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Bile duct cancer diagnosis and treatment molecular marker and application thereof

A technology of cholangiocarcinoma and drugs, applied in the biological field, can solve the problems that the early diagnosis of cholangiocarcinoma cannot be applied, and achieve the effect of timely gene diagnosis, lower mortality rate, and better gene diagnosis

Active Publication Date: 2015-09-02
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 method cannot be applied to the early diagnosis of cholangiocarcinoma, because the early symptoms of CCA are not obvious clinically, and the key to surgical treatment, which is currently the most effective treatment for CCA, lies in the early detection of the disease, so in order to more effectively improve the The cure rate of CCA, how to diagnose early has attracted more and more attention of scholars

Method used

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  • Bile duct cancer diagnosis and treatment molecular marker and application thereof
  • Bile duct cancer diagnosis and treatment molecular marker and application thereof
  • Bile duct cancer diagnosis and treatment molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Screening for gene markers associated with cholangiocarcinoma

[0051] 1.1 Sample collection

[0052] 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.

[0053] 1.2 RNA sample preparation and quality analysis

[0054] 1.2.1 RNA sample preparation

[0055] Normal cholangiocarcinoma tissue and cholangiocarcinoma tissue were subjected to RNA extraction to determine the quality of RNA samples by agarose gel electrophoresis, and qualified ones could be used for further transcriptome analysis.

[0056] 1.2.2 Quality analysis of RNA samples (NanoDrop1000 spectrophotometer)

[0057] NanoDrop1000 spectrophotometer detects RNA samples, and the sample requirements for RNA-seq sequencing: OD260 / OD280 is 1.8-2.2.

[0058] 1.2.3 Quality analysis of RN...

Embodiment 2

[0069] Example 2 RT-PCR sequencing to verify the differential expression of the MARCO gene

[0070] 1. According to the detection results of high-throughput sequencing, the MARCO gene was selected for large-sample RT-PCR verification. According to the sample collection method in Example 1, 80 cases of cholangiocarcinoma tissue and 80 cases of normal bile duct tissue were selected.

[0071] 2. The RNA extraction process is the same as in Example 1.

[0072] 3. Reverse transcription: TAKARA's reverse transcription kit is used for reverse transcription of RNA, and the specific operation steps are shown in the instruction manual.

[0073] 4. PCR amplification

[0074] The primer sequence of MARCO gene RT-PCR is as follows:

[0075] MARCO: The forward primer is 5'-AGCAAGGAGTAAAGGGAGAA-3' (SEQ ID NO.3);

[0076] MARCO: The reverse primer is 5'-GGTTACTACTGCCGACAATC-3' (SEQ ID NO.4).

[0077] The RT-PCR primer sequence of β-actin gene is as follows:

[0078] β-actin: the forward...

Embodiment 3

[0090] Example 3 MARCO gene overexpression

[0091] 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.

[0092] 2. Overexpression of MARCO gene

[0093] 2.1 Construction of MARCO gene expression vector

[0094] Amplification primers were designed according to the coding sequence of the MARCO gene (as shown in SEQ ID NO.1), and the primer sequences were as follows: the forward primer was 5'-ATGCGGCTGCCAGAGCAA-3' (SEQ ID NO.7), and the reverse primer was 5' - CGGAGTATCTGGTTGGCTG-3' (SEQ ID NO. 8). The coding sequence of the full-length MARCO gene was amplified from the cDNA library of adult fetal brain (clontech company, catalog number: 638831). The obtained recombinant vector pcDNA3.1-MARCO was connected to the eukaryotic cell ...

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Abstract

The invention discloses a molecular marker-MARCO gene used for early diagnosis of bile duct cancer. Experiments show that mRNA level of the MARCO gene in bile duct cancer tissue is remarkably lower than that in normal bile duct tissue, so that whether a test receiver has risk of suffering from the bile duct cancer or not or whether the test receiver suffers from the bile duct cancer or not can be judged by measuring expression level of MARCO in the bile duct tissue of the test receiver. The MARCO gene and an expressed product thereof can be prepared to be drug to be used for patients suffering from the bile duct cancer or patients having high risk of suffering from the same to treat or prevent the bile duct cancer. A novel diagnosis method is provided for clinical diagnosis of the bile duct cancer, and novel candidate drug is provided for treating the bile duct cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of human MARCO gene in the diagnosis and treatment of cholangiocarcinoma. Background technique [0002] Cholangiocarcinoma (CCA) is a malignant tumor originating from bile duct epithelial cells, ranking second in hepatobiliary malignant tumors, second only to hepatocellular carcinoma, accounting for about 3% of digestive system tumors. Cholangiocarcinoma can occur in various parts of the extrahepatic bile duct, among which the proximal bile duct (hilar bile duct) is the most common, accounting for about 58%, the middle and distal bile ducts account for 13% and 18% respectively, and those occurring in the cystic duct account for 4% , and another 7% occurred as diffuse. [0003] The current clinical methods for diagnosing cholangiocarcinoma include: 1) laboratory examination: using total blood bilirubin, direct bilirubin, alkaline phosphatase and γ-glutamyl transferase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/68G01N33/574A61K48/00A61K38/17A61K45/00A61P35/00
CPCA61K38/17A61K38/1709A61K45/00A61K48/00C12Q1/6837C12Q1/6841C12Q1/686C12Q1/6886C12Q2600/158G01N33/574G01N33/57407G01N33/57484G01N33/68G01N33/6893
Inventor 杨承刚果春青宋宏涛
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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