WO2020200323A1 - 一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用 - Google Patents

一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用 Download PDF

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WO2020200323A1
WO2020200323A1 PCT/CN2020/083410 CN2020083410W WO2020200323A1 WO 2020200323 A1 WO2020200323 A1 WO 2020200323A1 CN 2020083410 W CN2020083410 W CN 2020083410W WO 2020200323 A1 WO2020200323 A1 WO 2020200323A1
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early
gastric
gastric cancer
cancer
product
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PCT/CN2020/083410
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English (en)
French (fr)
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李梢
张鹏
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清华大学
李梢
张鹏
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Priority to CN202080001970.7A priority Critical patent/CN111936858B/zh
Publication of WO2020200323A1 publication Critical patent/WO2020200323A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

  • the invention relates to a cell marker for assisting in the very early diagnosis of gastric cancer and other digestive system tumors and a measurement system and method thereof, as well as a cell marker for early diagnosis of gastric precancerous lesions and a measurement system and method thereof.
  • Gastric cancer is a common malignant tumor of the digestive system and has a high mortality rate. Most gastric cancers undergo a series of precancerous lesions, including intestinal metaplasia and dysplasia. The risk of gastric cancer in patients with precancerous lesions is nearly three times that of patients with non-precancerous lesions [1]. Early diagnosis of gastric precancerous lesions is of great significance for the prevention of gastric cancer.
  • the very early diagnosis of gastric cancer is also of great significance for the prevention and treatment of gastric cancer. Studies have shown that the 5-year survival rate for patients with early gastric cancer can reach 96% [2]. However, the diagnosis of early gastric cancer is still based on imaging, endoscopy, and histopathology, all of which have certain limitations. Imaging-based diagnosis cannot accurately observe cell-level changes, and it is easy to miss key biological characteristics; while diagnosis methods based on histopathological observations and the expression of conventional tumor marker molecules are limited by sensitivity and specificity.
  • the classic gastric cancer marker CEACAM5 is highly expressed in gastric cancer cells and other types of cells adjacent to the cancer (such as intestinal cells and normal gastric epithelial cells), so it is difficult to become a specific marker for early screening of gastric cancer.
  • the key transformation point of gastritis cancer transformation that is, the "carcinogenesis starting point”. This is the key difficulty in the very early diagnosis of gastric cancer, and it also limits the very early diagnosis of clinical gastric cancer. The key bottleneck. Therefore, identifying high-precision very early cell markers of gastric cancer is of great significance for defining the "carcinogenesis starting point" of gastritis cancer transformation and realizing the very early clinical diagnosis of gastric cancer.
  • the inventors discovered a group of groups that began to appear in the low-grade gastric dysplasia stage and have specific molecular characteristics (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) and a cell population with a higher risk of canceration (the inventor named it "very early gastric cancer cells”), which can be used as a marker for very early diagnosis of gastric cancer.
  • KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B a cell population with a higher risk of canceration
  • the present inventors defined the stage at which this cell population appeared as the key point for the transformation of gastric precancerous lesions into gastric cancer, that is, the "starting point of cancer.”
  • the inventors also found that the very early cell markers of gastric cancer can distinguish the risk of gastric cancer recurrence after surgery, and assess the risk of transformation from precancerous lesions of digestive system organs such as intestines, pancreas, and esophagus to cancer.
  • the molecular characteristics of the very early cell markers of gastric cancer are applied to the preparation of kits for the early diagnosis of gastric cancer or other digestive system tumors based on gastric tissue or blood samples.
  • kits are expected to provide an effective basis for relevant treatment measures or decision-making for patients with digestive system tumors, and has a good clinical application prospect.
  • a reagent for detecting the expression level of markers for use in the preparation of products for determining very early gastric cancer wherein the markers include KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, At least one selected from the group consisting of KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B.
  • a product for determining the cell content of gastric cancer in the very early stage thereby diagnosing the gastric cancer in the very early stage.
  • the product is characterized in that the product includes a reagent for detecting the expression level of markers in a sample, wherein the markers include KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and At least one selected from the group consisting of KRT6B.
  • a reagent for detecting the expression level of HES6 is provided for use in preparing a product for determining the level of gastric precancerous lesions.
  • a product for determining the level of gastric precancerous lesions characterized in that the product includes a reagent for detecting the expression level of HES6 in a sample.
  • the present invention mainly includes the following aspects, namely:
  • the present invention provides 13 molecules (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) that can be used as the characteristics of the very early cell populations of gastric cancer and applied to the early stage New use for diagnosing gastric cancer.
  • 13 molecules KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B
  • the present invention also provides the molecule (HES6) as a feature of the cell population in the early stage of gastric precancerous lesions, and is applied to a new application for early diagnosis of gastric precancerous lesions.
  • HES6 the molecule
  • the present invention provides 13 molecules (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) in tissues such as pancreas, intestine and esophagus from precancerous lesions to early cancer
  • 13 molecules KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B
  • the present invention provides the association between the expression levels of 13 molecules (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) and the risk of recurrence and survival after gastric cancer surgery, and It is a new application for judging the risk of gastric cancer recurrence after surgery.
  • 13 molecules KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B
  • the present invention provides a detection kit, which can be used for immunohistochemical (IHC) staining of the above-mentioned protein molecules to obtain their expression in the gastric tissue to be tested, and then to evaluate the early stage cells of gastric precancerous lesions in the tissue to be tested As well as the content of early cancer cells; it can also be used to detect the expression of the protein molecules in the blood by enzyme-linked immunosorbent assay (ELISA).
  • IHC immunohistochemical
  • the present invention proposes a system and method for early diagnosis of gastric precancerous lesions, gastric cancer, pancreatic cancer, intestinal cancer, and esophageal cancer and assessment of the risk of recurrence after gastric cancer surgery.
  • the system evaluates the number of gastric precancerous lesions and early cancer cells in gastric tissue based on the expression of cell marker proteins in tissues or blood, and then provides the risk of gastric precancerous lesions and gastric cancer, and the recurrence of gastric cancer after surgery. risk.
  • the system can also give the risk of other tumors of the digestive system such as colon cancer, esophageal cancer, and pancreatic cancer based on the expression of cell marker proteins in the patient's tissue or blood.
  • the system includes the following two parts:
  • the gastric precancerous lesion and very early gastric cancer cell counting device (30) is used to determine the proportion of HES6-positive cells in the early stage of gastric precancerous lesions (31) and the very early cell markers of gastric cancer KLK10, SLC11A2, SULT2B1 , KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B positive cell percentage scores (32).
  • the counting device calculates the average value to determine the overall expression level of KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B in the serum (33) .
  • the system gives the patient's risk index of gastric precancerous lesions and the risk index of gastric cancer based on the ratio of cells in the early stage of gastric precancerous lesions to the total number of cells in the entire tissue to be tested; it can also be based on the proportion of very early gastric cancer cells in the entire tissue to be tested The proportion of the total gives the patient's risk index for gastric cancer and the risk index for gastric cancer recurrence after surgery; it can also be used to evaluate the esophageal cancer and pancreatic cancer based on the expression levels of the very early cell marker molecules of gastric cancer in the esophagus, pancreas and intestinal tissues And the risk index of bowel cancer.
  • the overall expression level of the marker molecules of very early cells of gastric cancer in the serum can also be used to assess the risk index of the patient to be tested for cancer of the digestive system
  • the present invention proposes to detect the above-mentioned early stage cell markers of gastric precancerous lesions HES6 (10) and 13 kinds of very early stage cell markers of gastric cancer (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1). , PDZK1IP1 and KRT6B) expression levels in order to obtain their expression in the tissue/blood to be tested for the preparation of a composition for early diagnosis of gastric precancerous lesions and/or gastric cancer and other digestive system tumors.
  • Fig. 1 is a schematic diagram of a system for assisting early diagnosis of gastric precancerous lesions, gastric cancer and other digestive system tumors based on cell markers according to an embodiment of the present invention.
  • Fig. 2A is a schematic diagram illustrating the specific expression of the cell marker HES6 in specific cell types of gastric precancerous lesions according to a typical embodiment of the present invention.
