3% or the total expression of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is greater than 40% or a combination of at least two ;
  • the imprinted genes of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, Z11, and Z13 are independently expressed, and the imprinted gene copy number abnormal expression amount and total expression amount are independent of each other.
  • the judging degree of benign and malignant thyroid tumors is divided into benign tumors, thyroid cancer potential, early thyroid cancer, intermediate thyroid cancer, and advanced thyroid cancer.
  • the result of judging the benign and malignant degree of thyroid tumor is the imprinted gene deletion expression and imprinted gene copy of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16.
  • the number of abnormal expression levels is less than level I or the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed the level of the imprinted gene.
  • Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have an abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene, which is a benign tumor;
  • the result of judging the benign and malignant degree of the thyroid tumor is that the expression level of the imprinted gene deletion of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade I, Imprinted gene Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class I or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, the expression level of the imprinted gene is level II and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any one of the class II, then the potential of the thyroid cancer is judged;
  • the result of judging the benign and malignant degree of the thyroid tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II,
  • the imprinted gene copy number of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is at least two or the imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes
  • the expression level of the imprinted gene is class III and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any of the class III, which is early thyroid cancer;
  • the result of judging the benign and malignant degree of the thyroid tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade III,
  • the imprinted gene copy number of at least two imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is the level III or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes
  • the expression level of the imprinted gene is level IV and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any one of grade IV, and it is intermediate stage thyroid cancer;
  • the result of judging the benign and malignant degree of the thyroid tumor is that at least two of the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have an expression level of deletion IV or imprinted
  • the abnormal expression level of the copy number of the imprinted gene of at least two imprinted genes among the genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV, which is advanced thyroid cancer.
  • the present disclosure provides a model or a device for detecting thyroid tumors.
  • the present disclosure provides the use of the model or the device for preparing a medicament or a device for treating thyroid cancer.
  • the degree of benign and malignant thyroid tumors is determined into benign tumors, thyroid cancer potential, early thyroid cancer, intermediate thyroid cancer, and advanced thyroid cancer.
  • the benign and malignant results of thyroid tumors were that the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 had less than the expression level of imprinted genes and the abnormal expression of imprinted gene copy numbers were less than grade I or imprinted.
  • the expression level of the imprinted gene deletion is not more than 1
  • the imprinted genes Z1, Z3, Z4, Z5, Z6, In Z8, Z10, Z11, Z13, and Z16 the abnormal expression level of the copy number of the imprinted gene of no more than one imprinted gene is Grade I, which is a benign tumor;
  • the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have a level I expression of imprinted genes
  • the imprinted gene Z1 , Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class I or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have no more than 1 imprinted gene with an expression level of imprinted gene deletion and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed 1
  • the imprinted gene copy number abnormal expression level of any imprinted gene is any one of grade II, it is judged as the potential of thyroid cancer;
  • the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have a level of expression of imprinted genes of class II and the imprinted gene Z1 , Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least 2 imprinted gene copy number abnormal expression levels of class II or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have no more than one imprinted gene with an expression level of imprinted gene class III and imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed 1
  • the abnormal expression of the imprinted gene copy number of each imprinted gene is any of grade III, which is early thyroid cancer;
  • the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have a level of expression of imprinted genes of class III
  • the imprinted gene Z1 , Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class III or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have no more than 1 imprinted gene with an expression level of deletion gene imprinted and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed 1
  • the abnormal expression level of the imprinted copy number of each imprinted gene is any one of grade IV, which is intermediate stage thyroid cancer;
  • At least two of the imprinted genes in the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 are expressed as a class IV or imprinted gene Z1.
  • Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of grade IV, which is advanced thyroid cancer.
  • the performance of imprinted genes on thyroid tumor patient samples is intuitively expressed, and the method of in situ labeling of imprinted genes is objective and intuitive.
  • changes in imprinted (trace) genes were accurately detected, and quantitative models could be provided to make a great contribution to the diagnosis of molecular pathology.
  • FIG. 1 is a pathological section of thyroid cancer with hematoxylin-stained nuclei according to the present invention, wherein a is that there is no marker in the nucleus and no imprinted gene is expressed after hematoxylin-stained cells; and b is that hematoxylin-stained cells.
  • a is that there is no marker in the nucleus and no imprinted gene is expressed after hematoxylin-stained cells
  • b is that hematoxylin-stained cells.
  • the c is two red / brown markers in the nucleus, and the imprinted gene is missing
  • the d is the nucleus of the cell after hematoxylin staining.
  • Figure 2 (a) is the expression status of 10 genes in the pathological section of grade 0 thyroid tumor
  • Figure 2 (b) is the expression status of 10 genes in the pathological section of grade I thyroid cancer
  • Figure 2 (c) is grade II
  • Figure 2 (d) is the expression status of 10 genes in the pathological section of class III thyroid cancer
  • Figure 2 (e) is the expression of 10 genes in the pathological section of class IV thyroid cancer.
  • Figure 3 (a) is the intensity of imprinted genes Z1, Z11, and Z16 on thyroid cancer
  • Figure 3 (b) is the intensity of imprinted genes Z1, Z11, and Z16 on thyroid cancer copy number abnormalities
  • Figure 3 (c) To indicate the intensity of the total expression of imprinted genes Z1, Z11, and Z16 on thyroid cancer
  • Figure 3 (d) is the intensity of imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, and Z13 on imprinted thyroid cancer
  • Fig. 3 (e) is the intensity of copy number abnormalities of imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, and Z13 on thyroid cancer
  • Figure 3 (f) is the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, and Z13.
  • the intensity of the total expression of thyroid cancer where LOI is the expression level of the imprinted gene deletion, CNV is the expression level of the imprinted gene copy number abnormal, and TE is the total expression level of the imprinted gene;
  • Figure 4 (a) is the intensity of the imprinted gene Z1 imprint, abnormal copy number, and the total expression level.
  • Figure 4 (b) is the intensity of the imprinted gene Z11 imprint, abnormal copy number, and the total expression level.
