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Application of product for detecting CALN1 gene expression in diagnosis of cholangiocarcinoma

A technology for cholangiocarcinoma and expression product, applied in the biological field, can solve problems such as inability to use early diagnosis of cholangiocarcinoma, and achieve the effects of timely gene diagnosis, lower mortality, and better genetic diagnosis.

Active Publication Date: 2016-01-13
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods cannot be applied to the early diagnosis of cholangiocarcinoma. Therefore, in order to more effectively improve the cure rate of CCA, how to diagnose early has attracted more and more attention from scholars.

Method used

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  • Application of product for detecting CALN1 gene expression in diagnosis of cholangiocarcinoma
  • Application of product for detecting CALN1 gene expression in diagnosis of cholangiocarcinoma
  • Application of product for detecting CALN1 gene expression in diagnosis of cholangiocarcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Screening of gene markers associated with cholangiocarcinoma

[0050] 1. Sample collection

[0051] Eight samples of normal bile duct tissue and cholangiocarcinoma tissue were collected. The above samples are surgical resection specimens of patients with cholangiocarcinoma, and all the above samples were obtained with the consent of the organizational ethics committee.

[0052] 2. Preparation of RNA samples (use TAKARA RNA extraction kit for operation)

[0053] 1) In a clean area with less RNase interference, use a mortar containing an appropriate amount of liquid nitrogen to weigh about 20 mg of an isolated lung adenocarcinoma tissue sample, and grind it to powder with a pestle;

[0054] 2) Transfer the sample to a RNase-free 2mL centrifuge tube;

[0055] 3) Add 300 μl Lysissolution, place in a homogenizer, and grind thoroughly for 1-5 minutes;

[0056] 4) 12000g, 4°C, centrifuge for 10min, transfer the supernatant to a new 1.5mL centrifuge tube;

[0057...

Embodiment 2

[0087] Example 2 QPCR sequencing to verify the differential expression of the CALN1 gene

[0088] 1. Large-sample QPCR verification of differential expression of CALN1 gene. According to the sample collection method in Example 1, 80 cases of cholangiocarcinoma tissue and 80 cases of normal bile duct tissue were selected.

[0089] 2. The specific operation steps of QPCR are as follows:

[0090] (1) RNA extraction

[0091] After collecting the samples, freeze them in liquid nitrogen. After taking them out, put the tissue into a pre-cooled mortar for grinding. After the tissue sample is powdered:

[0092] ① Add Trizol and store at room temperature for 5 minutes;

[0093] ② Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5min-10min;

[0094] ③After 12000rpm high-speed centrifugation for 15 minutes, suck the upper aqueous phase (absorb 70%) into another new centrifuge tube, and be careful not to suck the protein su...

Embodiment 3

[0106] Example 3 CALN1 gene overexpression

[0107] 1. Human cholangiocarcinoma cell line QBC939 was cultured at 37°C and 5% CO in DMEM (high glucose) medium containing 10% calf serum. 2 , Cultivated in an incubator with a relative humidity of 90%. The medium was changed once every 2-3 days, and 0.25% trypsin was used for routine digestion and passage.

[0108] 2. Overexpression of CALN1 gene

[0109] 2.1 Construction of CALN1 gene expression vector

[0110] Amplification primers were designed according to the coding sequence of the CALN1 gene (as shown in SEQIDNO.1), and the primer sequences were as follows: the forward primer was 5'-ATGCGGCTGCCAGAGCAA-3' (SEQIDNO.7), and the reverse primer was 5'-CGGAGTATCTGGTTGGCTG-3 '(SEQ ID NO. 8). The coding sequence of the full-length CALN1 gene was amplified from the cDNA library of adult fetal brain (clontech company, catalog number: 638831), and the above cDNA sequence was double-digested with restriction endonucleases BamHI and ...

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Abstract

The invention discloses CALN1 genes and an expression product thereof used as a molecular marker for the early diagnosis of cholangiocarcinoma. By utilizing RNA-sep screening and by virtue of large-sample RT-PCR verification, the abnormality of the CALN1 gene is associated to the occurrence and development of cholangiocarcinoma. According to a research result of the invention, a drug capable of improving the CALN1 gene expression level in cholangiocarcinoma tissues can be researched and developed, so that the cholangiocarcinoma can be clinically prevented and treated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of human CALN1 gene in the diagnosis and treatment of cholangiocarcinoma. Background technique [0002] The incidence of cholangiocarcinoma has been increasing year by year in recent years, accounting for about 58% to 75% of extrahepatic cholangiocarcinoma. In daily life, having cholangiocarcinoma has a great impact on the patient's health and daily life. Due to its characteristics of late discovery, early metastasis, poor prognosis, low surgical resection rate, and insensitivity to radiotherapy and chemotherapy, it has always been a difficult problem and a research hotspot in the field of surgery. Because it is not sensitive to non-surgical treatments such as chemotherapy and radiotherapy, comprehensive treatment methods still have no clear significance in improving the long-term efficacy. Therefore, the thoroughness of surgical treatment is the key to the treatment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/68G01N33/574A61K48/00A61K38/17A61P35/00
CPCA61K38/00A61K48/00C12Q1/6886C12Q2600/158G01N33/57484G01N33/68G01N2333/47G01N2800/08
Inventor 杨承刚果春青宋宏涛
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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