  • Fig. 2B shows the ratio distribution of positive cells marked by the early stage cell marker of gastric precancerous lesions (HES6) and positive cells marked by the late stage precancerous lesion marker MUC2 in the gastric precancerous lesion tissue in an embodiment according to the present invention.
  • Figure 2C shows the positive cells marked by the early stage cell markers (HES6) of gastric precancerous lesions in four stages of non-precancerous lesions, mild precancerous lesions, severe precancerous lesions, and early gastric cancer stages in an embodiment of the present invention.
  • HES6 early stage cell markers
  • FIG. 2D shows the proportion distribution of positive cells marked by the gastric precancerous lesion cell marker (HES6) in normal gastric tissue and gastric precancerous lesion tissue in an embodiment according to the present invention.
  • HES6 gastric precancerous lesion cell marker
  • Fig. 2E is a schematic diagram illustrating the partial co-expression of the cell marker HES6 and the classic marker MUC2 of gastric precancerous lesions in tissues according to a typical embodiment of the present invention.
  • Fig. 2F is used to illustrate a schematic diagram of demonstrating that the cell marker HES6 appears in the tissue along with the proliferative cell marker KI67, but is not co-expressed according to a typical embodiment of the present invention.
  • Figure 3A shows 13 early cancer cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in early gastric cancer tissues in an embodiment according to the present invention Expression patterns of different types of cells.
  • Figure 3B shows 13 early cancer cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent gastritis (19) in an embodiment according to the present invention.
  • Example gastric precancerous lesions (39 cases) and early gastric cancer (19 cases) samples.
  • Figure 3C shows 13 early cancer cell markers (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) in independent gastritis and gastric cancer in an embodiment according to the present invention.
  • Figure 3D shows the expression patterns of five representative marker molecules (KLK10, KRT17, TIMP1, MAP17, and KLK6) of gastric cancer very early cells in normal gastric tissues, dysplasia (precancerous lesions of the stomach) and early gastric cancer in an embodiment according to the present invention .
  • Fig. 3E shows the proportion distribution of positive cells marked by gastric cancer very early cell typical markers (KLK10) in gastric precancerous lesions, early gastric cancer, and advanced gastric cancer in an embodiment according to the present invention.
  • KLK10 very early cell typical markers
  • Figure 4 is used to illustrate the association between the representative components (KLK10, SLC11A2, KLK7 and TIMP1) of gastric cancer very early cell markers and the risk of recurrence after gastric cancer surgery according to a typical embodiment of the present invention.
  • Figure 5A shows 13 very early cell markers of gastric cancer (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) in an independent normal intestine in an embodiment of the present invention.
  • Figure 5B shows 13 very early cell markers of gastric cancer (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) in an independent normal esophagus in an embodiment according to the present invention.
  • KLK10 gastric cancer
  • Figure 5C shows the representative molecules (ECM1, KRT17, TIMP1 and MMP7) in the very early cell markers of gastric cancer in the example according to the present invention in independent normal pancreas (8 cases), intraepithelial neoplasia (6 cases) and pancreatic cancer (12 cases) The expression pattern in the sample.
  • Fig. 1 is a schematic diagram of a system for assisting early diagnosis of gastric precancerous lesions and gastric cancer based on cell markers according to an embodiment of the present invention.
  • the present invention first provides a detection kit based on 14 molecules.
  • the kit includes reagents for detecting the expression levels of molecules such as KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B, as well as blank solution, diluent, antigen retrieval solution, etc.
  • the reagents are antibodies for immunohistochemical detection methods, as shown in Table 1.
  • the reagent can also be an antibody for Western Blot or ELISA detection method.
  • the present invention also provides an immunohistochemical method for detecting the expression levels of the above 13 molecules in gastric tissue.
  • the detection method in addition to the above reagents included in the kit, the user can prepare or purchase the following reagents by himself:
  • gastric precancerous lesions In order to verify the role of the present invention in assisting early diagnosis of gastric precancerous lesions and digestive system tumors, and predicting the risk of gastric cancer recurrence, the inventors independently treated gastric precancerous lesions, early gastric cancer, gastric cancer recurrence, and other digestive system tumors (intestinal cancer, esophageal cancer). Carcinoma and pancreatic cancer) and typical cases of precancerous lesions were analyzed.
  • HES6 a very early cell marker of gastric precancerous lesions, in different types of cells in independent gastric precancerous lesions. The results are shown in Figure 2A. HES6 is specifically and highly expressed in gastric precancerous cells.
  • HES6 gastric precancerous lesions
  • MUC2 the late marker of gastric precancerous lesions
  • the inventors evaluated the proportion distribution of very early cells in gastric precancerous lesions in four stages of gastric tissue samples: non-precancerous lesions, mild precancerous lesions, severe precancerous lesions, and early canceration. The results are shown in Figure 2C. The very early cells of gastric precancerous lesions began to appear in the mild precancerous lesion stage, and their proportion gradually increased as the degree of precancerous lesions deepened.
  • the expression of HES6 in the gastric precancerous lesion samples is obtained according to the gastric precancerous lesion cell marker (10) in the immunohistochemical test kit (20) of the present invention.
  • the kit uses immunohistochemical staining (IHC) to measure the expression level of markers.
  • IHC immunohistochemical staining
  • the inventors also used fluorescence to co-stain MUC2, a classic marker of gastric precancerous lesions, and KI67, a cell proliferation marker, in the sample.
  • the paraffin-embedded surgical samples were fixed with 10% formalin buffer, and the tissue sections were 4 microns/sheet.
  • Reagent A blocking solution, 10% goat serum
  • Reagent B Diluted and ready-to-use anti-HES6 primary antibody
  • Reagent C anti-goat biotinylated secondary antibody
  • Reagent F 20 times concentrated DAB substrate buffer solution
  • Reagent G 20 times concentrated DAB chromogenic solution.
  • the reagent B is imported in original packaging and ready-to-use antibody, and the dilution factor is 1:200; reagent G is imported in original packaging and the ready-to-use antibody, the dilution factor is 1:400; reagent A, C , D, E, F, G are imported and subpackaged with original packaging.
  • reagents in addition to the above-mentioned reagents contained in the kit, users can prepare or purchase the following reagents by themselves:
  • Tissue embedding fix the tissue specimen to be tested with 10% neutral formalin for 2 hours, rinse with running water repeatedly to remove the fixative, place the specimen in 75% alcohol overnight, and then use alcohol gradient dehydration, 75% alcohol 1h, 1h with 85% alcohol, 1h with 95% alcohol, 2 times with anhydrous ethanol, 1.5h each time, then soak in xylene for 1.5h, immerse in wax in 60°C oven for 1h for embedding, and store at 4°C for later use after cooling;
  • Paraffin section trim the wax block, adjust the microtome (SLEE paraffin microtome CUT5062) to set the section thickness to 3 ⁇ 4 ⁇ m, slice continuously, float and flatten in 60°C warm water, and spread it on the carrier coated with cationic resin. On the slide
  • Antigen retrieval Add 1000ml of citrate buffer to the pressure cooker, immerse the slice rack with slices in the buffer, repair at high temperature and high pressure for 2 minutes and 45 seconds, wash with TBS 3 times, 2 minutes each time;
  • Color development Add the DAB color development solution above to the slices to completely cover the tissue slices, observe the color development under a microscope, and rinse with distilled water to stop the color development;
  • Figure 2D shows the expression of the cell marker HES6 in normal gastric tissues and precancerous gastric tissues. It can be seen that the proportion of HES6-positive cells in normal gastric tissue is 0, while the proportion in gastric precancerous lesions increases to 10%.
  • Figures 2E and 2F respectively show the co-expression patterns between the cell marker HES6 and the classic gastric precancerous lesion marker MUC2 and the proliferative cell marker KI67 in independent precancerous lesion samples.
  • HES6 is partially co-expressed with MUC2, a classic marker of gastric precancerous lesions
  • HES6 surrounds the proliferative cell marker KI67 in the tissue. Therefore, it can be considered that HES6 positive cells are cells in the early stage of gastric precancerous lesions.
  • the inventors assessed that 13 very early gastric cancer cell marker molecules (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) are different in independent early gastric cancer samples The expression of the cell type. The results are shown in Figure 3A. These cell markers are specifically and highly expressed in cancer cells.
