Use of set of biomarkers in preparation of colorectal cancer diagnostic reagents

A biomarker and colorectal technology, which is applied in the field of prediction or diagnosis of colorectal cancer, can solve the problem of undetermined biomarker colorectal cancer development stage information and guide treatment, etc., to achieve the effect of convenient individual examination and easy use

Inactive Publication Date: 2017-03-01
POOCHON SCI LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no biomarkers have been identified that can provide information on the stage of colorectal cancer development and guide treatment

Method used

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  • Use of set of biomarkers in preparation of colorectal cancer diagnostic reagents
  • Use of set of biomarkers in preparation of colorectal cancer diagnostic reagents
  • Use of set of biomarkers in preparation of colorectal cancer diagnostic reagents

Examples

Experimental program
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example 1

[0095] Example 1: Cancer and paracancerous tissue samples. All samples were collected from clinical patients in accordance with the regulations of the Medical Ethics and Human Clinical Trials Committee of the Affiliated Hospital of Nantong University. After the patient's operation, the cancer and paracancerous tissue samples were placed in different tubes and kept on ice during the subsequent sample transfer process; the samples were finally placed at -80°C for subsequent processing. Paracancerous tissue samples were obtained from at least 5 cm away from the cancerous tissue lesion. A total of 22 pairs of cancer and paracancerous tissue samples were obtained from 22 patients, of which 10 had lymph node metastasis and 12 had no metastasis (Table 1). All patients had been identified as colon or colorectal cancer by pathologists using histological means. Each patient had a detailed pathological record, and those who had various other malignant or infectious diseases, or underwe...

example 2

[0096] Example 2: Protein extraction and separation and in-gel trypsin hydrolysis. Total protein was extracted from fresh frozen tissue using the following method. Cut 0.05 to 0.1 g of frozen tissue into small pieces of 1 mm square with a clean blade, and then transfer to a 1.5 ml centrifuge tube; add 0.4 ml of lysate (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1mM Na2EDTA, 1mM EGTA, 1% Triton X-100, Protease inhibitor cocktail pill); Homogenize with Tang’s homogenizer; add 50ul 10% SDS and 50ul1M DTT to the tube after homogenization, heat at 95°C for 10min; then Sonication is performed to break up the DNA in the sample. The sonicated mixture was centrifuged at 15,000×g for 10 min; the supernatant was transferred to a new tube and stored at -80°C for subsequent analysis. The protein concentration of the supernatant was measured using BCA from Pierce / Thermo Scientific TM Kit OK.

[0097] Gradient gel electrophoresis separation (133ug protein, NuPAGE4-12% Bis-Tris Gel, Life Techno...

example 3

[0098] Example 3: LC / MS / MS analysis. Sixteen small tryptic peptides from one sample were sequentially analyzed by mass spectrometry; the equipment we used was a ThermoScientific Q-Exactive hybrid Quadrupole-Orbitrap mass spectrometer coupled with a Thermo Dionex UltiMate 3000RSLCnano system. The small peptide samples obtained by trypsin hydrolysis were added to a peptide collection tube at a rate of 5ul / min, and the collected peptides were collected at a rate of 0.3ul / min using a 3-36% linear acetonitrile (dissolved in 0.1% formic acid) gradient. Washed into a 25 cm long C18 reverse column (New Obietive, Woburn, MA) over 110 minutes. The small peptides eluted from the reverse column were ionized and sprayed into the mass spectrometer; we used Thermo's Nanospray Flex Ion Source ES071 for ionization under the following conditions: voltage, 1.6kV, temperature, 250°C. The QExactive mass spectrometer operates in a data-dependent mode and can automatically switch between full-scan ...

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Abstract

The invention discloses the use of a set of biomarkers in the preparation of colorectal cancer diagnostic reagents, and further proposes a method of using biomarkers for distinguishing between normal intestinal cells and colorectal cancer cells. The method comprises: a) separating a test sample from a biopsy specimen of a subject; b) separating biological samples containing normal colorectal tissue cells or tissues from the biopsy specimens or the subject or family members thereof; c) analyzing the content of biomarker proteins of the above samples in a) and b); and d) comparing the content of biomarker proteins in normal and colorectal cancer cells or tissues. The invention also discloses a kit for detecting the level of biomarkers in a specimen. The method of the invention can be combined with other diagnostic methods to detect colorectal adenoma / colorectal cancer and improve the overall sensitivity and specificity, and can be also applied to early diagnosis and monitoring.

Description

[0001] cross reference [0002] This patent application has priority U.S. Patent Serial No. 62 / 105642, filed January 20, 2015. technical field [0003] The present invention mainly relates to methods and kits for predicting or diagnosing colorectal cancer by detecting biomarkers. [0004] Background of the invention [0005] Colorectal cancer (also called colon cancer, rectal cancer or bowel cancer) is the development of cancer in the rectum or colon (part of the large intestine). Produced by abnormal growth of cells that have the ability to invade and spread to other parts of the body. Signs and symptoms may include blood in the stool, changes in stool, weight loss, and frequent fatigue. [0006] Most colorectal cancers are due to lifestyle and aging, and to a lesser extent genetic disorders. Risk factors include diet, obesity, smoking, and lack of physical activity. Dietary factors that increase risk include red meat, processed meat, and alcohol. Another risk-increasin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/68
CPCG01N33/57419G01N33/57446G01N33/5748G01N33/57484G01N33/6803G01N33/6848G01N2333/4703G01N2333/4725G01N2333/705G01N2333/71G01N2333/8121G01N2333/912G01N2333/948G01N2570/00
Inventor 郝建江朱建伟
Owner POOCHON SCI LLC
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