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Neutrophil-activating peptide 2 (72) taken as primary liver cancer markers and application thereof

A primary liver cancer and active peptide technology, applied in the biological field, can solve the problems of delaying the diagnosis of liver cancer, not reaching the diagnosis of liver cancer, missing the opportunity of radical cure, etc., and achieving the effect of improving the level of diagnosis and treatment

Inactive Publication Date: 2010-02-10
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, 30%-40% of liver cancer patients are negative for AFP, which often leads to two situations in early clinical diagnosis: one is that the APP concentration is higher than normal (20 μg / L) but does not reach the standard for diagnosing liver cancer (400 μg / L). If no space-occupying lesions in the liver are found in the imaging diagnosis, it may be regarded as active liver disease. Long-term follow-up in outpatient clinics will miss the opportunity for radical treatment
The second is AFP-negative cases. Real-time ultrasound shows suspicious space-occupying lesions and cannot be identified. Further tests such as CT, magnetic resonance imaging, and radionuclide scanning are often performed on such patients. There are often different opinions and conclusions, thus delaying the diagnosis of liver cancer diagnosis
For this reason, in recent years, the joint detection of multiple markers has been advocated to improve the sensitivity and specificity of liver cancer detection. The application of joint detection also has certain limitations

Method used

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  • Neutrophil-activating peptide 2 (72) taken as primary liver cancer markers and application thereof
  • Neutrophil-activating peptide 2 (72) taken as primary liver cancer markers and application thereof
  • Neutrophil-activating peptide 2 (72) taken as primary liver cancer markers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Identification of serum markers related to liver cancer

[0020] The sample used for separation is 1ml serum of liver cancer patients. Use Blue 3GA and Protein A to remove albumin and IgG in the serum, prepare Tricine-SDS-PAGE separating gel, gap gel and stacking gel, mix the serum sample without albumin and IgG with the sample buffer, boil for 5 minutes, Load the sample, use the vertical small electrophoresis tank for electrophoresis, 40V for about 1.5h, when the sample enters the separation gel, the voltage rises to 60V for about 1.5h, after the electrophoresis, Coomassie staining for 1h, wash the gel surface with double distilled water until the background is clear , cut off the target protein band of the gel with a 1.5mm gel cutting pen, place it in a centrifuge tube, perform in-gel enzymolysis after decolorization, and separate the enzymatically hydrolyzed sample with a C18 reversed-phase column, and directly perform high-performance liquid phase For tandem mass s...

Embodiment 2

[0024] Differential expression of neutrophil active peptide 2 (72) in liver cancer and control samples

[0025] Take out the serum sample from the -80°C refrigerator, put it on the ice box to dissolve, centrifuge at 10000rpm, 4°C for 5min, take 10μl of serum, add 2 times the volume of U9 buffer (containing DTT) to dilute. When diluting, be careful not to repeatedly blow and suck with the pipette to avoid a large number of air bubbles. Tap the bottom of the sample tube or a shaker with your fingers to mix the sample thoroughly. Take the above samples in an ice bath at 300rpm and shake for 30min or flick and mix with your fingers every 5 minutes. Add 30μl of the above-mentioned denatured samples to 360μl of 50mM NaAC, pH 4.0 binding buffer, so that the total dilution factor of the serum reaches about 40 times. . Mix as soon as possible to avoid protein denaturation and precipitation and air bubbles.

[0026]Carefully take out the WCX2 chip, put the chip into a bioprocessor (Bi...

Embodiment 3

[0030] Expression of Neutrophil Active Peptide 2(72) in Hepatocellular Carcinoma

[0031] 30 cases of liver cancer tissue and corresponding paracancerous tissue samples were all inpatients in the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Guangxi Medical University from January 2007 to January 2008. All patients underwent liver resection, did not receive chemotherapy, radiotherapy and / or other anti-tumor therapy before operation, and were confirmed to be primary hepatocellular carcinoma by postoperative pathological examination. Specimens were fixed in neutral formalin and embedded in paraffin.

[0032] Rabbit anti-human NAP-2 polyclonal antibody (Canada Immunechem Pharmaceuticals, INC), goat anti-rabbit HRP-labeled secondary antibody (Guangxi Nanning Zhongjia Bioimmune Technology Co., Ltd.), DAB kit (Fuzhou Maixin Company), human liver cancer cell line SMCC -7721 and normal human liver cell line HL-7702 were provided by the Tumor Laboratory of Me...

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Abstract

The invention discloses Neutrophil-activating peptide 2 (72) taken as primary liver cancer markers and application thereof. The Neutrophil-activating peptide 2 (72) consists in human blood serum and the tissues of liver cancer, is one of 11 peptide chains which are formed by cutting Platelet basic protein by means of enzyme with pancreatic enzyme outside cells, and has 7789 Dalton of molecular weight; and in the blood serum and the tissues of primary liver cancer patients, the expression of the Neutrophil-activating peptide 2 (72) obviously increases. The content of the Neutrophil-activating peptide 2 (72) in the blood serum of the liver cancer patients can be detected by a surface enhancing laser desorption ionization-time-of-flight mass spectrometer, the expression of the Neutrophil-activating peptide 2 (72) in the tissues of the liver cancer can be detected by means of immune chemistry, and the expression level of NAP-2 (72) mRNA in the SMMC-7721 cell strain of the liver cancer by means of real-time fluorescent quantification PCR, therefore, the detection results are taken as auxiliary index for detecting the liver cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of a polypeptide marker, in particular to a neutrophil active peptide 2 (72) as a primary liver cancer serum marker and its application. Background technique [0002] Primary hepatic cancer (hereafter referred to as liver cancer) is one of the most prevalent malignant tumors in the world. The International Cancer Research Center (IARC) estimated that in 2000, there were 564,000 cases of liver cancer worldwide, and 549,000 deaths from liver cancer. However, due to the large number of patients with hepatitis and liver cirrhosis in China, the incidence and mortality of liver cancer in my country are higher than the world average. Compared with the 1970s, the mortality rate of liver cancer in China has increased significantly. Compared with other countries in the world, the mortality rate of liver cancer in my country ranks first among all countries. Primary liver cancer is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435G01N27/62G01N33/574C12Q1/68G01N27/626
Inventor 何敏张志勇覃健蒋作祎胡天桥韦霄
Owner GUANGXI MEDICAL UNIVERSITY
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