Model for evaluating HR defect and application

A defect model and defect technology, applied in the evaluation of multi-molecular markers of homologous recombination defects, evaluation of HR defect models and application fields, can solve the problem that the evaluation system of multi-molecular markers has not yet been found, and the combination drug or sequential drug has not been found. Drug administration and other issues, to achieve the effect of improving anti-tumor efficacy, easy clinical test detection, and convenient use

Active Publication Date: 2019-12-13
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] So far, no multi-molecular marker evaluation system for judging homologous recombination deficiency has been found, and there have been no reports about individualized chemotherapy for gallbladder cancer, that is, the combined administration or sequential administration of CQ or PARP inhibitors and FAM

Method used

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  • Model for evaluating HR defect and application
  • Model for evaluating HR defect and application
  • Model for evaluating HR defect and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of Multimolecular Markers for Evaluation of Homologous Recombination Deficiency and HR Deficiency Model

[0044] 1. Assessing the Selection of Multimolecular Markers in the HR Deficiency Model

[0045] The DNA sequencing data of 553 gallbladder cancer patient samples were obtained from the database Catalog Of Somatic Mutations In Cancer (COSMIC, cancer.sanger.ac.uk / cancergenome / projects / cosmic), and the mutated genes were sorted according to the mutation frequency. Research on molecules related to the repair mechanism Selected 15 genes (M1-M15) that played a key role in them as multi-molecular markers for chemotherapy of gallbladder cancer targeting homologous recombination, as shown in Table 1.

[0046] Table 1. Multimolecular marker combinations for judging HR deficiency

[0047]

[0048] 2. Assessing Validation of Multimolecular Markers in HR Deficiency Models

[0049] 1) Puromycin concentration selection:

[0050] Gallbladder cancer GBC...

Embodiment 2

[0075] Example 2: Isolation and culture of primary gallbladder cancer cells and verification of the accuracy of the HR-deficient model

[0076] 1. Construction of gallbladder cancer GBC-SD and SGC-996 cell lines with stable knockdown of BRCA1 and BRCA2

[0077] (1) Lentivirus pre-infection experiment

[0078] Follow 3-5 x 10 3 GBC-SD and SGC-996 gallbladder cancer cells were pre-seeded in the wells of 96-well culture plate per well (1640 medium containing 10% FBS, volume 100 μl), and the cell fusion degree should be 30% during lentivirus infection -50% or so. 10 μl of virus solution (BRCA1, BRCA2 lentiviral particles are entrusted to Shanghai Jikai Biological Co., Ltd. to make the coating) according to the final concentration of 1×10 6 TU / ml, 1×10 5 TU / ml, 1×10 4 TU / ml 3 concentrations pre-infected cells. For example, the number of gallbladder cancer cells is about 1×10 4 1, the MOI of the above three wells is about 100, 10, 1. After culturing at 37°C for about 12 hours...

Embodiment 3

[0090] Example 3: The autotropic inhibitor chloroquine significantly enhances the killing effect of FAM on gallbladder cancer cells with normal HR

[0091] 1. Configure chloroquine:

[0092] An appropriate amount of chloroquine (Sigma, product number: C6628) was dissolved in double-distilled water to form a 10 mM stock solution, which was sterilized with a 0.22 μm filter and stored at 4°C. For in vitro cell experiments, it was diluted to 100 μM with 1640 medium for experiments. In vivo animal experiments were injected intraperitoneally according to the standard of 31.25ul / 10g at a concentration of 10mM.

[0093] The gallbladder cancer cells GBC-SD and SGC-996 used in this example were judged by the HR deficiency model described in Example 1 to have a T value of 0, that is, the HR was normal.

[0094] 2.5-FU treatment induces autophagy in gallbladder cancer cells:

[0095] (1) Gallbladder cancer cells GBC-SD and SGC-996 were treated with 5 μM 5-FU for 24 and 48 hours, respect...

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Abstract

The present invention discloses a model for evaluating an HR defect and its application. Based on a tumor individualized chemotherapy strategy, the method evaluates whether HR in gallbladder cancer cells is defective by using an existing HR-related indicator, and inhibits autophagy and PARP treatment according to normal HR or defective HR, improves the anti-tumor efficacy of a chemotherapy regimenFAM, and avoids the occurrence of resistance after multiple rounds of highly toxic medication for the patient. A multi-molecular marker used for detecting gallbladder cancer cells contains only 15 genes, is liable to clinical trials, reduces the cost of detection, and is not affected by the batch effect of experiments or the difference in the detection platforms, does not require data standardization between multiple samples before use, and is easy to use.

Description

[0001] (1) Technical field [0002] The invention relates to the fields of life science and biotechnology, in particular to a multimolecular marker for evaluating homologous recombination defects, a model for evaluating HR defects and its application. [0003] (2) Background technology [0004] Gallbladder cancer is one of the most common malignant tumors in the biliary system, and its incidence has shown an obvious upward trend in recent years. Malignant biliary tumors lack typical symptoms in the early stage and are difficult to diagnose. At the same time, because of the special anatomical structure and complex adjacent relationship, the radical resection rate is low and the prognosis is extremely poor. The 5-year survival rate is only 9% to 18%[Baiu I, Visser B. Gallbladder Cancer. JAMA. 2018;320(12):1294.]. At present, research on the prevention and treatment of gallbladder cancer is still in its infancy, mostly in the discussion of surgical methods, preoperative testing a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16H20/17G16B30/00C12Q1/6869
CPCG16H20/17G16B30/00C12Q1/6869C12Q2531/113
Inventor 汤佳城梁霄卫赛赛张占丰
Owner ZHEJIANG UNIV
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