Medulloblastoma/cell marker and application thereof

A medulloblastoma and cell technology, applied in the field of preparation of reagents for tumor diagnosis/prognosis, can solve problems such as diagnosis/prognosis of tumors/tumor cells, and achieve accurate diagnosis/prognosis and slow down drug resistance

Pending Publication Date: 2021-12-31
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reagents for diagnosing / prognosing tumors / tumor cells at the protein level. If the protein that directly transmits the signal pathway between SMOs is not known, it is impossible to accurately prepare reagents for diagnosing / prognosing tumors / tumors at the protein level. cell

Method used

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  • Medulloblastoma/cell marker and application thereof
  • Medulloblastoma/cell marker and application thereof
  • Medulloblastoma/cell marker and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0066] Table 3 Preparation of silver staining solution

[0067] ddH2O 35.0ml Silver complex solution 5.0ml Reduction Moderator 5.0ml Image development Reagent 5.0ml

[0068] ④ Termination of silver staining

[0069] Immediately pour off the silver staining reagent, 5% acetic acid, and keep it for 15 minutes, then pour it off and wash it twice with 100ml ddH2O double pure water, each time for 10 minutes.

[0070] In the embodiment of the present invention, the IP experiment process is:

[0071] ①For the collected ONS76-52, 53, and 54 cells (52 is the Smo W535L mutant type, 53 is the Smo D473H mutant type, and 54 is the Smo Wild type) (2 dishes of cells were collected for each treatment group), use freshly prepared The lysate (add 25×Complete inhibitor, 10×PhosphoStop inhibitor, and 100×PMSF before use) was used to lyse the cells on ice, add 800 μl IP lysate to each tube of cells, and repeatedly suck the lysate up and down with a pipette gun to...

Embodiment 1

[0081] (1) Cell treatment and experimental grouping

[0082] The 3rd to 10th generation medulloblasts in good growth state were subcultured, and after 12 hours of starvation on the second day, new serum-free medium was replaced with 8 dishes of each cell, and six groups of experiments were first divided into six groups as shown in Table 4, after 24 hours Then each group was divided into SAG stimulation group and non-SAG stimulation group as shown in Table 5.

[0083] Table 4 Cell Treatment Grouping

[0084]

[0085] Table 5 IP experiment grouping

[0086]

[0087]

[0088] (2) For the IP experiment, during the Western Blotting process, two gels were run at the same time, and the sample volume was the same. The sample volume of input and IP was 6 μl for each sample, and the sample volume of elution was 20 μl. One piece of glue was used for silver staining, and the other was used for WB chemiluminescence development (primary antibody flag-M2). WB chemiluminescence dev...

Embodiment 2

[0090] In conjunction with Example 1, ONS76-W535L (LDE225 / SAG) and ONS76-WT (LDE225 / SAG) were analyzed by Venn, and the results were as follows Figure 4 Shown: 65 of the SMO protein IP pull-down proteins in ONS76-W535L (LDE225 / SAG) are ONS76-W535L-specific proteins, such as: PFKP, PKM, PRKAA1, etc.; there are 117 proteins in ONS76-W535L and ONS76- All are shared in WT, such as kinases: CDC42BPA, CSNK2B, CDK1, DAPK3, etc.; there are 65 ONS76-W535L (LDE225 / SAG) independent proteins.

[0091] DAVID FunctionalAnnotation Bioinformatics Microarray Analysis analysis was performed on 65 independent proteins of ONS76-W535L (LDE225 / SAG), and the results are shown in Tables 6, 7, and 8. LDE225 / SAG) abnormal activation. In particular, RAB10, RHEB, PFKP, PRKAA1, PKM, and TPI1 in the AMPK signaling pathway and the Glycolysis / Gluconeogenesis signaling pathway are all target proteins for the treatment of drug-resistant W535L medulloblastoma.

[0092] Table 6 DAVID Functional Annotation Bio...

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Abstract

The invention belongs to the technical field of preparation of tumor diagnosis/prognosis reagents, and particularly relates to a medulloblastoma/cell marker and application thereof. The invention relates to the research and application of the protein in the SMO-activated mutant medulloblastoma/cell based on one or more of GPR37 protein, RAB35 protein, RPS10 protein, NUDT3 protein, USP6NL protein, REE protein, CACYBP protein and CKAP5 protein. The specific conjugates of the proteins are prepared into kits or reagents, so that the myeloblastoma/cells can be diagnosed/prognosed more simply, conveniently and accurately, and particularly, the drug-resistant myeloblastoma/cells can be screened more accurately, and then the drugs are applied according to the case.

Description

technical field [0001] The invention belongs to the technical field of reagent preparation for tumor diagnosis / prognosis, and specifically relates to a medulloblastoma / cell marker and its application. It is based on the SMO activating mutation of one or more of GPR37 protein, RAB35 protein, RPS10 protein, NUDT3 protein, USP6NL protein, RERE protein, CACYBP protein, CKAP5 protein in medulloblastoma / cell protein research and application. Background technique [0002] The Hedgehog (Hh) signaling pathway plays an important role in maintaining embryonic development and adult tissue homeostasis. Once the Hh pathway is abnormally regulated, body defects or tumors will occur. The Hh signaling pathway is extremely active in many tumors, and SMO is a key signal transduction molecule in the Hh signaling pathway. Targeting SMO has become a hot spot in drug research. At present, SMO inhibitors have entered clinical use to treat various tumors, such as Vismodegib (GDC-0449) has been app...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/573C12Q1/6886
CPCG01N33/57407G01N33/57488G01N33/573C12Q1/6886C12Q2600/158C12Q2600/118C12Q2600/106G01N2333/705G01N2333/47G01N2333/914
Inventor 姚月良王岩卞修武
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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