  • the inventors assessed 13 very early cell markers of gastric cancer (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1 and KRT6B) in independent gastritis (19 cases), The expression of gastric precancerous lesions (39 cases) and early gastric cancer (19 cases) samples. The results are shown in Figure 3B. The expression levels of these cell markers in gastritis are significantly lower than those in the dysplasia stage (precancerous lesions of the stomach) and early gastric cancer.
  • the average expression of the above-mentioned molecules was used as a molecular signature, and it was found that it showed a gradual and increasing trend in the transformation of gastritis-low-grade dysplasia-high-grade dysplasia-early gastric cancer, suggesting that the above-mentioned molecules are high in gastric tissue. Expression is closely related to the occurrence of gastric cancer, and can be used as an index to assess the risk of gastric cancer.
  • the sample data involved in this verification comes from public data (GSE55696).
  • the inventors evaluated 13 very early cell markers of gastric cancer (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent blood samples of gastritis and gastric cancer patients.
  • the expression situation in. The results are shown in Figure 3C.
  • the expression levels of these cell markers in the serum of gastric cancer patients are significantly increased.
  • the average expression level of the above-mentioned molecules was used as a molecular signature, and it was found that its value was significantly higher in cancer samples compared to normal samples.
  • the inventors obtained the five representative components (KLK10, KRT17, TIMP1, MAP17 and KLK6) in the early cancer cell markers according to the gastric precancerous cell markers (11) in the immunohistochemical test kit (20) of the present invention. Expression in gastric tissue, gastric precancerous lesions, early carcinogenesis and advanced carcinogenesis.
  • the kit uses immunohistochemical staining (IHC) to measure the expression level of markers.
  • IHC immunohistochemical staining
  • the paraffin-embedded surgical samples were fixed with 10% formalin buffer, and the tissue sections were 4 microns/sheet.
  • Reagent A blocking solution, 10% goat serum
  • Reagent B Diluted and ready-to-use anti-Klk10 primary antibody (it can also be any of the KRT17, TIMP1, MAP17 and KLK6 antibodies in Table 1);
  • Reagent C anti-goat biotinylated secondary antibody
  • Reagent F 20 times concentrated DAB substrate buffer solution
  • Reagent G 20 times concentrated DAB chromogenic solution.
  • the reagent B is imported in original packaging and ready-to-use antibody, and the dilution factor is 1:200; reagent G is imported in original packaging and the ready-to-use antibody, the dilution factor is 1:400; reagent A, C , D, E, F, G are imported and subpackaged with original packaging.
  • Tissue embedding fix the tissue specimen to be tested with 10% neutral formalin for 2 hours, rinse with running water repeatedly to remove the fixative, place the specimen in 75% alcohol overnight, and then use alcohol gradient dehydration, 75% alcohol 1h, 1h with 85% alcohol, 1h with 95% alcohol, 2 times with anhydrous ethanol, 1.5h each time, then soak in xylene for 1.5h, immerse in wax in 60°C oven for 1h for embedding, and store at 4°C for later use after cooling;
  • Paraffin section trim the wax block, adjust the microtome (SLEE paraffin microtome CUT5062) to set the section thickness to 3 ⁇ 4 ⁇ m, slice continuously, float and flatten in 60°C warm water, and spread it on the carrier coated with cationic resin. On the slide
  • Antigen retrieval Add 1000ml of citrate buffer to the pressure cooker, immerse the slice rack with slices in the buffer, repair at high temperature and high pressure for 2 minutes and 45 seconds, wash with TBS 3 times, 2 minutes each time;
  • Color development Add the DAB color development solution above to the slices to completely cover the tissue slices, observe the color development under a microscope, and rinse with distilled water to stop the color development;
  • Figure 3D shows the expression of five representative marker molecules (KLK10, KRT17, TIMP1, MAP17 and KLK6) in the very early cells of gastric cancer in normal gastric tissue, dysplasia (precancerous lesions of the stomach) and early gastric cancer.
  • KLK10, KRT17, TIMP1, MAP17 and KLK6 five representative marker molecules
  • the very early gastric cancer cells identified in the gastric precancerous lesions based on the above-mentioned representative marker molecules are circled. It can be seen that very early cells of gastric cancer begin to appear in the dysplasia stage and continue to exist in early cancer.
  • Figure 3E shows the proportion of positive cells marked by a typical cell marker (KLK10) of very early gastric cancer in gastric precancerous lesions, early gastric cancer and advanced gastric cancer. It can be seen that KLK10-positive cells did not appear in samples at the precancerous stage of gastric cancer, while the proportion in early gastric cancer samples was 5%, and the proportion in advanced samples increased to 35%.
  • KLK10-positive cells did not appear in samples at the precancerous stage of gastric cancer, while the proportion in early gastric cancer samples was 5%, and the proportion in advanced samples increased to 35%.
  • KLK10, SLC11A2, KLK7, and TIMP1 are significantly related to the survival risk of gastric cancer recurrence after surgery.
  • the average expression of the very early cell marker molecules of gastric cancer in the sample was used as a molecular marker, and it was found that it was also significantly related to the postoperative recurrence of gastric cancer (p ⁇ 0.01), indicating that the very early cell markers of gastric cancer can be used as a signature. Markers to assess the risk of gastric cancer recurrence.
  • the sample data involved in this verification comes from public data (TCGA).
  • the inventors also verified that the very early cell marker molecules of gastric cancer (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) are useful in evaluating other organs of the digestive system (pancreas, esophagus, and KRT6B). Intestine) the application of risk of transformation from precancerous lesions to cancer.
  • KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B are useful in evaluating other organs of the digestive system (pancreas, esophagus, and KRT6B).
  • Intestine the application of risk of transformation from precancerous lesions to cancer.
  • the average value of the expression of the above molecules in the sample was used as a molecular signature, and it was found that it showed a gradual increasing trend in the transformation process of normal intestine-low-grade dysplasia-high-grade dysplasia-intestinal cancer, suggesting that the above-mentioned molecules are in High expression in bowel tissue is closely related to the occurrence of bowel cancer, and can be used as an indicator to assess the risk of bowel cancer.
  • the sample data involved in this verification comes from public data (GSE37364).
  • the inventors evaluated 13 very early cell markers of gastric cancer (KLK10, SLC11A2, SULT2B1, KLK7, ECM1, LMTK3, KLK6, KIAA1199, KRT17, MMP7, TIMP1, PDZK1IP1, and KRT6B) in independent normal esophagus (5 cases) , Precancerous lesions (4 cases) and esophageal cancer (3 cases) samples.
  • the results are shown in Figure 5B.
  • the expression levels of these very early cell markers of gastric cancer in the normal esophagus are lower than those in the dysplasia stage (precancerous lesion) and esophageal cancer stage.
  • pancreatic cancer The average expression of the above-mentioned molecules was used as a molecular signature, and it was found that it showed a gradual and increasing trend in the transformation process of normal pancreas-pancreatic intraepithelial neoplasia-pancreatic cancer, suggesting that the high expression of the above-mentioned molecules in pancreatic tissue is related to The occurrence of pancreatic cancer is closely related and can be used as an indicator to assess the risk of pancreatic cancer.
  • the sample data involved in this verification comes from public data (GSE43288).

