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Application of EVI-1 inhibitor for preparing medicines for treating vascular anomaly remodeling diseases

A vascular abnormality, EVI-1 technology, applied in the field of biomedicine, can solve the problem of not inhibiting EVI-1 against abnormal vascular remodeling, and achieve the effect of reducing abnormal intimal neogenesis, reducing toxic side effects, and promoting specific gene expression

Active Publication Date: 2018-04-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there are still no reports of inhibiting EVI-1 against abnormal vascular remodeling at home and abroad.

Method used

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  • Application of EVI-1 inhibitor for preparing medicines for treating vascular anomaly remodeling diseases
  • Application of EVI-1 inhibitor for preparing medicines for treating vascular anomaly remodeling diseases
  • Application of EVI-1 inhibitor for preparing medicines for treating vascular anomaly remodeling diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 EVI-1 is involved in the process of smooth muscle cell phenotype transformation.

[0027] Primary mouse aortic smooth muscle cells were isolated and cultured by enzymatic digestion. The vascular smooth muscle cells used in the experiment were selected from P3-P12. Primary aortic smooth muscle cells were cultured in 10% FBS-DMEM medium (FBS was purchased from Gibco, and DMEM was purchased from Hyclone) in a 5% CO2 incubator at 37°C.

[0028] Vascular smooth muscle cell stimulation group: normal culture for 48 hours, serum-free starvation for 48 hours, and serum-free starvation for 24 hours + 5ng / ml TGF-β stimulation for 24 hours. After smooth muscle cells were treated according to the above grouping, cellular RNA was collected, and RT-qPCR was used to detect the expression changes of related target genes during the process of smooth muscle cell phenotype transformation. figure 1 The results of A showed that EVI-1 decreased significantly after 24 hours of seru...

Embodiment 2

[0031] Example 2 EVI-1 regulates phenotypic transformation of smooth muscle cells.

[0032] Establishment of EVI-1 gene stable knockdown cell lines. EVI-1 shRNA plasmid construction, shRNA sequence: Evi1-shRNA-F: 5'-CCGGCAGGTACTGTGGCAAGATATTCTCGAGAATATCTTGCCACAGTACCTGTTTTTG-3' (SEQ ID NO.2); Evi1-shRNA-R: 5'-AATTCAAAAACAGGTACTGTGGCAAGATATTCTCGAGAATATCTTGCCACAGTACCTG-3' (SEQ ID NO.3). After the construction of the plasmid is completed, further virus packaging is required to play a role: (1) 293T cells are routinely cultured and passaged, and the passage ratio is optimal to make the cell density reach 80% on the second day according to the experimental requirements. (2) Use TransIT-X2 (mirusbio company reagent to co-transfect Evi1-shRNA plasmid or control plasmid, pUMVC and pCMV-VSV-G into 293T cells (strictly follow the company's instructions). (3) Change to conventional culture after overnight transfection (4) After 48 hours, the cell supernatant was collected, centrifuged, f...

Embodiment 3

[0038] Example 3. EVI-1 transcriptional repression gene list of vascular smooth muscle cell-specific contraction.

[0039]Cell transfection: Use TransIT-X2TM transfection reagent to transfect relevant plasmids into vascular smooth muscle cells (one well in a 24-well culture plate). (1) The smooth muscle cells were routinely cultured and subcultured, and the subculture ratio was optimal to make the cell density reach 80% on the second day according to the experimental requirements. (2) Mix TransIT-X2TM Transfection Reagent before use. (3) Prepare the following reagents according to the protocol. Serum-free DMEM 50ul; plasmid 200ng; TransIT-X2 1.5ul (4) Stand at room temperature for 30 minutes or more to allow the mixed reagents to fully combine (5) Replace the cell medium and add freshly prepared 2% FBS+DMEM medium. (6) Evenly drop the transfection mixture into the well plate. (7) Collect cell samples for testing after 48 hours.

[0040] Luciferase detection: Use the Dual-L...

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Abstract

The invention discloses applications of a transcription factor and an oncoprotein-ecotropic viral integration site 1 (EVI-1) inhibitor in vascular remodeling, wherein the EVI-1 inhibitor employs vascular smooth muscle specific marker gene (SMA, SM22, etc) and a specific transcription factor (SRF, Myocardin) as direct target points, thus improving expression of the smooth muscle specific gene, significantly inhibiting conversion from contractile type to secreting type of vascular medial smooth muscle cells, and further reducing abnormal regeneration of inner membrane after vascular injury. Theapplication is carried out from cell functional test into molecular mechanism researching at gene and protein levels, wherein an in-vivo animal pathological model test is converted into specimen analysis of clinical patients; all the results prove that the EVI-1 inhibitor is a key regulation factor for phenotypic transformation of smooth muscle cells; so there are reasons to believe that the EVI-1inhibitor is highly possible to be used as a novel anti-vascular anomaly remodeling medicine for treatment of cardiovascular and cerebrovascular diseases in future.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to the application of EVI-1 inhibitors in abnormal vascular remodeling diseases, in particular to the application of miR-22-3p in the preparation of abnormal vascular remodeling caused by abnormal transformation of vascular smooth muscle phenotype Application in the medicine of disease. Background technique [0002] Atherosclerosis (Atherosclerosis, AS) is a common disease in clinical practice, and it is the main cause of cardiovascular and cerebrovascular diseases such as acute coronary syndrome (Acute Coronary Syndrome, ACS), acute myocardial infarction (Acute Myocardial Infarction, AMI) and ischemic stroke. basis of disease. The morbidity and mortality of this disease rank first among all kinds of diseases all the time in the world. [0003] Percutaneous transluminal coronary angioplasty (PTCA) is currently one of the most effective methods for the treatment of coronary heart...

Claims

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Application Information

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IPC IPC(8): A61K31/7105A61P9/00A61P9/14
CPCA61K31/7105
Inventor 张力杨峰陈齐山杨眉
Owner ZHEJIANG UNIV
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