Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for preparing specific vascular smooth muscle cells and vascular endothelial cells of CADASIL patients

A vascular smooth muscle and vascular endothelium technology, applied in the biological field, can solve the problems of limitations, different genetic backgrounds, and inability to completely simulate the clinical symptoms of patients, achieving good safety, broad application prospects, and avoiding rejection effects.

Pending Publication Date: 2019-04-26
INST OF ZOOLOGY CHINESE ACAD OF SCI
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The study of CADASIL pathogenesis is largely limited by existing disease models
Although the existing CADASIL mouse model can simulate the abnormalities of vascular function and structure of CADASIL patients, it cannot fully simulate all the clinical symptoms of patients, and the genetic background is also different from that of patients
Immortalized primary vascular smooth muscle cells cannot represent normal human cells due to virus infection or plasmid transfection, and it is difficult to obtain a large number of primary cells because CADASIL is a rare disease

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing specific vascular smooth muscle cells and vascular endothelial cells of CADASIL patients
  • Method for preparing specific vascular smooth muscle cells and vascular endothelial cells of CADASIL patients
  • Method for preparing specific vascular smooth muscle cells and vascular endothelial cells of CADASIL patients

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1. Obtainment of induced pluripotent stem cell lines carrying CADASIL patient-specific gene mutations

[0159] 1. Preparation of induced pluripotent stem cell lines carrying CADASIL patient-specific gene mutations

[0160] The present invention is to carry CADASIL-related NOTCH3 gene heterozygous mutations (in this case, c.3226C> T, p.R1076C, CADASIL patient-derived skin fibroblasts with heterozygous mutations in the NOTCH3 gene that have been reported in other literature or clinically confirmed to cause the disease can be reprogrammed to obtain induced pluripotent stem cell lines to normal healthy people The skin fibroblasts were used as controls (WT#1, WT#2).

[0161] The specific method is as follows:

[0162] 1. It will be derived from heterozygous mutations carrying NOTCH3 gene (c.3226C> T, p. R1076C) the ex vivo skin fibroblasts of CADASIL patients and the ex vivo skin fibroblasts derived from normal healthy people were expanded.

[0163] 2. When the cells grow to ...

Embodiment 2

[0196] Example 2: CADASIL-specific induced pluripotent stem cells differentiate into vascular smooth muscle cells and vascular endothelial cells

[0197] In the present invention, the induced pluripotent stem cells derived from WT#1, the induced pluripotent stem cells derived from WT#2, and the induced pluripotent stem cells derived from CADASIL in Example 1 are differentiated into vascular smooth muscle cells or vascular endothelial cells. The specific methods are as follows:

[0198] 1. CADASIL-specific induced pluripotent stem cell line differentiates into vascular smooth muscle cells

[0199] The induced pluripotent stem cells derived from WT#1, induced pluripotent stem cells derived from WT#2, and induced pluripotent stem cells derived from CADASIL obtained in Example 1 were seeded as small clones on a six-well plate coated with Matrigel, and mTESR was used. Culture the medium for about 5 days, and when the density reaches 70% or more, digest the cells with TryPLE to make the cl...

Embodiment 3

[0225] Example 3. Identification of the disease-related cell phenotype of CADASIL-specific vascular smooth muscle cells

[0226] The NF-κB pathway up-regulation and abnormal proliferation occur in the specific vascular smooth muscle cells of the CADASIL patient of the present invention.

[0227] 1. Up-regulation of NF-κB pathway appears in the specific vascular smooth muscle cells of CADASIL patients

[0228] The up-regulation of vascular inflammation plays a key role in the occurrence and development of many vascular diseases. Inflammation is involved in the occurrence and development of atherosclerosis, hypertension-related vascular disease, and diabetic vascular disease. I want to use the CADASIL patient-specific vascular smooth muscle cell model to detect whether there is inflammation in the occurrence and development of CADASIL vascular disease. Up. The NF-κB pathway is a key pathway to regulate inflammation. Therefore, the activation of NF-κB pathway in the vascular smooth mu...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for preparing specific vascular smooth muscle cells and vascular endothelial cells of CADASIL patients. The method for preparing a CADASIL disease cell model providedby the invention comprises the following steps: a) reprogramming in vitro fibroblasts from a CADASIL patient carrying NOTCH3 gene heterozygous mutation, thereby acquiring an induced pluripotent stem cell line; b) directionally differentiating the induced pluripotent stem cell line into vascular wall cells, thereby acquiring the CADASIL disease cell model. According to the invention, a reprogramming technique and a directional differentiation technique are utilized to in vitro acquire the specific induced pluripotent stem cells of CADASIL patients, the specific induced pluripotent stem cells are directionally differentiated into vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC) which are used for researching pathogenesis of CADASIL arteriole lesion, and a platform issupplied for drug screening.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to a preparation method of CADASIL patient-specific vascular smooth muscle cells and vascular endothelial cells. Background technique [0002] Cerebralautosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) with subcortical infarction and leukoencephalopathy is a hereditary cerebrovascular disease caused by heterozygous mutations in the NOTCH3 gene. The clinical manifestations are repeated Onset of transient ischemic attack (TIA) and stroke, progressive cognitive decline and mental disorders. Due to the onset and progressive aggravation of the disease in the youth and middle age, CADASIL can have a serious impact on the quality of life and life span of patients. The pathogenesis of the disease is currently unclear. The establishment of a disease cell model will facilitate further research on the pathogenesis and development of disease treatments. [0003] The...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N5/10C12N15/85C12Q1/02
CPCC12N5/0691C12N5/069C12N15/85C12N2503/02C12N2506/45C12N2510/00C12N2800/107G01N33/5061G01N33/5064G01N2500/10
Inventor 曲静刘光慧令晨王思
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com