  • Figure 4 (c) is The imprinted gene Z16 imprint deletion, copy number abnormality, and the intensity of total expression.
  • Figure 4 (d) is the imprinted gene Z3 imprint deletion, copy number abnormality, and the total expression intensity.
  • Figure 4 (e) is the imprinted gene Z4 imprint deletion, Copy number abnormality and the intensity of total expression.
  • Figure 4 (f) is the intensity of the imprinted gene Z5 imprint, the copy number is abnormal and the intensity of the total expression
  • Figure 4 (g) is the imprinted gene Z6 imprint, the copy number is abnormal and the total expression
  • Figure 4 (h) shows the intensity of the imprinted gene Z8 signature deletion, copy number abnormality, and the total expression level
  • Figure 4 (i) shows the intensity of the imprinted gene Z10 signature deletion, copy number abnormality, and the total expression level
  • Figure 4 (j) is the intensity of the imprinted gene Z13 imprint deletion, copy number abnormality, and the total expression level
  • LOI is the imprinted gene deletion gene expression level
  • CNV is the imprinted gene copy number abnormal gene expression level
  • TE is the imprinted gene total expression level.
  • Figure 5 (a) shows the distribution range and grading standard of imprinted gene Z1 in 41 cases of thyroid cancer pathological sections.
  • Figure 5 (b) shows the imprinted gene Z11 in 41 cases of thyroid cancer pathological sections. The distribution range and grading standard of imprint deletion and copy number abnormality.
  • Figure 5 (c) is the distribution range and grading standard of imprint gene Z16 applied to 41 thyroid cancer pathological sections.
  • Figure 5 (d) ) Is the distribution range and grading standard of the imprinted gene Z3 in 41 cases of thyroid cancer pathological sections.
  • Figure 5 (e) is the imprinted gene Z4 in 41 cases of thyroid cancer pathological sections. Distribution range and grading standard of copy number abnormalities.
  • Figure 5 (f) is the distribution range and grading standard of imprinted gene Z5 applied to 41 cases of thyroid cancer pathological sections.
  • Figure 5 (g) is the imprinted gene.
  • Z6 was applied to 41 cases of thyroid cancer pathological sections, and the distribution range and grading standard of imprint deletion and copy number abnormalities were shown in Fig. 5 (h).
  • the imprinted gene Z8 was used in 41 cases of thyroid cancer pathological sections, and the imprint was missing. Distribution range and grading standard of copy number abnormalities.
  • Figure 5 (i) is the distribution range and grading standard of imprinted gene Z10 applied to 41 cases of thyroid cancer pathological sections.
  • Figure 5 (j) is the imprinted gene.
  • Z13 was used in 41 cases of thyroid cancer pathological sections.
  • the distribution range and grading standard of imprint deletion and copy number abnormality where LOI is the gene expression of imprint gene deletion, CNV is the gene expression of imprint gene copy number abnormal, and TE is imprint Total gene expression.
  • Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by methylating alleles from a particular parent so that only one allele is expressed in one gene and the other is in a state of gene silencing. This type of gene is called an imprinted gene. Blot deletion is an epigenetic change in which imprinted gene demethylation causes silencing of alleles to be activated and gene expression to begin. A large number of studies have shown that this phenomenon (missing imprinting) is common in various types of cancer and occurs earlier than changes in cell and tissue morphology. At the same time, in healthy cells, the proportion of missing blots is extremely low, in stark contrast to cancer cells. Therefore, the methylation status of imprinted genes can be used as a pathological marker to analyze the abnormal state of cells through specific molecular detection techniques.
  • the detection model and device described in the present disclosure express the performance of the lack of imprints on the samples of patients with thyroid tumors in an intuitive way.
  • the imprints traces can be detected objectively, intuitively, and early.
  • Gene changes and can provide a quantified model, making a huge contribution to the diagnosis of thyroid tumors;
  • the detection device of the present disclosure can determine the benign and malignant degree of thyroid tumors by puncture biopsy before surgery for patients with thyroid tumors, thereby providing a basis for surgery and accurate treatment, which is a revolutionary breakthrough in the diagnosis of thyroid tumors in the field of cell molecules;
  • the present disclosure can accurately determine the type of thyroid tumors, and clearly classify the malignant degree of thyroid tumors through the combined detection of imprinted genes, which greatly improves the early and clear diagnosis of thyroid cancer, especially for early screening and cancer surgery.
  • Follow-up follow-up especially for follow-up follow-up of patients with suspected relapse, can gain time and make a significant contribution to saving patients' lives;
  • the present disclosure can accurately distinguish benign and malignant thyroid eosinophilic tumors (HCT) from the molecular level, and provides a solution to the current problem that it is difficult to distinguish benign and malignant thyroid eosinophilic tumors with histological morphology.
  • the detection method of the present disclosure is different from the immunohistochemical method, and reduces false positives and other negative effects. Not only that, the target drug or technology that causes the gene to be silenced, eliminated, or rearranged through the missing site of the thyroid tumor-related imprinted gene is found. Methods can be used to guide the later treatment and medication.
  • the method for detecting an imprinted gene includes the following steps:
  • Design probe design specific primers based on the imprinted gene sequence
  • the design probe is based on the imprinted genes Z1 (Gnas), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce) and Z16 (Snrpn / Snurf) were designed, and a specific sequence was selected as a probe in the inner rotator of each gene.
  • the specific probe was designed by Advanced Cell Diagnostics.
  • the formula for calculating the expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression level of the imprinted gene copy number in the model are as follows:
  • FIG. 1 a, b, c, and d are shown in FIG. 1, where a is a cell nucleus after hematoxylin staining, there is no marker in the nucleus, and imprinted genes are not expressed; and b is a cell after hematoxylin staining, There is a red / brown mark in the nucleus, and the nucleus of the gene is imprinted; c is the red / brown mark in the nucleus of the cell, and there are two red / brown markers in the nucleus of the cell, and the d is hematoxylin After staining, there were more than two red / brown markers in the nucleus, marking the nucleus with abnormal gene copy number.
