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Application of miR-375 inhibitor in preparation of vascular senescence resistant medicines

A mir-375, 1.mir-375 technology, applied in the application field of miR-375 inhibitors in the preparation of anti-vascular aging drugs, to achieve the effect of promoting vascular cell aging and slowing down vascular cell aging

Inactive Publication Date: 2017-12-29
田小利 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the cell-specific regulation of miRNA
The inventors found that there are few reports on the systematic evaluation of the regulatory role of miRNA in the aging process of blood vessel-related cells, especially vascular endothelial cells

Method used

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  • Application of miR-375 inhibitor in preparation of vascular senescence resistant medicines
  • Application of miR-375 inhibitor in preparation of vascular senescence resistant medicines
  • Application of miR-375 inhibitor in preparation of vascular senescence resistant medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Systematic screening of miRNAs that promote vascular endothelial cell aging

[0049] 1. The materials used in this example are as follows:

[0050] 1.1 cells

[0051] The human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) used in this example were all isolated in the inventor's laboratory.

[0052] 1.2 Main reagents

[0053] 1.2.1 Cell isolation and culture

[0054] Endothelial Cell Medium (ECM) (Sciencell)

[0055] Pancreatin (Amresco)

[0056] EDTANa 2 (Beijing Chemical Plant)

[0057] 1.2.2 Cell transfection

[0058] Lipo2000 liposome transfection reagent (Invitrogen company)

[0059] 1.2.3 β-galactosidase (β-galactosidase, β-gal) detection

[0060] Aging-related β-gal detection kit (Beiyuntian Biotechnology Research Institute)

[0061] 2. The specific operation steps of this embodiment are as follows:

[0062] 2.1 HUVEC isolation and culture

[0063] 1) Quickly move the taken umbilical cord into a clean opera...

Embodiment 2

[0079] Example 2 Validation of mir-375 in other types of blood vessel-related cell models

[0080] Due to the strong heterogeneity of endothelial cells, the endothelial cell properties of different blood vessels may be completely different. Therefore, the inventors selected human coronary artery endothelial cells (Human Coronary Artery Endothelial Cells, HCAEC) and human aortic smooth muscle cells (Human Aortic Smooth Muscle Cells, HASMC) to repeat the above experiment as described in Example 1.

[0081] The main experimental reagents and test methods used in this example are the same as those in Example 1 except that the HCAEC and HASMC cells used were purchased from ScienCell, USA. I won't repeat them here.

[0082] The experimental results are as follows:

[0083] Such as figure 2 As shown, the HCAEC after miR-375 transfection, that is, the miR-375 transfection group was compared with the negative control group (ie, HUVEC cells transfected with scamble), β-gal staining ...

Embodiment 3

[0085] Example 3 Changes of other aging-related molecules or phenotypes when miR-375 is overexpressed

[0086] Since β-gal staining is only one of the characteristics of senescent cells. In order to further confirm that miR-375 is related to vascular aging, the inventors tested the effects of miR-375 on other aging-related molecules or phenotypes in the HUVEC model.

[0087] The HUVEC cells and main reagents used in this example are the same as those in Example 1. The specific operation steps are as follows:

[0088] 1. Western blot

[0089] 1) 48 hours after transfection of HUVEC cells, the cells were lysed, and the total protein of the cells was extracted.

[0090] 2) The total protein is subjected to polyacrylamide gel denaturing electrophoresis.

[0091] 3) The protein was transferred to the membrane and blocked with 5% BSA.

[0092] 4) Incubate p16 and p21 protein antibodies.

[0093] 5) Secondary antibody incubation.

[0094] 6) Elution and band development, where...

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Abstract

The invention discloses application of an miR-375 inhibitor in preparation of vascular senescence resistant medicines, and belongs to the field of biological medicines. According to the application, miRNA promoting vascular cell senescence is systematically screened, which discovers that miR-375 has a remarkable promoting effect for senescence vascular cells, especially vascular endothelium; then, miR-375 is verified in other types of vascular related cell model, and the effect of promoting vascular cell senescence of the miR-375 can be determined by acquiring the changing result of other senescence related molecules or phenotype when miR-375 is overexpressed. On the basis, experiments further determine that the miR-375 inhibitor is favorable to endothelial cell senescence induced by cell resisting oxidative stress, and can be used for effectively retarding vascular cell senescence. A novel scientific accordance is provided to researches and development of targeted vascular senescence prevention and treatment method, and a new thread is provided to development of new vascular senescence resistant medicines, especially vascular cell senescence resistant medicines and novel vascular senescence molecular mechanisms.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of miR-375 inhibitors in the preparation of anti-vascular aging drugs. Background technique [0002] Aging is a basic feature of life. Aging can occur at different levels such as individuals, tissues, organs, and cells. It is a process determined by internal programming and affected by environmental factors. William Osler (William Osler) pointed out after completing a large number of human anatomy that "vascular aging is aging", which illustrates the important role of vascular aging in aging. Vascular aging is closely related to various age-related vascular diseases, such as hypertension, coronary heart disease, stroke, vascular dementia, atherosclerosis and stroke. These diseases seriously affect the health and quality of life of the elderly, and bring a heavy burden to patients and society. Therefore, the study of vascular aging is an important breakthrough in analy...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K45/06A61K48/00A61K31/7105A61P9/00
CPCA61K45/00A61K31/7105A61K45/06
Inventor 田小利李扬
Owner 田小利