Abstract

本发明人发现了一群在胃低级别异型增生阶段开始出现、具有特定分子特征、且具有高度癌变风险的细胞群(命名为"胃癌极早期细胞")，可作为极早期诊断胃癌的标志。胃癌极早期细胞的分子标志被应用到制备基于胃组织或血液样本实现胃癌或其他消化系统肿瘤早期诊断的试剂盒中。该胃癌极早期细胞标志还可区分胃癌术后复发风险，以及肠、胰腺以及食管等消化系统器官肿瘤发生的风险。另外，本发明人还发现一群在胃癌前病变早期出现、具有特定分子特征的细胞群，可作为早期诊断胃癌前病变的标志，也被集成到试剂盒中。本发明可用于临床上早期诊断胃癌及其他消化道肿瘤的发生，也可作为上述消化道肿瘤防治的干预靶点，应用前景良好。

Description

一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用 技术领域
本发明涉及一种用于辅助极早期诊断胃癌及其他消化系统肿瘤中的细胞标志及其测定系统和方法，同时涉及一种用于早期诊断胃癌前病变的细胞标志及其测定系统和方法。
背景技术
胃癌是消化系统常见的恶性肿瘤，具有较高的死亡率。大多数胃癌的发生会经历一系列癌前病变的过程，包括肠上皮化生以及异型增生等。癌前病变患者罹患胃癌的风险是非癌前病变患者的近3倍[1]。早期诊断胃癌前病变对于胃癌的预防具有重要的意义。
除胃癌前病变外，胃癌的极早期诊断对于胃癌的防治同样具有重要意义。研究表明，胃早癌患者的5年术后生存率可达96％[2]。然而，胃早癌的诊断目前依然以影像学、内镜、病理组织学为主，均存在一定的局限。基于影像学的诊断无法精确地观测到细胞层次的变化，容易遗漏关键生物特征；而基于病理组织学观测和常规肿瘤标志物分子表达的诊断方式会受限于灵敏度和特异度。例如，经典的胃癌标志物CEACAM5，在胃癌细胞及其癌旁的其他类型细胞(如肠细胞和正常胃上皮细胞)中均表现出高表达，因此很难成为胃癌早期筛查的特异标志物。更为重要的是，由于胃炎癌转化时程长，难以界定胃炎癌转化的关键转化点、亦即“癌变起始点”，这是胃癌极早期诊断的关键难点，也是限制临床胃癌极早期诊断的关键瓶颈。因此，识别高精度的胃癌极早期细胞标志对于界定胃炎癌转化的“癌变起始点”、实现胃癌临床极早期诊断具有十分重要的意义。
与胃癌发生发展类似的是，消化系统其他器官如肠、食管以及胰腺 中肿瘤的发生与炎症的恶性转化密切相关，也会经历包括异型增生在内的癌前病变过程。因此，以胃癌极早期细胞标志的识别为切入点，找到消化系统肿瘤共性的早癌标志物对于提高消化系统肿瘤早期诊断率也具有十分重要的意义。
发明内容
在本发明中，本发明人发现了一群在胃低级别异型增生阶段开始出现、具有特定分子特征(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)、且具有较高癌变风险的细胞群(发明人将其命名为“胃癌极早期细胞”)，可作为极早期诊断胃癌的标志。本发明人将出现该细胞群的阶段定义为胃癌前病变向胃癌转化的关键点、亦即“癌变起始点”。此外，本发明人还发现该胃癌极早期细胞标志可区分胃癌术后复发风险，以及评估肠、胰腺以及食管等消化系统器官癌前病变向癌症转化的风险。在本发明中，该胃癌极早期细胞标志的分子特征被应用到制备基于胃组织或血液样本实现胃癌或其他消化系统肿瘤早期诊断的试剂盒中。另外，本发明人还发现了一群在胃癌前病变早期阶段开始出现、具有特定分子特征(HES6)的细胞群，可作为早期诊断胃癌前病变的标志，也被集成到试剂盒中。该试剂盒有望为消化系统肿瘤患者采取相关治疗措施或决策提供有效依据，临床应用前景良好。
根据本发明的一个方面，提供了检测标志物的表达水平的试剂在制备确定极早期胃癌的产品中应用，其中所述标志物包括从由KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B组成的组中选出的至少一种。
根据本发明的另一个方面，提供了一种用于确定胃癌极早期细胞含量、进而诊断极早期胃癌的产品。其特征在于，所述产品包括检测样本中标志物表达水平的试剂，其中所述标志物包括从由KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B组成的组中选出的至少一种。
根据本发明的另一个方面，提供了检测HES6表达水平的试剂在制备确定胃癌前病变水平的产品中应用。
根据本发明的另一个方面，提供了一种用于确定胃癌前病变水平的产品，其特征在于，所述产品包括检测样本中HES6表达水平的试剂。
本发明主要包括以下方面，即：
1)本发明提供了13个分子(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)可作为胃癌极早期细胞群的特征，并应用于早期诊断胃癌的新用途。
2)本发明还提供了分子(HES6)作为胃癌前病变早期阶段细胞群的特征，并应用于早期诊断胃癌前病变的新用途。
本发明提供了13个分子(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在胰腺、肠和食管等组织中从癌前病变到早癌的变化特征，并应用于早期诊断胰腺癌、肠癌和食管癌的新用途。
本发明提供了13个分子(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)的表达水平与胃癌术后复发生存风险之间的关联，并应用于判断胃癌术后复发风险的新用途。
3)本发明提供了一种检测试剂盒，既可用于免疫组化(IHC)染色上述蛋白分子获取其在待测胃组织中的表达情况，进而评估待测组织中的胃癌前病变早期阶段细胞以及早癌细胞含量；也可以用于通过酶联免疫吸附测定(ELISA)检测上述蛋白分子在血液中的表达情况。
4)本发明提出了一种胃癌前病变、胃癌、胰腺癌、肠癌以及食管癌早期诊断和胃癌术后复发风险评估的系统和方法。对于该系统依据组织或血液中细胞标志蛋白的表达情况，来评估胃组织中胃癌前病变细胞以及早癌细胞数量，进而给出待检测者罹患胃癌前病变以及胃癌的风险、胃癌术后复发的风险。该系统还可以依据患者组织或血液中细胞标志蛋白的表达情况，给出待检测者罹患肠癌、食管癌、胰腺癌等消化系统其他肿瘤的风险。该系统包括以下两部分：
检测试剂盒(20)，用于分别免疫组化染色上述胃癌前病变早期阶段细胞标志HES6(10)以及13种胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)的表达水平(11)从而获取其在待测组织中的表达情况；也可以用于通过酶联免疫吸附测定检测获取待测患者血液样本中胃癌极早期细胞标志的含量。
若是待测组织样本，则胃癌前病变及胃癌极早期细胞计数装置(30)，用于确定胃癌前病变早期阶段细胞标志HES6阳性细胞比例分数(31)以及胃癌极早期细胞标志KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B阳性细胞比例分数(32)。若是待测血液样本，则该计数装置通过计算平均值进而确定血清中KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B的总体表达水平(33)。
系统根据胃癌前病变早期阶段细胞占整个待测组织中细胞总数的比例给出患者罹患胃癌前病变的风险指数以及罹患胃癌的风险指数；也可根据胃癌极早期细胞比例占整个待测组织中细胞总数的比例给出患者罹患胃癌的风险指数、以及胃癌术后复发风险指数；也可根据食管、胰腺以及肠道组织中胃癌极早期细胞标志物分子的表达水平来分别评估罹患食管癌、胰腺癌以及肠癌的风险指数。同时，胃癌极早期细胞的标志物分子在血清中的总体表达水平也可以用来评估待测患者罹患消化系统肿瘤的风险指数
5)本发明提出了分别检测上述胃癌前病变早期阶段细胞标志HES6(10)以及13种胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)的表达水平从而获取其在待测组织/血液中的表达情况的试剂在制备用于早期诊断胃癌前病变和/或胃癌及其他消化系统肿瘤中的组合物中的应用。
附图说明
图1是根据本发明的一个实施例的基于细胞标志的胃癌前病变及胃癌及其他消化系统肿瘤辅助早期诊断系统的原理图。
图2A用于说明根据本发明的一个典型实施例的，论证细胞标志HES6在胃癌前病变特定细胞类型中特异表达的示意图。
图2B是在根据本发明的实施例中胃癌前病变早期阶段细胞标志(HES6)所标记的阳性细胞与癌前病变晚期阶段标志MUC2所标记的阳性细胞在胃癌前病变组织中的比例分布。
图2C是在根据本发明的实施例中胃癌前病变早期阶段细胞标志(HES6)所标记的阳性细胞在非癌前病变阶段、轻度癌前病变、重度癌前病变以及胃癌早期阶段等四个阶段胃组织中的比例分布。
图2D是在根据本发明的实施例中胃癌前病变细胞标志(HES6)所标记的阳性细胞在正常胃组织以及胃癌前病变组织中的比例分布。
图2E用于说明根据本发明的一个典型实施例的、论证细胞标志HES6与胃癌前病变经典标志MUC2在组织中部分共表达的示意图。
图2F用于说明根据本发明的一个典型实施例的、论证细胞标志HES6在组织中伴随着增值细胞标志KI67出现，但不共表达的示意图。