  • the thyroid puncture biopsy sample was obtained by puncturing the suspected diseased tissue, and the 10% neutral formalin solution was fixed for more than 24 hours.
  • the other detection methods were the same as in Example 1.
  • Figs. 4 (a)-4 (j) The specific sensitivity of each imprinted gene to thyroid cancer is shown in Figs. 4 (a)-4 (j). It can be seen from Figs. 4 (a)-4 (c) that the imprinted gene Z1 has an imprinted deletion and increased expression. It starts to appear in the malignant potential stage, and it rapidly rises to very high levels in early thyroid cancer, and continues to be maintained in advanced thyroid cancer. The copy number abnormality of the imprinted gene Z1 rises rapidly to very high levels in the malignant potential stage.
  • the imprint of the imprinted gene Z11 has a low level of imprinting, abnormal copy number, and increased expression in the malignant potential stage, and it slowly rises in the early to middle stage of thyroid cancer, and rapidly rises to very high in the stage of advanced thyroid cancer. High levels; the imprinting gene Z16 has imprinted deletions, abnormal copy numbers, and increased expression levels that rapidly rise from the malignant potential stage to the intermediate stage of thyroid cancer, and maintain high levels to the advanced stage of thyroid cancer;
  • the increase in the expression level of the imprinted gene Z4 began to appear in the stage of malignant potential, and did not increase significantly in early stage thyroid cancer. Moderately accelerated rise, but reached a higher sensitivity in the advanced stage of thyroid cancer; the imprint of the imprinted gene Z5 and copy number abnormalities began to appear in the intermediate stage of thyroid cancer, and the level of the imprinted gene Z5 was still not high, and the expression level of the imprinted gene Z5
  • the increase begins in the early stages of thyroid cancer and gradually progresses during the development of thyroid cancer
  • the level of the imprinted gene Z6 is still not high; the imprint of the imprinted gene Z6 appears in intermediate thyroid cancer, which has increased to a certain extent in advanced thyroid cancer, but the level is not high, and the copy number of the imprinted gene Z6 is abnormal in the thyroid
  • the potential stage of cancer began to appear, and it remained stable in early and middle stage thyroid cancer, and then rose rapidly to a higher level in the advanced stage of thyroid cancer.
  • the increase in the expression level of the imprinted gene Z6 began to appear in the early stage of thyroid cancer and maintained in the middle stage of thyroid cancer. Stable, rapidly rising to a higher level in the advanced stage of thyroid cancer; the absence of the imprint of the imprinted gene Z8 rises rapidly in the middle stage of thyroid cancer, and continues to rise to a higher level in the advanced stage of thyroid cancer, and the abnormal copy number of the imprinted gene Z8 is in the middle stage of the thyroid The cancer stage rapidly rises, and continues to rise to a high level in the advanced stage of thyroid cancer.
  • the increase in the expression level of the imprinted gene Z8 appears in the advanced stage of thyroid cancer and reaches a higher level; the imprint of the imprinted gene Z10 is missing, the copy number is abnormal, and the expression level Increased in the advanced stage of thyroid cancer, and To a very high level; the imprint of the imprinted gene Z13 begins to appear in the early stage of thyroid cancer, and it slowly rises in the middle and advanced stages of thyroid cancer.
  • the abnormal copy number of the imprinted gene Z13 begins to appear in the early stage of thyroid cancer, and slowly in the middle stage of thyroid cancer.
  • Tissues of 41 thyroid cancer patients were obtained, including puncture biopsy cell samples.
  • the detection method was the same as in Example 1.
  • any one of the imprinted gene deletion expression amount is less than 12%, the imprinted gene copy number abnormal expression amount is less than 1%, or the total imprinted gene expression amount is less than 25%. Or at least two of them are grade 0, any one or at least two of the imprinted gene deletion expression amount is 12-20%, the imprinted gene copy number abnormal expression amount is 1-2%, or the total imprinted gene expression amount is 25-35%.
  • the species is class I. Any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 35-45%.
  • any one or at least two of the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 3-5% or the total imprinted gene expression level is 45-60% is level III, imprinted Any one or at least two of the gene deletion expression levels greater than 30%, the imprinted gene copy number abnormal expression levels greater than 5%, or the total imprinted gene expression levels greater than 60% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 0.5%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • the expression level of the imprinted gene deletion is less than 12%, the abnormal expression amount of the imprinted copy number is less than 1%, or the total expression level of the imprinted gene is less than 25%. Or at least two of them are grade 0, any one or at least two of the imprinted gene deletion expression amount is 12-20%, the imprinted gene copy number abnormal expression amount is 1-2%, or the total imprinted gene expression amount is 25-35%.
  • the species is class I. Any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 35-45%.
  • any one or at least two of the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 3-5% or the total imprinted gene expression level is 45-60% is level III, imprinted Any one or at least two of the gene deletion expression levels greater than 30%, the imprinted gene copy number abnormal expression levels greater than 5%, or the total imprinted gene expression levels greater than 60% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 0.5%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 0.5%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 0.5%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 0.5%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 0.5%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • the expression level of the imprinted gene deletion is less than 10%, the abnormal expression amount of the imprinted copy number is less than 0.5%, or the total expression level of the imprinted gene is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression is 0.5-1%, or total imprinted gene expression is 15-20% The species is Class I. Any one or at least two of the imprinted gene deletion expression is 15-20%, the imprinted gene copy number abnormal expression is 1-2%, or the total imprinted gene expression is 20-30% is II.
  • any one or at least two of the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2-3%, or the total imprinted gene expression level is 30-40%, level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 3%, or the total imprinted gene expression levels greater than 40% are grade IV.
  • the judgment of the benign and malignant degree of thyroid tumors is divided into benign tumors, thyroid cancer potential, early thyroid cancer, intermediate thyroid cancer and advanced thyroid cancer;
  • the results of judging the benign and malignant degree of thyroid tumors are that the imprinted gene deletion expression amount and imprinted gene copy number abnormal expression amount of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 are less than 1 Class 1 or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16.
