Application of peripheral blood exosome miRNA combined marker to preparation of kit for detecting HBV positive liver cirrhosis early liver cancer

A technology for detecting early and early stage liver cancer, applied in the fields of molecular biology and medical diagnosis, can solve the problems of lack of pertinence and complex pathogenesis of liver cancer, and achieve the effect of excellent sensitivity and specificity, convincing and accurate results.

Active Publication Date: 2021-12-07
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pathogenesis of primary liver cancer is more complex, and different types of patients may have different biomarkers. Existing panels are generally aimed at all types of liver cancer patients and lack specificity.

Method used

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  • Application of peripheral blood exosome miRNA combined marker to preparation of kit for detecting HBV positive liver cirrhosis early liver cancer
  • Application of peripheral blood exosome miRNA combined marker to preparation of kit for detecting HBV positive liver cirrhosis early liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Subject and Sample Extraction

[0022] The samples were provided by Singapore General Hospital (approved by Singapore SingHealth CIRB B, approval number CIRB Ref: 2007 / 447 / B). Among them, 31 were LC+ / HBV+ HCC patients, and 59 were LC+ / HBV+ non-HCC patients.

Embodiment 2

[0023] Example 2 Extraction of exosomes

[0024] Collect 2ml of peripheral blood from the subject with a disposable syringe, quickly transfer it to an EDTA anticoagulant tube, and mix well. Separate the plasma within 2 hours of collection. The specific steps are: 1) transfer the whole blood to a 2ml centrifuge tube, and centrifuge at 500g for 10min at room temperature; 2) take the upper plasma, and centrifuge at 2000g for 10min at 4°C; 3) take the upper plasma, 4°C, Centrifuge at 16,000 g for 10 min; 4) The separated plasma is directly used for exosome extraction or frozen in a -80°C refrigerator.

[0025] Exosomes were extracted according to the instructions of the exosome extraction kit Exo-Quick exosome precipitation solution (EXOQ5SA-1, SBI, United States). 1) Add the plasma to a centrifuge tube, and add Exo-Quick at a ratio of 63 μl for every 250 μl of plasma; 2) Centrifuge at 1500 g at 4°C for 30 minutes, discard the supernatant; 3) Centrifuge at 1500 g at 4°C for 5 min...

Embodiment 3

[0026] Example 3 Extraction of exosome miRNA

[0027]miRNA was extracted according to the operating instructions of the miRNeasy Serum / PlasmaKit kit (217184, QIAGEN, Germany). The specific steps are: 1) add 5 times the volume of QIAzol Lysis Reagent to the plasma separated in Example 2, vortex or pipette to mix; 2) incubate at room temperature for 5 minutes; 3) add an equal volume of chloroform, and shake for 15 seconds; 4) Incubate at room temperature for 2-3 minutes; 5) Centrifuge at 12,000 g for 15 minutes at 4°C; 6) Transfer the upper layer to a new centrifuge tube, add 1.5 times the volume of absolute ethanol, and mix by pipetting; 7) Take 700 μl of the sample to the RNeasy MinElute spin column ( Place in a 2ml collection tube), cover the lid, centrifuge at room temperature>8000g for 15s, discard; 8) repeat step 7 once; 9) add 700μl RWTbuf to RNeasy MinElute spincolumn, centrifuge at room temperature>8000g for 15s, discard; 10) add Add 500μl RPEbuf to RNeasy MinElute spi...

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Abstract

The invention discloses an application of a peripheral blood exosome miRNA combined marker to preparation of a kit for detecting early liver cancer. The miRNA combined marker is selected from hsa-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p, hsa-miR-150-5p, hsa-miR-134-5p, hsa-miR-4433b-3p, and hsa-miR-222-3p which are derived from a plasma exosome. Early liver cancer is primary HCC of HBV positive (HBV +) and liver cirrhosis (LC +). According to the invention, the primary HCC of HBV positive (HBV +) and liver cirrhosis (LC +) is detected, so that the pertinence is strong, and the detection result is persuasive.

Description

technical field [0001] The invention relates to the fields of molecular biology and medical diagnosis, in particular to the application of a peripheral blood exosome miRNA combined marker in the preparation of a kit for detecting early liver cancer. Background technique [0002] Primary liver cancer is one of the malignant tumors with the highest morbidity and mortality worldwide. Hepatocellular carcinoma (HCC) is the main form of primary liver cancer, which usually evolves from chronic liver diseases such as cirrhosis and HBV infection. The five-year survival rate of early HCC is 50%-70%, while that of patients with advanced liver cancer is only 10%-19%. Therefore, early diagnosis and screening is the key to the treatment of liver cancer. Due to the lack of early diagnostic methods to effectively distinguish between early liver cancer patients and chronic liver disease patients, the current diagnosis of HCC mainly relies on imaging, serum AFP and tissue biopsy, which has a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/113
CPCC12Q1/6886C12N15/113C12Q2600/158C12Q2600/178
Inventor 陈健翔常存杰李骞乔逸婷董衡孙孟情陈保东孟瑶
Owner HANGZHOU NORMAL UNIVERSITY
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