Application of chaperonin CCTgamma in preparation of tumour diagnosis reagent

A preparation and protein inhibitor technology, applied in the field of biomedicine, can solve the problems of high mortality, confusion of trauma and great harm, and achieve the effects of good repeatability, clear results and rapid response

Active Publication Date: 2015-06-24
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Osteosarcoma has no obvious clinical symptoms at the onset, and is easily confused with trauma, and lung met

Method used

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  • Application of chaperonin CCTgamma in preparation of tumour diagnosis reagent
  • Application of chaperonin CCTgamma in preparation of tumour diagnosis reagent
  • Application of chaperonin CCTgamma in preparation of tumour diagnosis reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 High-throughput sequencing and analysis

[0033] Collect 5 cases of osteosarcoma samples and 3 cases of normal control tissues for RNA extraction. After RNA extraction, agarose gel electrophoresis can be used to preliminarily determine whether the quality of the extracted RNA samples is qualified or not, and whether they can be used for further transcription group analysis. Then, the extraction of RNA samples was detected by NanoDrop1000 spectrophotometer, and the sample requirements for RNA-seq sequencing: OD260 / OD280 was 1.8-2.2.

[0034] The sequencing platform is the HiSeq 2500 high-throughput sequencing platform of Illumina, which performs high-throughput transcriptome deep sequencing. After sequencing, we use Fast-QC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) The software evaluates the quality of sequencing data as a whole, including base quality value distribution, quality value position distribution, GC content, PCR duplication conten...

Embodiment 2

[0035] Example 2 Osteosarcoma tissue and normal tissue CCTγ gene expression

[0036] 1. Materials and methods

[0037] 1. Materials

[0038] 65 cases of osteosarcoma tissues and 25 cases of normal tissues were collected, grouped and numbered.

[0039] 2. Method

[0040] 2.1 Extraction of total RNA from osteosarcoma tissue and normal tissue

[0041] use Reagent (invitrogen, Cat. No. 15596-018) was used to extract sample RNA, and the experimental operation was carried out according to the product manual. The specific operation was as follows:

[0042] 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:

[0043] ① Add Trizol and store at room temperature for 5 minutes;

[0044] ② Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5-10 minutes;

[0045] ③ 12000rpm high-speed centrifugation f...

Embodiment 3

[0070] Embodiment 3 The cultivation of osteosarcoma cell line MG-63

[0071] 1. Materials

[0072] (1) Source of specimens

[0073] The human osteosarcoma cell line MG-63 was purchased from the Shanghai Cell Institute of the Chinese Academy of Sciences.

[0074] (2) Main reagents

[0075] DMEM / HIGH Glucose (1×) (Thermo Fisher Biochemicals (Beijing) Co., Ltd.)

[0076] (3) Main solution

[0077] 1. Cell culture medium

[0078] DMEM medium + 10% standard fetal bovine serum.

[0079] 2. PBS (balanced salt solution)

[0080] Dissolve 8g NaCl, 0.25g KCl, 1.44g Na in 800ml distilled water 2 HPO 4 and 0.24g KH 2 PO 4 Adjust the pH value of the solution to 7.4 with HCl, add water to make up to 1 L, autoclave, and store at room temperature.

[0081] 3. 0.25% trypsin digestion solution

[0082] Add 0.25g of trypsin to 100ml of deionized water, filter and sterilize it, and distribute it for use.

[0083] 2. Experimental method

[0084] (1) Cell culture

[0085] 1. Cell pas...

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Abstract

The invention relates to an application of chaperonin CCTgamma in preparation of a tumour diagnosis reagent and in particular relates to a new application of the chaperonin CCTgamma in preparation of an osteosarcoma diagnosis reagent. The inventor adopts bioinformatics method analysis for carrying out gene screening based on high-throughput sequencing results, a candidate gene CCTgamma is picked out, and further molecular cell biology experiments prove that CCTgamma has a good correlation with osteosarcoma, can be used for preparing an auxiliary osteosarcoma diagnosis and treatment preparation and has important clinical application value.

Description

technical field [0001] The invention relates to the field of biomedicine, and the application of chaperone CCTγ in the preparation of tumor diagnostic reagents. Specifically, the invention relates to the new application of chaperone CCTγ in the preparation of osteosarcoma diagnostic reagents. Background technique [0002] Chaperonins are cyclic oligomer complexes composed of about 60kDa subunits, widely present in cells, expressed in large quantities and related in sequence, and mediate the folding of polypeptide chains through an ATP-dependent reaction, converting monomers into and oligomeric subunits into fully functional native proteins. Eukaryotic chaperones are called TCP-1ring complex (TRiC), cytosolic chaperonin (c-cpn) or cpn-containing TCP-1 (CCT). Chaperone CCT is a large cylindrical multi-subunit complex with a central cavity structure for binding unfolded and denatured polypeptides. Each ring of CCT is composed of eight different molecular weights between 52-65k...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K31/7088C12Q1/68A61P35/00
Inventor 杨承刚崔双双边洋孙耀兰
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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