  • the expression level of the imprinted gene deletion is not more than 1 and the imprinted genes Z1, Z3, Z4, Z5.
  • the abnormal expression level of the copy number of the imprinted gene of not more than 1 imprinted gene in Z6, Z8, Z10, Z11, Z13, and Z16 is Grade I, and it is a benign tumor;
  • the result of judging the benign and malignant degree of the thyroid tumor is that the expression level of the imprinted gene deletion of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade I, Imprinted gene Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class I or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, the expression level of the imprinted gene is level II and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 The abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any one of the class II, then the potential of the thyroid cancer is judged;
  • the result of judging the benign and malignant degree of the thyroid tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II,
  • the imprinted gene copy number of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is at least two or the imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes
  • the expression level of the imprinted gene is class III and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any of the class III, which is early thyroid cancer;
  • the result of judging the benign and malignant degree of the thyroid tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade III,
  • the imprinted gene copy number of at least two imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is the level III or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes
  • the expression level of the imprinted gene is level IV and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any one of grade IV, and it is intermediate stage thyroid cancer;
  • the result of judging the benign and malignant degree of the thyroid tumor is that at least two of the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have an expression level of deletion IV or imprinted
  • the abnormal expression level of the copy number of the imprinted gene of at least two imprinted genes among the genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV, which is advanced thyroid cancer.
  • the detection model and system according to the present invention express the performance of the lack of imprints on samples of patients with thyroid tumors in an intuitive way.
  • the method of in situ marking of imprinted genes it is objective, intuitive, early and accurate. Detects changes in imprinted (trace) genes and can provide a quantified model, making a significant contribution to the diagnosis of thyroid tumors.
  • the present invention illustrates the detailed method of the present invention through the foregoing embodiments, but the present invention is not limited to the detailed method, which does not mean that the present invention must rely on the detailed method to be implemented.
  • Those skilled in the art should know that any improvement to the present invention, equivalent replacement of the raw materials of the products of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

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Abstract

一种用于检测甲状腺肿瘤良恶性程度的分级模型、装置及其应用，该模型通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量对印记基因在甲状腺肿瘤中的变化进行分级，展示了印记缺失在甲状腺肿瘤病人的组织和细胞样本上的表现，通过对印记基因原位标记的方法，客观、直观、早期、精确地检测出印记基因的变化，并可以提供量化的模型，对甲状腺肿瘤的诊断具有意义。

Description

一种用于检测甲状腺肿瘤良恶性程度的分级模型及其应用 技术领域
本公开涉及生物技术领域，例如基因诊断领域，例如一种分级模型及其应用，例如一种用于检测甲状腺肿瘤良恶性程度的分级模型及其应用，例如一组印记基因在检测甲状腺肿瘤良恶性程度中的分级模型及其组成的装置。
背景技术
根据世界卫生组织统计，2012年甲状腺癌全球新增病例298,100例，中国新诊断病例约90,000例，美国新诊断病例约25,520例。大多数甲状腺癌分化程度较高，具有极好预后，5年生存率高于95％，但是一部分甲状腺癌在肿瘤很小的时期就发生转移。此外，分化较低的甲状腺甲状腺癌可以是致命的，其生存时间中值范围为4个月到5年。目前甲状腺癌的病理诊断难点包括良性的滤泡性甲状腺瘤(FTA)和恶性的滤泡性甲状腺癌(FTC)之间的鉴别，以及甲状腺嗜酸细胞肿瘤(HCT)的良恶性判断，目前仅有的BRAF基因检测只能用于甲状腺***状癌的检测，对FTC和HCT的诊断没有作用。我国在过去的十年间，甲状腺癌的发病率增加了4.6倍，并且还在继续增长中，目前每年的新增的甲状腺癌的患者约有10万人左右，其中难以诊断的滤泡状甲状腺腺癌和嗜酸性细胞甲状腺瘤约占10％左右，加上3-4倍的疑似病例，预计每年大约有4-5万病例需要明确诊断，再加上全球的患者，这一数字将增加到10万以上，因此亟需开发一种准确率更高的检测技术。
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。
癌症的产生是随时间推移而累积的表观遗传改变和基因上的变异所导致的不受控制的细胞生长/***。传统病理学诊断根据细胞和组织的大小，形态和结构上的变异，从而做出甲状腺肿瘤良恶性判断。随着分子生物学的发展与深入，越来越多的分子检测技术被应用于甲状腺癌症的检测。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。
基于上述原因，目前的甲状腺癌诊断需要新的检测***和检测模型，基于患者活检样本，解析甲状腺癌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
本公开提供了一种用于检测甲状腺肿瘤良恶性程度的印记基因分级模型和诊断方法及其应用。
为达到上述目的，本申请采用以下技术方案：
本公开提供了一种用于甲状腺肿瘤的印记基因分级模型，所述模型通过计算印记基因的总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量在甲状腺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、Z11或Z16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Z11为Grb10，所述印记基因Z16为Snrpn/Snurf。
所述印记(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，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。
发明人发现通过计算Z1、Z11和Z16中任意一个印记基因在甲状腺肿瘤中的印记基因缺失表达量和印记基因拷贝数异常表达量，对甲状腺癌的诊断敏感度可以达到64.3％以上。
在本文一实施例中，若初步检测只检测一个印记基因，可以检测Z1、Z11和Z16中的任意一个印记基因。
在本文一实施例中，若初步检测只检测一个印记基因，可以检测Z1或Z11中的任意一个印记基因。
在本文一实施例中，若初步检测只检测一个印记基因，可以检测Z1。
发明人发现，若单独检测一个Z1印记基因，对甲状腺癌的诊断敏感度可以达到84.8％，若单独检测一个Z11印记基因，对甲状腺癌的诊断敏感度可以达到66.7％，若单独检测一个Z16印记基因，对甲状腺癌的诊断敏感度可以达到64.3％。
