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.
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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...
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