图3A是在根据本发明的实施例中13个早癌细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在胃早癌组织中不同类型细胞的表达模式。
图3B是在根据本发明的实施例中13个早癌细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、 KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立的胃炎(19例)、胃癌前病变(39例)及胃早癌(19例)样本中的表达模式。
图3C是在根据本发明的实施例中13个早癌细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立的胃炎、胃癌患者血液样本中的表达模式。
图3D是在根据本发明的实施例中胃癌极早期细胞五个代表性标志分子(KLK10、KRT17、TIMP1、MAP17和KLK6)在正常胃组织、异型增生(胃癌前病变)以及早期胃癌的表达模式。
图3E是在根据本发明的实施例中胃癌极早期细胞典型标志(KLK10)所标记的阳性细胞在胃癌前病变、早期胃癌以及进展期胃癌中的比例分布。
图4用于说明根据本发明的一个典型实施例的胃癌极早期细胞标志代表性成分(KLK10、SLC11A2、KLK7和TIMP1)与胃癌术后复发风险的关联。
图5A是在根据本发明的实施例中13个胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立的正常肠组织(38例)、癌前病变(29例)及肠癌(27例)样本中的表达模式。
图5B是在根据本发明的实施例中13个胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立的正常食管(5例)、癌前病变(4例)及食管癌(3例)样本中的表达模式。
图5C是在根据本发明的实施例中胃癌极早期细胞标志中代表性分子(ECM1、KRT17、TIMP1以及MMP7)在独立的正常胰腺(8例)、上皮内瘤变(6例)及胰腺癌(12例)样本中的表达模式。
具体实施方式
图1是根据本发明的一个实施例的基于细胞标志的胃癌前病变及胃癌辅助早期诊断系统的原理图。
具体地，本发明首先提供了一种基于14个分子的检测试剂盒。该试剂盒包括检测KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B等分子表达水平的试剂，还包括空白液、稀释液、抗原修复液等。作为优选实施方案之一，所述试剂为免疫组化检测方法用抗体，具体如表1所示。所述试剂也可以为Western Blot或ELISA检测方法用抗体。
表1.待测分子所用免疫组化检测抗体列表及配制浓度比
Figure PCTCN2020083410-appb-000001
其次，本发明还提供了一种胃组织中上述13个分子表达水平的免疫组化检测方法。在该检测方法的一个实施例中，除试剂盒中包含的上述试剂外，用户可自行配制或购买以下试剂：
(1)蒸馏水或去离子水；
(2)3％H 2O 2；
(3)二甲苯；
(4)75％、85％、95％酒精及无水乙醇；
(5)10mM TBS溶液(pH7.2～7.4)：三羟基氨基甲烷1.21g，氯化钠7.6g，加蒸馏水800mL，浓盐酸调pH值至7.2～7.4，最后定容至1000mL；
(6)10mM pH6.0柠檬酸缓冲液：柠檬酸0.38g，柠檬酸三钠2.45g，加蒸馏水900mL，浓盐酸调pH值至6.0，最后定容至1000mL；
(7)苏木精溶液；
(8)中性树脂。
实施例1
为了验证本发明在辅助早期诊断胃癌前病变以及消化系统肿瘤、预测胃癌复发风险的作用，本发明人分别独立地对胃癌前病变、胃早癌、胃癌复发、其他消化系统肿瘤(肠癌、食管癌和胰腺癌)及其癌前病变典型病例进行了分析。
胃癌前病变极早期细胞的诊断
首先，发明人评估了胃癌前病变极早期细胞标志HES6在独立胃癌前病变样本不同类型细胞中的表达情况。结果如图2A所示，HES6在胃癌前病变细胞中特异高表达。
其次，发明人评估了胃癌前病变极早期细胞标志HES6与晚期标志MUC2在独立的胃癌前病变样本中的共表达情况。结果如图2B所示，HES6与MUC2呈互斥的表达模式，提示HES6所标记的细胞不在癌前病变晚期出现。
再次，发明人评估了胃癌前病变极早期细胞在非癌前病变阶段、轻度癌前病变阶段、重度癌前病变阶段以及早期癌变等四个阶段胃组织样本中的比例分布。结果如图2C所示，胃癌前病变极早期细胞在轻度癌前病变阶段中开始出现，并随着癌前病变程度的加深，其比例逐渐增加。
再次，依据本发明的免疫组化测试试剂盒(20)中胃癌前病变细胞标志(10)来获取HES6在胃癌前病变样本中的表达。该试剂盒利用免疫组化染色(IHC)来衡量标志物的表达水平。作为补充分析，发明人同时在样本中利用荧光共染了胃癌前病变经典标志MUC2以及细胞增值标志KI67。采用10％福尔马林缓冲液固定石蜡包埋手术样本，组织切片为4微米/张。
本实施例中的试剂盒，包括以下组分：
(1)试剂A：封闭液，为10％山羊血清；
(2)试剂B：已稀释的即用型抗HES6一抗；
(4)试剂C：抗山羊生物素化二抗；
(5)试剂D：链亲和素标记的HRP；
(6)试剂E：20倍浓缩DAB底物溶液；
(7)试剂F：20倍浓缩DAB底物缓冲溶液；
(8)试剂G：20倍浓缩DAB显色溶液。
根据本发明的一个具体实施例，上述试剂B原装进口分装即用型抗体，稀释倍数为1：200；试剂G原装进口分装即用型抗体，稀释倍数为1：400；试剂A、C、D、E、F、G为原装进口分装。
根据本发明的一个具体实施例，除试剂盒中包含的上述试剂，用户可自行配制或购买以下试剂：
(1)蒸馏水或去离子水；
(2)3％H2O2；
(3)二甲苯；
(4)75％、85％、95％酒精及无水乙醇；
(5)10mM TBS溶液(pH7.2～7.4)：三羟基氨基甲烷1.21g，氯化钠7.6g，加蒸馏水800mL，浓盐酸调pH值至7.2～7.4，最后定容至1000mL；
(6)10mM pH6.0柠檬酸缓冲液：柠檬酸0.38g，柠檬酸三钠2.45g，加蒸馏水900mL，浓盐酸调pH值至6.0，最后定容至1000mL；
(7)苏木精溶液；
(8)中性树脂。
利用上述试剂盒检测组织中标志物的表达：
(1)组织包埋：10％的中性福尔马林固定待测组织标本2h，用流水反复冲洗以去除固定液，将标本放置入75％酒精过夜，然后采用酒精梯度脱水，75％酒精1h，85％酒精1h，95％酒精1h，无水乙醇2次，每次1.5h，然后置于二甲苯中浸泡1.5h，60℃烘箱中浸蜡1h包埋，冷却后4℃保存备用；
(2)石蜡切片：修整蜡块，调整切片机(SLEE石蜡切片机CUT5062)将切片厚度设为3～4μm，连续切片，置于60℃温水漂浮展平，平铺于涂有阳离子树脂的载玻片上；
(3)烤片：将待做切片置于切片架上，于60℃恒温烤箱中至少烤1h；
(4)脱蜡：切片放入盛有二甲苯的容器中脱蜡3次(即二甲苯Ⅰ、Ⅱ、Ⅲ)，每次10min；
(5)水化：切片经下行酒精水化，无水乙醇5min，95％乙醇2次(每次2min)，85％乙醇2min；75％乙醇2min，蒸馏水冲洗1分钟；
(6)抗原修复：高压锅中加入柠檬酸缓冲液1000ml，将装有切片的切片架浸入缓冲液中，高温高压修复2分45秒，用TBS洗涤3次，每次2min；
(7)3％H2O2滴加在切片上，室温静置15min，TBS洗涤3次，每次2min；
(8)封闭：滴加试剂A于切片上，需完全覆盖组织切片，室温孵育10min后吸干液体，无需冲洗；
(9)加一抗：不同的切片分别滴加试剂B(抗Hes6一抗)，需完全覆盖组织切片，37℃湿盒孵育2hr或4℃过夜；
(10)洗涤：TBS-T洗涤(3×5min)；
(11)加二抗：试剂C(滴加生物素化二抗)，需完全覆盖组织切片，37℃湿盒中孵育30min；
(12)洗涤：TBS洗涤3次，每次5min；
(13)加HRP-SA：滴加试剂D(链亲和素标记的HRP)，需完全覆盖组织切片，37℃湿盒中孵育30min；
(14)洗涤：TBS洗涤3次，每次5min；
(15)制备DAB显色液：需现用现配，以染一张切片为例，取2.5ul试剂E加入到50ul蒸馏水中并混匀，再分别向上述液体中加入2.5ul试剂F及2.5ul试剂G，混匀；
(16)显色：滴加上述DAB显色液于切片上，需完全覆盖组织切片，显微镜下观察显色，蒸馏水冲洗终止显色；
(17)复染：苏木精复染3min，盐酸酒精分化；
(18)封片：75％酒精浸泡2min，85％酒精浸泡2min，95％酒精浸泡2min，无水乙醇浸泡2min，然后置于二甲苯中浸泡15min，更换二甲苯后再浸泡15min，中性树脂封片；
(19)结果判读：显微镜下观察染色的待测组织切片，阳性结果成棕黄色颗粒样染色，随机选择5个高倍视野(10*40)估计阳性细胞比例。
图2D展示了细胞标志HES6在正常胃组织、癌前病变胃组织中的表达情况。可以看出，在正常胃组织中HES6阳性细胞占比为0，而在胃癌前病变中其比例增加到10％。
最后，作为进一步的实施例材料，图2E和图2F分别展示了在独立的癌前病变样本中细胞标志HES6与经典的胃癌前病变标志MUC2以及增值细胞标志KI67之间共表达模式。。从图2E中可以看出，，HES6与胃癌前病变经典标志MUC2部分共表达；而从图2F中可以看出，HES6在组织中环绕在增值细胞标志KI67周围。因此可以认为HES6阳性细胞即为胃癌前病变早期阶段细胞。