在本文一实施例中，所述模型计算印记基因的方法为：若检测印记基因的两个印记基因的组合，所述组合可以是Z1、Z11和Z16中的任意两个，优选为Z1和Z11的组合，Z1和Z16的组合。
发明人发现通过计算两个或两个以上的印记基因的总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量可以进一步提高敏感度，检测印记基因Z1、Z11和Z16中的任意两个印记基因的组合，对甲状腺癌的诊断敏感度可以达到84.6％以上，检测Z1和Z16的组合时， 对甲状腺癌的诊断敏感度可以达到92.9％，检测Z1和Z11的组合时，对甲状腺癌的诊断敏感度可以达到97.5％以上。
在本文一实施例中，所述印记基因还包括Z3、Z4、Z5、Z6、Z8、Z10或Z13中的任意一个或至少两个的组合；其中，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z10为Gatm，所述印记基因Z13为Sgce。
发明人发现在使用Z1、Z11和Z16基因检测的基础上再增加Z3、Z4、Z5、Z6、Z8、Z10、Z13基因进行联合诊断，不仅有助于增加检测的准确度，而且增加其他探针辅助诊断可以进一步避免假阳性的出现，能够将检测准确度进一步提高，从而能够实现所有甲状腺肿瘤样本的精确分级和判断。
在本文一实施例中，所述模型计算印记基因的方法为：计算印记基因的组合，计算Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16基因的组合。
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。
在本文一实施例中，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(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，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/棕色标记，印记基因拷贝数异常的细胞核。
在本文一实施例中，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。
在本文一实施例中，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10μm，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。
在本文一实施例中，所述印记基因总表达量、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，通过每个探针在样本表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分别进行划分的五个不同的等级。
在本文一实施例中，所述针对Z1和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z1和Z16的印记基因缺失表达量小于12％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于1％或所述印记基因Z1和Z16的总表达量小于25％中的任意一种或至少两种的组合；
I级：所述印记基因Z1和Z16的印记基因缺失表达量为12-20％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为1-2％或所述印记基因Z1和Z16的总表达量为25-35％中的任意一种或至少两种的组合；
II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-3％或所述印记基因Z1和Z16的总表达量为35-45％中的任意一种或至少两种的组合；
III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为3-5％或所述印记基因Z1和Z16的总表达量为45-60％中的任意一种或至少两种的组合；
IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于5％或所述印记基因Z1和Z16的总表达量大于60％中的任意一种或至少两种的组合；
所述印记基因Z1和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
在本文一实施例中，所述针对Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失 表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量小于10％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量小于0.5％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量小于15％中的任意一种或至少两种的组合；
I级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为10-15％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为0.5-1％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为15-20％中的任意一种或至少两种的组合；
II级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为15-20％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为1-2％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为20-30％中的任意一种或至少两种的组合；
III级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为20-25％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为2-3％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为30-40％中的任意一种或至少两种的组合；
IV级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量大于25％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量大于3％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量大于40％中的任意一种或至少两种的组合；
所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
在本文一实施例中，本公开提供了一种检测甲状腺肿瘤良恶性程度的装置，其采用上述模型，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总表达量、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甲状腺肿瘤的良恶性程度。
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红色/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。
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记基因总表达量、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。
所述检测装置是用于细胞和组织水平下早期直观地观察甲状腺肿瘤的印记(迹)基因的变化从而判断肿瘤的良恶性程度，为早期甲状腺肿瘤患者提供最有利的治疗机会。
在本文一实施例中，本公开提供一种检测甲状腺肿瘤良恶性程度的方法，采用所述的模型或所述的装置，包括如下步骤：
(1)获取待测样本；
(2)根据印记基因序列设计特异性引物；
(3)将步骤(2)的探针与待测样本进行原位杂交；
(4)显微镜成像分析印记基因的表达情况，从而诊断甲状腺肿瘤的良恶性程度；
其中，所述分析单元通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量，通过所述的模型，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诊断甲状腺肿瘤的良恶性程度。
在本文一实施例中，步骤(1)所述的待测样本来自于人的组织和/或细胞。
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，本发明所述待测样本包括组织的石蜡切片、甲状腺穿刺细胞涂片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***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。
所述甲状腺穿刺细胞涂片具体操作步骤为通过穿刺获取人体细胞，及时用10％中性***固定即可。
由于穿刺活检对病人伤害小，取样过程简单，相较于血液的循环特性，穿刺活检还能定位，穿刺活检作为实验样本有其特殊的优势。
在本文一实施例中，所述待测样本为甲状腺穿刺细胞涂片。
在本文一实施例中，所述印记基因为Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16，所述印记基因Z1为Gnas，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z10为Gatm，所述印记基因Z11为Grb10，所述印记基因Z13为Sgce，所述印记基因Z16为Snrpn/Snurf。
所述印记基因Z1(Gnas)，Z3(Peg10)，Z4(Igf2r)，Z5(Mest)，Z6(Plagl1)，Z8(Dcn)，Z10(Gatm)，Z11(Grb10)，Z13(Sgce)，Z16(Snrpn/Snurf)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。
所述设计探针是根据印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16，即Gnas，Peg10，Igf2r，Mest，Plagl1，Dcn，Gatm，Grb10，Sgce和Snrpn/Snurf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，具体的探针由Advanced Cell Diagnostics公司设计。
在本文一实施例中，所述原位杂交采用RNAscope原位杂交方法。
在本文一实施例中，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。
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。
在本文一实施例中，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(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，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/棕色标记， 印记基因拷贝数异常的细胞核。
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。
