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Application of PZP gene in diagnosis and treatment of biliary duct cancer

A cholangiocarcinoma and gene technology, applied in the direction of gene therapy, medical preparations containing active ingredients, biochemical equipment and methods, etc., can solve problems such as inability to provide diagnosis

Active Publication Date: 2016-02-17
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of the above methods cannot provide a deliberate and sensitive diagnosis
Although some tumor markers are now clinically used for inspection, because these tumor markers are non-specific to cholangiocarcinoma, they only have a certain reference value, so it is important to find a specific cholangiocarcinoma marker the meaning of

Method used

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  • Application of PZP gene in diagnosis and treatment of biliary duct cancer
  • Application of PZP gene in diagnosis and treatment of biliary duct cancer
  • Application of PZP gene in diagnosis and treatment of biliary duct cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Screening of gene markers associated with cholangiocarcinoma

[0059] 1. Sample collection

[0060] 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.

[0061] 2. Preparation of RNA samples (operated using QIAGEN tissue RNA extraction kit)

[0062] 1) Tissue extraction

[0063] 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, grind it to powder with a pestle, and then transfer the sample to a 2 ml RNase-free in a centrifuge tube. Add 300 μl lysate, place in a homogenizer, grind thoroughly for 1-5min, centrifuge at 12000g, 4°C for 10min, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 600 μl ...

Embodiment 2

[0081] Example 2 QPCR sequencing to verify the differential expression of the PZP gene

[0082] 1. Large-sample QPCR verification of differential expression of PZP genes. According to the sample collection method in Example 1, 80 cases of cholangiocarcinoma tissue and 80 cases of normal bile duct tissue were selected.

[0083] 2. The specific operation steps of QPCR are as follows:

[0084] (1) RNA extraction

[0085] 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:

[0086] 1) Add Trizol and place at room temperature for 5 minutes;

[0087] 2) Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5-10min;

[0088] 3) Centrifuge at 12000rpm for 15min, transfer the upper aqueous phase to another new centrifuge tube (be careful not to absorb the protein material between the two aqueous phases)...

Embodiment 3

[0101] Example 3 PZP gene overexpression

[0102] 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.

[0103] 2. Overexpression of PZP gene

[0104] 2.1 Construction of PZP gene expression vector

[0105] According to the coding sequence of PZP gene (as shown in SEQIDNO.1) design amplification primer, primer sequence is as follows: forward primer is 5'-CCGGGATCCGCCACCATGCGGAAAGACAGA-3' (SEQIDNO.7), reverse primer is 5'-CGGCTCGAGAACATTTCCATGCTC-3 '(SEQ ID NO. 8). The coding sequence of the full-length PZP 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 XhoI and inserted into the Th...

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Abstract

The invention discloses application of a PZP gene in diagnosis and treatment of a biliary duct cancer. The PZP gene is low-expressed in tissue of a biliary duct cancer patient, and growth and invasion of biliary duct cancer cells can be restrained by increasing expression of PZP. According to the application of the PZP gene in the diagnosis and treatment of the biliary duct cancer, the sensibility and the specificity of biliary duct cancer diagnosis are greatly improved, and meanwhile a new target site is provided for gene treatment of the biliary duct cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of PZP gene in the diagnosis and treatment of cholangiocarcinoma. Background technique [0002] Cholangiocarcinoma refers to malignant tumors of the extrahepatic bile duct that originate from the confluence of the left and right hepatic ducts to the lower end of the common bile duct. Infection, choledochal cyst, etc., these factors can increase the risk of cholangiocarcinoma. The main clinical manifestations are progressive jaundice accompanied by epigastric discomfort, loss of appetite, weight loss, and itching. Such as tumor ulceration and bleeding, there may be melena or positive fecal occult blood test, anemia and other manifestations. [0003] The malignant degree of cholangiocarcinoma is extremely high, and the incidence rate in Europe and the United States is very low, but the incidence rate in Southeast Asia and my country is on the rise, and the overall tre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/574A61K45/00A61K48/00A61K31/7088A61P35/00
CPCA61K31/7088A61K45/00C12Q1/6886C12Q2600/158G01N33/57407
Inventor 高倩倩杨承刚孙耀兰
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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