胃癌极早期细胞在胃组织、血液样本中的诊断
首先，发明人评估了13个胃癌极早期细胞标志物分子(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立胃早癌样本中不同细胞类型的表达情况。结果如图3A所示，这些细胞标志在癌细胞中特异高表达。
其次，本发明人评估了13个胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立的胃炎(19例)、胃癌前病变(39例)及胃早癌(19例)样本中的表达情况。结果如图3B所示，这些细胞标志在胃炎中表达量显著低于异型增生阶段(胃癌前病变)和胃早癌阶段。将上述分子的均值表达作为一个分子标记(signature),发现其在胃炎-低级别异型增生-高级别异型增生-早期胃癌的转化中呈逐渐递增的变化趋势，提示上述分子在胃组织中的高表达与胃癌的发生具有密切关联，可作为评估胃癌发生风险的指标。本验证中涉及的样本数据来自于公共数据(GSE55696)。
再次，本发明人评估了13个胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立的胃炎、胃癌患者血液样本中的表达情况。结果如图3C所示，相比于正常对照样本，这些细胞标志物在胃癌患者血清中的表达水平显著升高。将上述分子的均值表达水平作为分子标记(signature),发现其数值相比正常样本，在癌症样本中也显著升高。提示上述分子在血清中的高表达也与胃癌的发生具有密切关联，也可作为评估胃癌发生风险的指标。本验证涉及的样本数据来自于公共数据(GSE64916)。
实施例2
本发明人依据本发明的免疫组化测试试剂盒(20)中胃癌前病变细胞标志(11)来获取早癌细胞标志中五个代表性成分(KLK10、KRT17、TIMP1、MAP17和KLK6)在正常胃组织、胃癌前病变、早期癌变以及进展期癌变中的表达。该试剂盒利用免疫组化染色(IHC)来衡量标志物的表达水平。采用10％福尔马林缓冲液固定石蜡包埋手术样本，组织切片为4微米/张。
本实施例中的试剂盒，包括以下组分：
(1)试剂A：封闭液，为10％山羊血清；
(2)试剂B：已稀释的即用型抗Klk10一抗(也可以是Table 1中KRT17,TIMP1,MAP17以及KLK6抗体的任意一只)；
(4)试剂C：抗山羊生物素化二抗；
(5)试剂D：链亲和素标记的HRP；
(6)试剂E：20倍浓缩DAB底物溶液；
(7)试剂F：20倍浓缩DAB底物缓冲溶液；
(8)试剂G：20倍浓缩DAB显色溶液。
根据本发明的一个具体实施例，上述试剂B原装进口分装即用型抗体，稀释倍数为1：200；试剂G原装进口分装即用型抗体，稀释倍数为1：400；试剂A、C、D、E、F、G为原装进口分装。
除试剂盒中包含的上述试剂，本发明人还自行配制了以下试剂(这些试剂也可以在市面上购买到)：
(1)蒸馏水或去离子水；
(2)3％H2O2；
(3)二甲苯；
(4)75％、85％、95％酒精及无水乙醇；
(5)10mM TBS溶液(pH7.2～7.4)：三羟基氨基甲烷1.21g，氯化钠7.6g，加蒸馏水800mL，浓盐酸调pH值至7.2～7.4，最后定容至1000mL；
(6)10mM pH6.0柠檬酸缓冲液：柠檬酸0.38g，柠檬酸三钠2.45g，加蒸馏水900mL，浓盐酸调pH值至6.0，最后定容至1000mL；
(7)苏木精溶液；
(8)中性树脂。
利用上述试剂盒检测组织中标志物的表达：
(1)组织包埋：10％的中性福尔马林固定待测组织标本2h，用流水反复冲洗以去除固定液，将标本放置入75％酒精过夜，然后采用酒精梯度脱水，75％酒精1h，85％酒精1h，95％酒精1h，无水乙醇2次，每次1.5h，然后置于二甲苯中浸泡1.5h，60℃烘箱中浸蜡1h包埋，冷却后4℃保存备用；
(2)石蜡切片：修整蜡块，调整切片机(SLEE石蜡切片机CUT5062)将切片厚度设为3～4μm，连续切片，置于60℃温水漂浮展平，平铺于涂有阳离子树脂的载玻片上；
(3)烤片：将待做切片置于切片架上，于60℃恒温烤箱中至少烤1h；
(4)脱蜡：切片放入盛有二甲苯的容器中脱蜡3次(即二甲苯Ⅰ、Ⅱ、Ⅲ)，每次10min；
(5)水化：切片经下行酒精水化，无水乙醇5min，95％乙醇2次(每次2min)，85％乙醇2min；75％乙醇2min，蒸馏水冲洗1分钟；
(6)抗原修复：高压锅中加入柠檬酸缓冲液1000ml，将装有切片的切片架浸入缓冲液中，高温高压修复2分45秒，用TBS洗涤3次，每次2min；
(7)3％H2O2滴加在切片上，室温静置15min，TBS洗涤3次，每次2min；
(8)封闭：滴加试剂A于切片上，需完全覆盖组织切片，室温孵育10min后吸干液体，无需冲洗；
(9)加一抗：不同的切片分别滴加试剂B，需完全覆盖组织切片，37℃湿盒孵育2hr或4℃过夜；
(10)洗涤：TBS-T洗涤(3×5min)；
(11)加二抗：试剂C(滴加生物素化二抗)，需完全覆盖组织切片，37℃湿盒中孵育30min；
(12)洗涤：TBS洗涤3次，每次5min；
(13)加HRP-SA：滴加试剂D(链亲和素标记的HRP)，需完全覆盖组织切片，37℃湿盒中孵育30min；
(14)洗涤：TBS洗涤3次，每次5min；
(15)制备DAB显色液：需现用现配，以染一张切片为例，取2.5ul试剂E加入到50ul蒸馏水中并混匀，再分别向上述液体中加入2.5ul试剂F及2.5ul试剂G，混匀；
(16)显色：滴加上述DAB显色液于切片上，需完全覆盖组织切片，显微镜下观察显色，蒸馏水冲洗终止显色；
(17)复染：苏木精复染3min，盐酸酒精分化；
(18)封片：75％酒精浸泡2min，85％酒精浸泡2min，95％酒精浸泡2min，无水乙醇浸泡2min，然后置于二甲苯中浸泡15min，更换二甲苯后再浸泡15min，中性树脂封片；
(19)结果判读：显微镜下观察染色的待测组织切片，阳性结果成棕黄色颗粒样染色，随机选择5个高倍视野(10*40)估计阳性细胞比例。
如图3D展示了胃癌极早期细胞中五个代表性标志分子(KLK10、KRT17、TIMP1、MAP17和KLK6)在正常胃组织、异型增生(胃癌前病变)以及早期胃癌的表达情况。其中，在胃癌前病变中依据上述代表性标志分子所识别出的胃癌极早期细胞用圆圈圈了出来。可以看出，胃癌极早期细胞开始出现于异型增生阶段，并在早癌中继续存在。
进而，利用本发明的早期诊断系统中胃癌前病变及早癌细胞计数装置(32)，计算了在各个阶段中胃癌极早期细胞的含量。如图3E展示了胃癌极早期细胞典型标志(KLK10)所标记的阳性细胞在胃癌前病变、早期胃癌以及进展期胃癌中的比例分布。可以看出KLK10阳性细胞未出现在胃癌前病变阶段样本，而在早期胃癌样本中其比例为5％，在进展期样本中其比例增加到35％。
胃癌极早期细胞标志物分子在评估胃癌术后复发风险中的应用
发明人评估了胃癌极早期细胞标志中的代表性成分(KLK10、SLC11A2、KLK7和TIMP1)与胃癌术后复发风险的关联。这些数据来源于431例记录术后复发时间信息的胃癌患者。结果如图4所示，上述细胞标志与胃癌术后复发生存风险呈显著相关(p<0.05)。将胃癌极早期细胞标志分子在样本中的均值表达量作为一个分子标记(signature),发现其也与胃癌的术后复发呈显著相关(p<0.01)，提示了该胃癌极早期细胞标志可作为评估胃癌复发风险的标志物。本验证中涉及的样本数据来自于公共数据(TCGA)。
胃癌极早期细胞标志物分子在评估胰腺癌、肠癌以及食管癌风险中的应用
本发明人还验证了胃癌极早期细胞标志分子(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在评估消化系统其他器官(胰腺、食管以及肠)从癌前病变向癌症转化风险的应用。
1.胃癌极早期细胞标志物分子在评估肠癌风险中的应用。本发明人评估了13个胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1 和KRT6B)在独立的正常肠组织(38例)、癌前病变(29例)及肠癌(27例)样本中的表达情况。结果如图5A所示，这些胃癌极早期细胞标志在正常肠组织中的表达量显著低于异型增生阶段(肠癌前病变)和肠癌阶段(p<0.05)。将上述分子在样本中表达量的均值作为分子标记(signature),发现其在正常肠-低级别异型增生-高级别异型增生-肠癌的转化过程中呈逐渐递增的变化趋势，提示上述分子在肠组织中的高表达与肠癌的发生具有密切关联，可作为评估肠癌发生风险的指标。本验证中涉及的样本数据来自于公共数据(GSE37364)。
2.胃癌极早期细胞标志物分子在评估食管癌风险中的应用。
其次，本发明人评估了13个胃癌极早期细胞标志(KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B)在独立的正常食管(5例)、癌前病变(4例)及食管癌(3例)样本中的表达情况。结果如图5B所示，这些胃癌极早期细胞标志在正常食管中的表达量低于异型增生阶段(癌前病变)和食管癌阶段。提示上述分子在食管中的高表达与食管癌的发生具有密切关联，可作为评估食管癌发生风险的指标。本验证中涉及的样本数据来自于公共数据(GSE19529)。