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，通过计算印记基因总表达量、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。
在本文一实施例中，所述印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量分成五个不同的等级。
所述五个不同的等级为在样本每个探针表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量分别进行划分。
所述针对Z1和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z1和Z16的印记基因缺失表达量小于12％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于1％或所述印记基因Z1和Z16的总表达量小于25％中的任意一种或至少两种的组合；
I级：所述印记基因Z1和Z16的印记基因缺失表达量为12-20％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为1-2％或所述印记基因Z1和Z16的总表达量为25-35％中的任意一种或至少两种的组合；
II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-3％或所述印记基因Z1和Z16的总表达量为35-45％中的任意一种或至少两种的组合；
III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为3-5％或所述印记基因Z1和Z16的总表达量为45-60％中的任意一种或至少两种的组合；
IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于5％或所述印记基因Z1和Z16的总表达量大于60％中的任意一种或至少两种的组合；
所述印记基因Z1和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
所述针对Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量小于10％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量小于0.5％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量小于15％中的任意一种或至少两种的组合；
I级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为10-15％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为0.5-1％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为15-20％中的任意一种或至少两种的组合；
II级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为15-20％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为1-2％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为20-30％中的任意一种或至少两种的组合；
III级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为20-25％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为2-3％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为30-40％中的任意一种或至少两种的组合；
IV级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量大于25％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量大于3％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量大于40％中的任意一种或至少两种的组合；
所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
在本文一实施例中，所述判断甲状腺肿瘤的良恶性程度分为良性肿瘤、甲状腺癌潜能、早期甲状腺癌、中期甲状腺癌和晚期甲状腺癌。
在本文一实施例中，所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为良性肿瘤；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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II级中的任意一种情况，则判断为甲状腺癌潜能；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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III级中的任意一种情况，则为早期甲状腺癌；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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IV级中的任意一种情况，则为中期甲状腺癌；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为晚期甲状腺癌。
在本文一实施例中，本公开提供一种所述的模型或所述的装置用于甲状腺肿瘤检测。
在本文一实施例中，本公开提供一种所述的模型或所述的装置用于制备治疗甲状腺癌的 药物或器械的用途。
在本文一实施例中，判断甲状腺肿瘤的良恶性程度分为良性肿瘤、甲状腺癌潜能、早期甲状腺癌、中期甲状腺癌和晚期甲状腺癌。
甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为良性肿瘤；
甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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II级中的任意一种情况，则判断为甲状腺癌潜能；
甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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III级中的任意一种情况，则为早期甲状腺癌；
甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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IV级中的任意一种情况，则为中期甲状腺癌；
甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13 和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为晚期甲状腺癌。
与相关技术相比，本文实施例中，利用所述检测模型和装置，以直观的方法表现了印记基因在甲状腺肿瘤病人样本上的表现，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。
附图说明
图1是本发明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2(a)为0级甲状腺肿瘤的病理切片中10个基因的表达状态，图2(b)为I级甲状腺癌的病理切片中10个基因的表达状态，图2(c)为II级甲状腺癌的病理切片中10个基因的表达状态，图2(d)为III级甲状腺癌的病理切片中10个基因的表达状态，图2(e)为IV级甲状腺癌的病理切片中10个基因的表达状态；
图3(a)为印记基因Z1、Z11和Z16对甲状腺癌的印记缺失的强度，图3(b)为印记基因Z1、Z11和Z16对甲状腺癌的拷贝数异常的强度，图3(c)为印记基因Z1、Z11和Z16对甲状腺癌的总表达量的强度，图3(d)为印记基因Z3、Z4、Z5、Z6、Z8、Z10和Z13对甲状腺癌的印记缺失的强度，图3(e)为印记基因Z3、Z4、Z5、Z6、Z8、Z10和Z13对甲状腺癌的拷贝数异常的强度，图3(f)为印记基因Z3、Z4、Z5、Z6、Z8、Z10和Z13对甲状腺癌的总表达量的强度，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，TE为印记基因总表达量；
图4(a)为印记基因Z1印记缺失、拷贝数异常和总表达量的强度，图4(b)为印记基因Z11印记缺失、拷贝数异常和总表达量的强度，图4(c)为印记基因Z16印记缺失、拷贝数异常和总表达量的强度，图4(d)为印记基因Z3印记缺失、拷贝数异常和总表达量的强度，图4(e)为印记基因Z4印记缺失、拷贝数异常和总表达量的强度，图4(f)为印记基因Z5印记缺失、拷贝数异常和总表达量的强度，图4(g)为印记基因Z6印记缺失、拷贝数 异常和总表达量的强度，图4(h)为印记基因Z8印记缺失、拷贝数异常和总表达量的强度，图4(i)为印记基因Z10印记缺失、拷贝数异常和总表达量的强度，图4(j)为印记基因Z13印记缺失、拷贝数异常和总表达量的强度，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，TE为印记基因总表达量；
图5(a)为印记基因Z1应用于41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5(b)为印记基因Z11应用于41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5(c)为印记基因Z16应用于41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5(d)为印记基因Z3应用于41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5(e)为印记基因Z4应用于41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5(f)为印记基因Z5应用于41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5(g)为印记基因Z6应用于41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5(h)为印记基因Z8应用于41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5(i)为印记基因Z10应用于41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5(j)为印记基因Z13应用于41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，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，TE为印记基因总表达量。
具体实施方式
为更进一步阐述本发明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发明的技术方案，但本发明并非局限在实施例范围内。
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。
