Application of substances that reduce the expression of usp1 in the preparation of medicines for the treatment of childhood T-lineage acute lymphoblastic leukemia

A technology of acute lymphocytes and leukemia cells, applied in the field of biomedicine, to achieve the effect of inhibiting cell proliferation and promoting cell apoptosis

Active Publication Date: 2022-07-12
BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of action of USP1 in the development of T-ALL, especially in children, has not been reported yet.

Method used

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  • Application of substances that reduce the expression of usp1 in the preparation of medicines for the treatment of childhood T-lineage acute lymphoblastic leukemia
  • Application of substances that reduce the expression of usp1 in the preparation of medicines for the treatment of childhood T-lineage acute lymphoblastic leukemia
  • Application of substances that reduce the expression of usp1 in the preparation of medicines for the treatment of childhood T-lineage acute lymphoblastic leukemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: The deubiquitinase USP1 is overexpressed in childhood T-ALL

[0058] 1. mRNA level overexpression

[0059] The NCBI GEO database (https: / / www.ncbi.nlm.nih.gov / geo) downloads the gene expression profiling results (GSE13159) of bone marrow specimens from healthy children and children with T-ALL. Using T-Test analysis, we found that in The expression level of USP1 mRNA in bone marrow specimens of children with T-ALL (n=169) was significantly higher than that of bone marrow specimens of healthy children (n=73, pfigure 1 shown in A.

[0060] 2. Protein level overexpression

[0061] Use a heparin anticoagulant tube to extract 2ml of bone marrow cells from children with T-ALL, add 5ml of red blood cell lysate and mix well, let stand for 2 minutes, then centrifuge at 100g for 5 minutes, discard the supernatant and repeat the above steps. The cells were washed twice, the supernatant was removed, and the cells were collected.

[0062] Total protein was extracted wit...

Embodiment 2

[0064] Example 2: Construction of RNA interference recombinant expression vector for deubiquitinating enzyme USP1

[0065] 1. Selection of RNA interference target sequences

[0066] For the full-length cDNA sequence (SEQ ID NO: 14) of the deubiquitinase USP1-encoding gene USP1, the following two DNA sequences were selected as target sequences for RNA interference:

[0067] sh-1: Positions 2574-2594 of SEQ ID NO: 14 (i.e. 5'-CCAGTGACCAAACAGGCATTA-3')

[0068] sh-2: Positions 2030-2050 of SEQ ID NO: 14 (i.e. 5'-GCTAGTGGTTTGGAGTTTGAT-3')

[0069] Positions 422-2779 in SEQ ID NO:14 are open reading frames encoding the deubiquitinating enzyme USP1 shown in SEQ ID NO:15.

[0070] 2. Small interfering RNA (siRNA)

[0071] Two siRNAs, siRNA-1 and siRNA-2, were designed for the two target sequences of the deubiquitinase USP1 in step 1. The target sequence of siRNA-1 was sh-1, and the target sequence of siRNA-2 was sh- 2.

[0072] siRNA-1

[0073] siRNA-1-F: 5'-ccagugaccaaacaggcau...

Embodiment 3

[0112] Example 3: Deubiquitinase USP1 RNA interference recombinant expression vector lentivirus infection of leukemia cells and Western blot detection of the expression of deubiquitinase USP1 in recombinant leukemia cells

[0113] 1. Deubiquitinase USP1 RNA interference recombinant expression vector lentiviral infection of leukemia cells

[0114] (1) RNA interference recombinant expression vector lentiviral packaging

[0115] The target gene GV493-USP1-sh-1, GV493-USP1-sh-2 or the control sequence GV493-sh-Control plasmid, virus packaging helper plasmids pHelper 1.0 and pHelper2.0 (purchased from Shanghai Jikai Gene Medical Technology Co., Ltd. ) co-transfected 293T cells, and the supernatant was extracted 48-72 h after the transfection was completed for virus harvesting. Centrifuge at 4°C and 4000g for 10min to remove cell debris; filter the supernatant with a 0.45μm filter and put it in a 40ml ultracentrifuge tube; balance the samples respectively, and put the ultracentrifu...

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Abstract

The invention discloses the application of a substance that reduces the expression of USP1 in the preparation of a medicament for treating childhood T-lineage acute lymphoblastic leukemia. The invention discloses a short hairpin RNA forming a stem-loop structure, which is shRNA-1 or shRNA-2. The deubiquitinating enzyme USP1 has the functions of inhibiting cell apoptosis and promoting cell proliferation in T-line acute lymphoblastic leukemia cells. The present invention proves that inhibiting the expression of USP1 can promote the apoptosis of tumor cell leukemia cells and inhibit the proliferation of leukemia cells. The invention provides a new way for the treatment of T-line acute lymphoblastic leukemia, and the deubiquitinating enzyme USP1 is expected to become a potential target of anti-leukemia treatment, and has a very broad application prospect in the medical field.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to the application of a substance that reduces the expression of deubiquitinase USP1 in the preparation of a medicament for treating childhood T-lineage acute lymphoblastic leukemia. Background technique [0002] Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and the leading cause of childhood disease-related death. T-lineage acute lymphoblastic leukemia (T-ALL) is a very aggressive subtype of ALL, and up to 30% of children with T-ALL are still refractory to treatment or relapse. The mechanism of occurrence and development is not yet clear, which is the bottleneck for the further improvement of the cure rate of T-ALL. Therefore, in-depth study of the molecular mechanisms of the occurrence and development of T-ALL in children, especially refractory and relapsed T-ALL, is crucial for exploring new targeted treatment options and improving survival rates. [0003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11A61K31/7088A61K31/713A61K45/00A61K31/506A61P35/02
CPCC12N15/113A61K31/7088A61K31/713A61K45/00A61K31/506A61P35/02C12N2310/14C12N2310/531
Inventor 刘曙光高超郑胡镛张瑞东
Owner BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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