3.胃癌极早期细胞标志中代表性分子在评估胰腺癌风险中的应用。
本发明人评估了胃癌极早期细胞标志中代表性分子(ECM1、KRT17、TIMP1和MMP7)在独立的正常胰腺(8例)、上皮内瘤变(6例)及胰腺癌(12例)样本中的表达情况。结果如图5C所示，这些胃癌极早期细胞代表性标志物分子在正常胰腺组织中的表达量显著低于在上皮内瘤变阶段(癌前病变)和胰腺癌中。将上述分子的均值表达作为一个分子标志(signature),发现其在正常胰腺-胰腺上皮内瘤变-胰腺癌的转化过程中呈逐渐递增的变化趋势，提示上述分子在胰腺组织中的高表达与胰腺癌的发生具有密切关联，可作为评估胰腺癌发生风险的指标。本验证中涉及的样本数据来自于公共数据(GSE43288)。
参考文献:
1.de Vries AC,van Grieken NCT,Looman CWN,Casparie MK,de Vries E,Meijer GA,Kuipers EJ.(2008)Gastric cancer risk in patients with premalignant gastric lesions:A nationwide cohort study in the Netherlands.GASTROENTEROLOGY,134,945-952.
2.ITOH H,OOHATA Y,NAKAMURA K,NAGATA T,MIBU R,NAKAYAMA F.(1989)COMPLETE 10-YEAR POSTGASTRECTOMY FOLLOW-UP OF EARLY GASTRIC-CANCER.AMERICAN JOURNAL OF SURGERY,158,14-16.

Claims (20)

  1. 检测标志物的表达水平的试剂在制备确定胃癌极早期细胞含量从而确定极早期胃癌的产品中应用，其中所述标志物包括从由KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B组成的组中选出的至少一种。
  2. 根据权利要求1所述的应用，其特征在于：
    所述试剂利用免疫组化染色或酶联免疫吸附来衡量所述标志物的表达水平。
  3. 根据权利要求1或2所述的应用，其特征在于：
    所述产品是从芯片、制剂、试剂盒中选出的一种。
  4. 一种用于确定胃癌极早期细胞含量的产品，其特征在于，所述产品包括检测样本中标志物表达水平的试剂，其中所述标志物包括从由KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B组成的组中选出的至少一种。
  5. 根据权利要求4所述的产品，其特征在于所述产品是从芯片、制剂、试剂盒中选出的一种。
  6. 检测标志物的表达水平的试剂在制备用于消化系统肿瘤的辅助极早期诊断的产品中应用，其中所述标志物包括从由KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B组成的组中选出的至少一种。
  7. 根据权利要求6所述的应用，其特征在于：
    所述试剂利用免疫组化染色来衡量所述标志物的表达水平。
  8. 根据权利要求6或7所述的应用，其特征在于：
    所述产品是从芯片、制剂、试剂盒中选出的一种。
  9. 一种用于消化系统肿瘤的辅助极早期诊断的产品，其特征在于：
    所述产品包括检测样本中标志物表达水平的试剂，其中所述标志物 包括从由KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B组成的组中选出的至少一种。
  10. 根据权利要求9所述的产品，其特征在于所述试剂利用免疫组化染色来衡量所述标志物的表达水平。
  11. 根据权利要求9或10所述的应用，其特征在于：
    所述产品是从芯片、制剂、试剂盒中选出的一种。
  12. 检测HES6表达水平的试剂在制备确定胃癌前病变水平的产品中应用。
  13. 根据权利要求12所述的应用，其特征在于：
    所述试剂利用免疫组化染色或酶联免疫吸附来衡量HES6的表达水平。
  14. 根据权利要求12或13所述的应用，其特征在于：
    所述产品是从芯片、制剂、试剂盒中选出的一种。
  15. 一种用于确定胃癌前病变水平的产品，其特征在于所述产品包括检测样本中HES6表达水平的试剂。
  16. 根据权利要求15所述的产品，其特征在于：
    所述试剂利用免疫组化染色或酶联免疫吸附来衡量HES6的表达水平。
  17. 根据权利要求15或16所述的产品，其特征在于所述产品是从芯片、制剂、试剂盒中选出的一种。
  18. 检测标志物的表达水平的试剂在制备用于辅助评估胃癌复发风险的产品中应用，其中所述标志物包括从由KLK10、SLC11A2、SULT2B1、KLK7、ECM1、LMTK3、KLK6、KIAA1199、KRT17、MMP7、TIMP1、PDZK1IP1和KRT6B组成的组中选出的至少一种。
  19. 根据权利要求18所述的应用，其特征在于：
    所述试剂利用免疫组化染色来衡量所述标志物的表达水平。
  20. 根据权利要求18或19所述的应用，其特征在于：
    所述产品是从芯片、制剂、试剂盒中选出的一种。
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Publication number Priority date Publication date Assignee Title
CN111781356A (zh) * 2019-04-04 2020-10-16 清华大学 一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008118915A (ja) * 2006-11-10 2008-05-29 Kazuto Nishio 胃癌高発現遺伝子特定による胃癌診断および創薬への利用
WO2010100899A1 (ja) * 2009-03-02 2010-09-10 株式会社ジーンサイエンス 血液試料に含まれる単核球細胞を用いた癌関連遺伝子の発現解析による癌の遺伝子検査方法
CN105572354A (zh) * 2014-10-17 2016-05-11 广州瑞博奥生物科技有限公司 一种检测早期胃癌的抗体芯片试剂盒

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050244843A1 (en) * 2001-11-16 2005-11-03 Wen-Tien Chen Blood test prototypes and methods for the detection of circulating tumor and endothelial cells
CN100388925C (zh) * 2005-06-02 2008-05-21 成都中医药大学 一种益气健脾的药物组合物及其制备方法和用途
CA2724638C (en) * 2008-05-27 2020-02-18 Dako Denmark A/S Hybridization compositions and methods comprising a polar aprotic solvent
FR2934278B1 (fr) * 2008-07-22 2013-12-06 Oreal Signature moleculaire representative de dysfonctionnements de l'homeostasie epidermique
WO2010028658A1 (en) * 2008-09-15 2010-03-18 Herlev Hospital, Region Hovedstaden Ykl-40 as a marker for gastrointestinal cancers
US9310370B2 (en) * 2009-05-15 2016-04-12 Pacific Edge Limited Markers for detection of gastric cancer
US20120269721A1 (en) * 2009-10-12 2012-10-25 The Regents Of The University Of California Targeted nanoclusters and methods of their use
CN103237901B (zh) * 2010-03-01 2016-08-03 卡里斯生命科学瑞士控股有限责任公司 用于治疗诊断的生物标志物
KR101115441B1 (ko) * 2011-03-17 2012-03-15 한국생명공학연구원 대장암 진단용 조성물 및 그 용도
CN106048050B (zh) * 2011-03-25 2020-01-07 生物梅里埃公司 体外确定个体患结直肠癌概率的方法及试剂盒