本公开所述检测模型和装置，以直观的方法表现了印记缺失在甲状腺肿瘤病人的样本上 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，为甲状腺肿瘤的诊断做出巨大贡献；
本公开检测装置，可以在甲状腺肿瘤病人手术前通过穿刺活检得出甲状腺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甲状腺肿瘤的革命性突破；
本公开可以精确的判断甲状腺肿瘤的类型，通过印记基因的组合检测对甲状腺肿瘤的恶性程度进行明确地分级，极大地提高了对甲状腺癌的早期、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；
本公开可以从分子层面精确地区分甲状腺嗜酸细胞肿瘤(HCT)的良恶性，为目前组织细胞形态学难以区分甲状腺嗜酸细胞肿瘤良恶性的问题提供了解决方案，同时还可以对滤泡性甲状腺瘤(FTA)和滤泡性甲状腺癌(FTC)进行区分，解决国际上形态学诊断的难点。
本公开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，通过发现的甲状腺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。
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***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)、Z3(Peg10)、Z4(Igf2r)、Z5(Mest)、Z6(Plagl1)、Z8(Dcn)、Z10(Gatm)、Z11(Grb10)、Z13(Sgce)和Z16(Snrpn/Snurf)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，具体的探针由Advanced Cell Diagnostics公司设计。
(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；
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(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，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/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甲状腺穿刺活检样本的印记基因分析
所述甲状腺穿刺活检样本是，通过穿刺取出可疑病变组织，10％中性***溶液固定24h以上，其他检测方法同实施例1。
从图3(a)-图3(f)可以看出，Z1，Z3，Z4，Z5，Z6，Z8，Z10，Z11，Z13，Z16每个基因对甲状腺癌的反应敏感性或者说对应于甲状腺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甲状腺癌的敏感度如图4(a)-图4(j)，从图4(a)-图4(c)可以看出，印记基因Z1的印记缺失和表达量增加在恶性潜能阶段开始出现，在早期甲状腺癌中迅速上升到很高的水平，并在中晚期甲状腺癌中继续维持，印记基因Z1的拷贝数异常在恶性潜能阶段迅速升高到很高的水平，在早期到晚期甲状腺癌中继续维持；印记基因Z11的印记缺失、拷贝数异常和表达量增加在恶性潜能阶段水平较低，在甲状腺癌早期到中期缓慢上升，在晚期甲状腺癌阶段迅速上升到很高的水平；印记基因Z16的印记缺失、拷贝数异常和表达量增加从恶性潜能阶段到中期甲状腺癌阶段迅速上升，到晚期甲状腺癌阶段维持很高的水平；
从图4(d)-图4(j)可以看出，印记基因Z3的印记缺失和拷贝数异常在恶性潜能和早期甲状腺癌阶段上升不明显，到中期甲状腺癌阶段缓慢增加，到晚期甲状腺癌中上升速度加快，达到较高的水平，印记基因Z3的表达量增加在恶性潜能阶段开始出现，在甲状腺癌的发 展过程中缓慢上升，到晚期甲状腺癌阶段仍然很低；印记基因Z4的印记缺失和拷贝数异常在甲状腺癌的发展过程中缓慢上升，到晚期甲状腺癌阶段仍然很低，印记基因Z4的表达量增加在恶性潜能阶段开始出现，在早期甲状腺癌中没有明显上升，在中期甲状腺癌中加速上升，但晚期甲状腺癌阶段达到较高的敏感度；印记基因Z5的印记缺失和拷贝数异常在中期甲状腺癌阶段开始出现，到晚期甲状腺癌阶段水平仍然不高，印记基因Z5的表达量增加在早期甲状腺癌阶段开始出现，在甲状腺癌发展过程中逐渐上升，到晚期甲状腺癌阶段水平仍然不高；印记基因Z6的印记缺失在中期甲状腺癌中出现，在晚期甲状腺癌中有一定程度的上升，但是水平不高，印记基因Z6的拷贝数异常在甲状腺癌潜能阶段开始出现，在早期和中期甲状腺癌中维持稳定，到晚期甲状腺癌阶段又快速上升到较高水平，印记基因Z6的表达量增加在早期甲状腺癌阶段开始出现，在中期甲状腺癌中维持稳定，到晚期甲状腺癌阶段快速上升到较高水平；印记基因Z8的印记缺失在中期甲状腺癌阶段迅速上升，到晚期甲状腺癌阶段继续上升到较高水平，印记基因Z8的拷贝数异常在中期甲状腺癌阶段迅速上升，到晚期甲状腺癌阶段继续上升到很高水平，印记基因Z8的表达量增加在晚期甲状腺癌阶段出现，并达到较高水平；印记基因Z10的印记缺失、拷贝数异常和表达量增加在晚期甲状腺癌阶段出现，并达到很高水平；印记基因Z13的印记缺失在早期甲状腺癌阶段开始出现，在中期和晚期甲状腺癌阶段缓慢上升，印记基因Z13的拷贝数异常在早期甲状腺癌阶段开始出现，在中期甲状腺癌阶段缓慢上升，到晚期甲状腺癌阶段加速上升并达到较高水平，印记基因Z13的表达量增加在恶性潜能阶段开始出现，在早期和中期甲状腺癌阶段没有明显上升，在晚期甲状腺癌迅速上升到较高水平。
实施例341例甲状腺肿瘤样本的印记基因分析
获取41例甲状腺癌病人的组织包括穿刺活检细胞样本，检测方法同实施例1。
从图5(a)-图5(j)可以看出，41例甲状腺肿瘤组织样本中10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。
具体的分级如下：
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12％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于25％中的任意一种或至少两种为0级，印记基因缺失表达量为12-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为25-35％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为35-45％中的任意一种或至少两种为II级，印记基因 缺失表达量为25-30％、印记基因拷贝数异常表达量为3-5％或印记基因总表达量为45-60％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于5％或印记基因总表达量大于60％中的任意一种或至少两种为IV级；
从图5(b)可以看出，对于所述印记基因Z11，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(c)可以看出，对于所述印记基因Z16，印记基因缺失表达量小于12％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于25％中的任意一种或至少两种为0级，印记基因缺失表达量为12-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为25-35％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为35-45％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为3-5％或印记基因总表达量为45-60％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于5％或印记基因总表达量大于60％中的任意一种或至少两种为IV级；
从图5(d)可以看出，对于所述印记基因Z3，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(e)可以看出，对于所述印记基因Z4，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为 15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(f)可以看出，对于所述印记基因Z5，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(g)可以看出，对于所述印记基因Z6，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(h)可以看出，对于所述印记基因Z8，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(i)可以看出，对于所述印记基因Z10，印记基因缺失表达量小于10％、印记基 因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(j)可以看出，对于所述印记基因Z13，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于0.5％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为0.5-1％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-3％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于3％或印记基因总表达量大于40％中的任意一种或至少两种为IV级。
从这61个甲状腺癌肿瘤的样本综合分析可以得出：
所述判断甲状腺肿瘤的良恶性程度分为良性肿瘤、甲状腺癌潜能、早期甲状腺癌、中期甲状腺癌和晚期甲状腺癌；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为良性肿瘤；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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II级中的任意一种情况，则判断为甲状腺癌潜能；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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III级中的任意一种情况，则为早期甲状腺癌；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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IV级中的任意一种情况，则为中期甲状腺癌；
所述判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为晚期甲状腺癌。
综上所述，本发明所述检测模型和***，以直观的方法表现了印记缺失在甲状腺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，为甲状腺肿瘤的诊断做出巨大贡献。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明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，对本发明的任何改进，对本发明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，均落在本发明的保护范围和公开范围之内。

