Stromal cell protein CCN5 composition and application thereof

A composition and protein technology, applied in the field of biomedicine, can solve the problems of inhibited growth of vascular endothelial cells, lack of endothelium, affecting the effect of drug treatment, etc.

CN112190709APending Publication Date: 2021-01-08SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2021-01-08

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Abstract

A cell communication network factor 5 (CCN5, alias WISP2, CTGFL, CT58) is a member of a stromal cell protein CCN family, and is expressed in various cells and other tissue cells constituting a blood vessel. It is proved in the patent that vascular endothelium and smooth muscle cells CCN5 play different roles in injured angiogenesis intimal hyperplasia, endothelial cells CCN5 can promote injured endothelial cell repair to inhibit intimal hyperplasia by combining an integrin signal path and other mechanisms, and can also inhibit neogenesis intimal hyperplasia by regulating the function of smoothmuscle cells through paracrine; the smooth muscle CCN5 can regulate and control the functions of vascular smooth muscle cells through Filamin A or Thymosin beta 4 or other mechanisms to inhibit neogenesis intimal hyperplasia; and in addition, the recombinant CCN5 protein can also inhibit platelet aggregation. The invention provides application of a CCN5 gene, CCN5 protein, CCN5 active polypeptideor an accelerant thereof, and the CCN5 gene, CCN5 protein, CCN5 active polypeptide or the accelerant thereof are used for preparing a composition or a preparation, and the composition or the preparation is used for treating cardiovascular diseases such as vascular restenosis after coronary heart disease interventional therapy.
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Description

technical field

[0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to the extracellular matrix signal transduction molecule CCN5 gene, protein, related peptides and compositions thereof involved in cell response and the use of these substances in the treatment of vascular endothelial injury, damaged vascular proliferative diseases, Methods and applications in thrombosis, ischemic diseases and diseases related to vascular inflammation. Background technique

[0002] Coronary atherosclerotic heart disease is a major disease that threatens human health worldwide, ranking first among the causes of death. With the wide application and development of percutaneous coronary intervention, PCI (Percutaneous coronary intervention) has become one of the most effective methods for coronary revascularization. Since the world's first coronary intervention was carried out in 1977, PCI surgery has made great progress, from the birth ...

Examples

Embodiment 1

[0098] Example 1 Construction of CCN5 Knockdown Endothelial Cell Stable Strain, Detection of its Effect on Endothelial Cell Proliferation and Migration

[0099] (i) Establishment of mouse CCN5 knockdown cell lines: CCN5 shRNA lentiviral plasmids and control lentiviral plasmids, as well as packaging plasmids pCMV.DR8, pMD2.G (addgene) were transfected into human embryonic kidney via X-tremeGENE9 (Roche) ( HEK)293T cells. After 48 hours, the virus supernatant was collected and concentrated to infect human umbilical vein endothelial cells (HUVECs) to construct stable strains of CCN5 knockdown and control endothelial cells.

[0100] (ii) MTT assay to detect cell proliferation function: Spread the above-mentioned endothelial cells in a 96-well plate, add MTT at 0, 24, 48 and 72 hours, add DMSO after 4 hours, and measure the absorbance at a wavelength of 450 nm on a microplate reader. Such as figure 1 As shown in A, the proliferation ability of CCN5 knockdown stable cells was sig...

Embodiment 2

[0102] Example 2 Construction of vascular endothelial cell CCN5 specific knockout transgenic mice, detection of its effect on neointimal hyperplasia after vascular injury

[0103] CCN5 flox / flox Strain transgenic mice with endothelial cell-specific tamoxifen-inducible Cre recombinase expressing mouse Cdh5(PAC)-Cre ERT2 Hybridization was carried out to construct tamoxifen-inducible CCN5 endothelial cell-specific knockout transgenic mice. Such as figure 2 As shown, the femoral artery guidewire injury model showed that the neointimal hyperplasia area, intima / media ratio and vascular occlusion rate were significantly increased after endothelial cell CCN5 knockout, and the neointimal hyperplasia was significantly increased after endothelial cell CCN5 knockout.

Embodiment 3

[0104] Example 3 Constructing CCN5 Overexpressed Endothelial Cell Stable Strains, and Testing Its Effects on Endothelial Cell Proliferation and Migration

[0105] (i) Establishment of mouse CCN5 overexpression cell line: Transfect human embryonic kidney with CCN5 overexpression lentiviral plasmid, control lentiviral plasmid, and packaging plasmid pCMV.DR8, pMD2.G (addgene) via X-tremeGENE9 (Roche) (HEK) 293T cells. After 48 hours, the virus supernatant was collected and concentrated to infect human umbilical vein endothelial cells (HUVECs) to construct stable strains of CCN5 overexpression and control endothelial cells.

[0106] (ii) MTT assay to detect cell proliferation function: Spread the above-mentioned endothelial cells in a 96-well plate, add MTT at 0, 24, 48 and 72 hours, add DMSO after 4 hours, and measure the absorbance at a wavelength of 450 nm on a microplate reader. Such as image 3 As shown in A, the proliferation ability of CCN5 overexpressed stable strain cel...