EP2680003A1 (en) * 2012-06-28 2014-01-01 Fundació Institut d'Investigació Biomèdica de Bellvitge Serum biomarker for diagnosing colorectal cancer
CN105393117A (zh) * 2013-01-31 2016-03-09 凯普里昂蛋白质组学公司 2型糖尿病生物标志物及其用途
CR20200523A (es) * 2015-06-19 2020-12-23 Immatics Biotechnologies Gmbh NUEVOS PÉPTIDOS Y NUEVAS COMBINACIONES DE PÉPTIDOS PARA EL USO EN LA INMUNOTERAPIA Y MÉTODOS PARA CREAR SOPORTES PARA EL USO CONTRA EL CÁNCER DE PÁNCREAS Y OTROS TIPOS DE CÁNCER (Divisional 2018-0040)
KR101847815B1 (ko) * 2015-12-17 2018-04-12 바이오코아 주식회사 삼중음성유방암의 아형 분류 방법
CN106996975B (zh) * 2016-01-22 2019-05-31 益善生物技术股份有限公司 循环肿瘤细胞蛋白分型试剂盒
SE540173C2 (en) * 2016-04-20 2018-04-24 Hiloprobe Ab Marker genes for colorectal cancer classification, method for judging lymph node metastasis for prognosis of colorectal cancer and kit therefor
CN107475356A (zh) * 2016-06-08 2017-12-15 益善生物技术股份有限公司 骨肉瘤循环肿瘤细胞鉴定试剂盒
CN105986034A (zh) * 2016-06-15 2016-10-05 南京卡迪睿伯生物技术有限公司 一组胃癌基因的应用
CN106367527A (zh) * 2016-11-17 2017-02-01 苏州大学附属第二医院 直肠癌放化疗疗效相关靶基因的鉴定
CN106526203B (zh) * 2016-11-22 2018-10-09 汉氏联合（天津）干细胞研究院有限公司 一种用于左半结肠癌检测的试剂盒
CN109142738A (zh) * 2017-06-16 2019-01-04 中国科学院上海巴斯德研究所 Ecm1作为血清学检测肝纤维化的标志物及其应用
CN107460244A (zh) * 2017-08-30 2017-12-12 南通大学附属医院 一种用于检测卵巢癌的试剂盒
CN109457032B (zh) * 2018-12-20 2022-04-12 北京优迅医学检验实验室有限公司 甲状腺癌分子诊断试剂盒
CN111781356A (zh) * 2019-04-04 2020-10-16 清华大学 一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008118915A (ja) * 2006-11-10 2008-05-29 Kazuto Nishio 胃癌高発現遺伝子特定による胃癌診断および創薬への利用
WO2010100899A1 (ja) * 2009-03-02 2010-09-10 株式会社ジーンサイエンス 血液試料に含まれる単核球細胞を用いた癌関連遺伝子の発現解析による癌の遺伝子検査方法
CN105572354A (zh) * 2014-10-17 2016-05-11 广州瑞博奥生物科技有限公司 一种检测早期胃癌的抗体芯片试剂盒

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ANDREA GRIN MD ET AL.: "Evaluation of human tissue kallikrein-related peptidases 6 and 10 expression in early gastroesophageal adenocarcinoma", HUMAN PATHOLOGY, vol. 46, no. 4, 30 April 2015 (2015-04-30), XP029212082, ISSN: 0046-8177, DOI: 20200601161837 *
CARLA OLIVEIRA ET AL.: "Familial gastric cancer: genetic susceptibility, pathology, and implications for management", THE LANCET ONCOLOGY, vol. 16, no. 2, 28 February 2015 (2015-02-28), XP055740436, ISSN: 1470-2045, DOI: 20200601164019X *
GANGSHI WANG ET AL.: "Comparison of Global Gene Expression of Gastric Cardia and Noncardia Cancers from a High-Risk Population in China", PLOS ONE, vol. 8, no. 5, 22 May 2013 (2013-05-22), XP055740439, ISSN: 1932-6203, DOI: 20200601172400X *
GUI-FENG ZHUANG ET AL.: "Expression level and clinical significance of KLK7 and E-CAD in gastric cancer", JOURNAL OF HAINAN MEDICAL UNIVERSITY, vol. 23, no. 6, 24 March 2017 (2017-03-24), XP055740443, DOI: 20200601161231X *
MIHAELA CHIVU-ECONOMESCU ET AL.: "Knockdown of KRT17 by siRNA induces antitumoral effects on gastric cancer cells", GASTRIC CANCER, vol. 20, no. 6, 15 March 2017 (2017-03-15), XP036457290, ISSN: 1436-3305, DOI: 20200601163004X *
NORITAKA OYAMA ET AL.: "The Extracellular Matrix Protein 1 (ECM1) in Skin Biology: An Update for the Pleiotropic Action", THE OPEN DERMATOLOGY JOURNAL, vol. 7, 28 August 2013 (2013-08-28), XP055705347, ISSN: 1874-3722, DOI: 20200602094513X *
PENG ZHANG ET AL.: "A comprehensive analysis of single-cell transcriptome network underlying gastric premalignant lesions and early gastric cancer", BIORXIV, 5 November 2018 (2018-11-05), XP055740432, DOI: 20200601194051X *
WEN CHEN ET AL.: "Overexpression of SULT2B1b Promotes Angiogenesis in Human Gastric Cancer", CELLULAR PHYSIOLOGY AND BIOCHEMISTRY, vol. 38, no. 3, 4 March 2016 (2016-03-04), XP055740442, ISSN: 1421-9778, DOI: 20200601185633X *
ZHU, YABI ET AL.: "Non-official translation: Importance of KIAA1199 Gene Expression in Human Gastric Cancer", INTERNATIONAL JOURNAL OF DIGESTIVE DISEASES, vol. 36, no. 5, 25 October 2016 (2016-10-25), ISSN: 1673-534X, DOI: 20200601160943X *

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