Claims (20)

  1. 一种用于甲状腺肿瘤的印记基因分级模型，其通过计算印记基因的总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量在甲状腺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、Z11或Z16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Z11为Grb10，所述印记基因Z16为Snrpn/Snurf。
  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、Z11或Z16中的任意一个，优选为Z1或Z16，进一步优选为Z1。
  3. 根据权利要求1或2所述的模型，其中，所述模型计算印记基因的方法为：计算Z1、Z11或Z16中的任意两个印记基因的组合，优选为Z1和Z16的组合或Z1和Z11的组合。
  4. 根据权利要求1-3中任一项所述的模型，其中，所述印记基因还包括Z3、Z4、Z5、Z6、Z8、Z10或Z13中的任意一个或至少两个的组合；其中，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z10为Gatm，所述印记基因Z13为Sgce。
  5. 根据权利要求1-4中任一项所述的模型，其中，所述模型计算印记基因的方法为：计算印记基因的组合，计算Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的组合。
  6. 根据权利要求1-5中任一项所述的模型，其中，所述计算印记基因的总表达量、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，细胞核内存在一个红色/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，细胞核内存在两个红色/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红色/棕色标记，印记基因拷贝数异常的细胞核。
  7. 根据权利要求1-6中任一项所述的模型，其中，所述印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分成五个不同的等级。
  8. 根据权利要求7所述的模型，其中，所述五个不同的等级为针对Z1、Z3、Z4、Z5、 Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分别进行划分的五个不同的等级；
    所述针对Z1和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
    0级：所述印记基因Z1和Z16的印记基因缺失表达量小于12％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于1％或所述印记基因Z1和Z16的总表达量小于25％中的任意一种或至少两种的组合；
    I级：所述印记基因Z1和Z16的印记基因缺失表达量为12-20％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为1-2％或所述印记基因Z1和Z16的总表达量为25-35％中的任意一种或至少两种的组合；
    II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-3％或所述印记基因Z1和Z16的总表达量为35-45％中的任意一种或至少两种的组合；
    III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为3-5％或所述印记基因Z1和Z16的总表达量为45-60％中的任意一种或至少两种的组合；
    IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于5％或所述印记基因Z1和Z16的总表达量大于60％中的任意一种或至少两种的组合；
    所述针对Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
    0级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量小于10％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量小于0.5％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量小于15％中的任意一种或至少两种的组合；
    I级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为10-15％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为0.5-1％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为15-20％中的任意一种或至少两种的组合；
    II级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为 15-20％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为1-2％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为20-30％中的任意一种或至少两种的组合；
    III级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量为20-25％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量为2-3％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量为30-40％中的任意一种或至少两种的组合；
    IV级：所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因缺失表达量大于25％、所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的印记基因拷贝数异常表达量大于3％或所述印记基因Z3、Z4、Z5、Z6、Z8、Z10、Z11和Z13的总表达量大于40％中的任意一种或至少两种的组合。
  9. 一种检测甲状腺肿瘤良恶性程度的装置，采用如权利要求1-8中任一项所述的模型，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、印记基因拷贝数异常表达量和总表达量，通过权利要求1-8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诊断甲状腺肿瘤的良恶性程度。
  10. 一种检测甲状腺肿瘤良恶性程度的方法，采用如权利要求1-8中任一项所述的模型或如权利要求9所述的装置，包括如下步骤：
    (1)获取待测样本；
    (2)根据印记基因序列设计特异性引物；
    (3)将步骤(2)的探针与待测样本进行原位杂交；
    (4)显微镜成像分析印记基因的表达情况，从而诊断肺肿瘤的良恶性程度；
    其中，所述分析单元通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量，通过权利要求1-8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诊断肺肿瘤的良恶性程度。
  11. 根据权利要求10所述的方法，其中，步骤(1)所述的待测样本来自于人的组织和/ 或细胞。
  12. 根据权利要求10或11所述的方法，其中，所述待测样本为组织的石蜡切片和/或甲状腺穿刺细胞涂片。
  13. 根据权利要求10-12中任一项所述的方法，其中，所述原位杂交采用RNAscope原位杂交方法。
  14. 根据权利要求10-13中任一项所述的方法，其中，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。
  15. 根据权利要求10-14中任一项所述的方法，其中，所述诊断甲状腺肿瘤的良恶性程度分为良性肿瘤、甲状腺癌潜能、早期甲状腺癌、中期甲状腺癌和晚期甲状腺癌。
  16. 根据权利要求10-15中任一项所述的方法，其中，所述诊断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为良性肿瘤；
    所述诊断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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II级中的任意一种情况，则判断为甲状腺癌潜能；
    所述诊断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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III级中的任意一种情况，则为早期甲状腺癌；
    所述诊断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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IV级中的任意一种情况，则为中期甲状腺癌；
    所述诊断甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为晚期甲状腺癌。
  17. 一种如权利要求1-8中任一项所述的模型或如权利要求9所述的装置用于甲状腺肿瘤检测的用途。
  18. 一种如权利要求1-8中任一项所述的模型或如权利要求9所述的装置用于制备治疗甲状腺肿瘤的药物或器械的用途。
  19. 根据权利要求17或18所述的用途，其中，甲状腺肿瘤的良恶性程度分为良性肿瘤、甲状腺癌潜能、早期甲状腺癌、中期甲状腺癌和晚期甲状腺癌。
  20. 根据权利要求17-19中任一项所述的用途，其中，甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为良性肿瘤；
    甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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II级中的任意一种情况，则判断为甲状腺癌潜能；
    甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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III级中的任意一种情况，则为早期甲状腺癌；
    甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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IV级中的任意一种情况，则为中期甲状腺癌；
    甲状腺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16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，则为晚期甲状腺癌。
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