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Pulmonary artery hypertension treatment drug stem cell screening model and application thereof

A technology for pulmonary arterial hypertension and human embryonic stem cells, which is applied in the direction of cells modified by introducing foreign genetic material, the determination/inspection of microorganisms, and medical preparations containing active ingredients, etc., which can solve problems such as no reports.

Active Publication Date: 2015-11-18
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Human embryonic stem cells have advantages that tumor cells do not have, but whether the cells can be successfully used for anti-pulmonary hypertension drug screening has not been reported yet

Method used

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  • Pulmonary artery hypertension treatment drug stem cell screening model and application thereof
  • Pulmonary artery hypertension treatment drug stem cell screening model and application thereof
  • Pulmonary artery hypertension treatment drug stem cell screening model and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0069] Example 2: Construction of Human Embryonic Stem Cell Id1 Gene Targeting Vector

[0070] 1. Find the genome sequence of the Id1 gene from the database, determine the target site as exon 1, and retrieve the BAC plasmid containing the complete sequence of the Id1 gene [provided by the Sanger Institute] (5' and 3' upstream and downstream 5Kb sequences).

[0071] 2. Bioinformatics analysis: use the AOS computer program to design and select the optimal nucleotide fragment in the candidate region indicated by the arrow for recombination targeting. A total of 4 recombinant targeting sites were designed ( image 3 ): U5 and D3 loci are located 231bp downstream of ATG in exon 1 of Id1 gene; G5 is located about 6kb upstream of U5; G3 is located about 4kb downstream of D3.

[0072] 3. PCR reaction to generate gene-specific recombination fragment process:

[0073] The primers used to amplify the recombinant fragment consisted of 70 bases. The 20bp at the 3' end of the primer is t...

Embodiment 3

[0085] Example 3: Construction of human embryonic stem cell Id1 gene targeting double reporter gene Id1-Venus-Luc cell line

[0086] After being linearized with AsiSI, the plasmid of Id1-Venus-Luc-MC1-DTA was electrotransfected into P57 human embryonic stem cell H9 (a passaged human embryonic stem cell, which can be purchased from the market). Under the condition of 37°C and 25% CO, culture medium was changed every day on DR4MEF-trophoblast cells-(Applied Stem Cell Co., Ltd., USA) in the medium containing serum replacement. Use BioRad electroporation instrument (ZAP: 250V, uF500, TC8.6) electroporation, then use puromycin screening to get 19 clones in total, pick out the green fluorescent integrated plasmid clone (cells with MC1-DTA plasmid type can not survive), detect Consistency between fluorescein activity and endogenous Id gene expression ( Figure 8 ).

[0087] We found that clone 2 was stimulated by the same amount of BMP4, and the luciferase expression level was cons...

Embodiment 4

[0088] Embodiment four: the application of Venus-Luci dual reporter gene driving Id1 promoter system human embryonic stem cell strain model

[0089] The ID1-V-LUChES model stem cells were inoculated in a 96-well plate, and when the cell colonies covered about 80% of the whole well, the original medium was removed, gently rinsed once with PBS, and human embryonic stem cell mTeSR medium (Hangzhou, China) was added. Belden Biotechnology Company) after starvation for 6h, 1H-pyrazol[3,4-d]pyrimidine, 1-benzene-4-(1-piperidinyl)-(CasNo:23000-46-6), 2- Acetylbenzothiophene (CasNo: 22720-75-8), formononetin (CasNo: 485-72-3). Use the above-mentioned medium to dilute to 5, 2.5, 1.25, 0.625, 0.3 μg / mL step by step, add to the 96-well plate inoculated with model stem cells in sequence, do three replicate wells in parallel for each concentration, and at the same time use only 0.1‰ DMSO culture medium was used as blank control. After culturing at 37°C and 5% CO2 for 18-24 hours, the cell...

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Abstract

The invention belongs to a novel pulmonary artery hypertension treatment drug screening technology established by applying a stem cell technology; with a BMP signal downstream key gene Id1 as a target spot, a double-report-gene original is designed and inserted into an Id1 genome to construct a restructuring carrier, the carrier is transferred into a human embryonic stem cell strain, and a stable-expression CGMCC11091 cell strain is obtained; the CGMCC11091 cell strain is stimulated by a to-be-tested anti pulmonary artery hypertension compound, then cells are cracked by a cell cracking fluid, according to a genetic testing system, the method detects luciferase expression activity, with the Id1 promoter driven double-report-gene expression quantity as an indicator, and a pulmonary artery hypertension treating compound screening model having Id1 expression up-regulated is obtained. The invention also discloses an application of the model in screening regulators for up-regulation of bone morphogenetic protein (BMP) signals, regulators for up-regulation of expression level of BMP-2 acceptors, preparations for improving the pulmonary artery blood hemodynamics, preparations for improving pulmonary vascular reconstitution, pulmonary artery hypertension treatment drugs and cardiopulmonary vascular disease treatment drugs.

Description

technical field [0001] The invention belongs to the technical field of using stem cell technology to establish a screening method for new therapeutic drugs for pulmonary arterial hypertension. Background technique [0002] Pulmonary arterial hypertension (PAH) is an increase in pulmonary arterial resistance due to primary pulmonary arterioles. It is a rare disease with a high fatality rate. Pulmonary vascular resistance increased progressively, eventually leading to right heart failure and death of the patient. Its main pathological feature is severe pulmonary vascular remodeling, including intimal fibrosis of small pulmonary arteries caused by endothelial dysfunction and smooth muscle cell proliferation, medial hypertrophy, adventitial hyperplasia, plexiform lesions, and arterial lumen occlusion. In the past 20 years, several new drugs including prostacyclin and its analogues, endothelin receptor antagonists, and phosphodiesterase inhibitors have been approved, but the 3-y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/68C12Q1/66C12Q1/02
CPCC12Q1/02C12Q1/66C12Q1/68C12N5/10C12N15/85A61K31/519G01N33/5023G01N2333/90241
Inventor 杨隽周芳宫世强韦清霞赵双司书毅
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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