Method for establishing disease model of 'human-derived' short QT syndrome

A technology for QT syndrome and disease model, which is applied in the establishment of "human-derived" short QT syndrome disease models, can solve problems such as endogenous gene expression changes and mutations, and achieve accurate research results and increased current density. Effect

Pending Publication Date: 2019-01-25
ZHEJIANG UNIV
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Problems solved by technology

In addition, retroviral delivery of Yamanaka factors into somatic cells has the potential to result in random integration of vector-encoded genes into the host genome, leading to changes in endogenous gene expression and unforeseen mutations

Method used

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  • Method for establishing disease model of 'human-derived' short QT syndrome
  • Method for establishing disease model of 'human-derived' short QT syndrome
  • Method for establishing disease model of 'human-derived' short QT syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Acquisition and cultivation of SQTs patient fibroblasts

[0044] Disinfect the skin surface of the patient with povidone iodine, put the skin piece into the prepared DMEM culture solution under aseptic conditions immediately after removing the skin piece, place the centrifuge tube containing the skin piece on the test tube rack of the ultra-clean bench, and prepare a large 60mm cell culture dish, add 5ml DPBS, transfer the skin piece to the Petri dish for rinsing. Cut the skin block into small pieces of about 1 mm to facilitate the digestion of collagenase. Equipped with 1mg / ml type Ⅰ collagenase solution. Prepare two 1.5ml centrifuge tubes, add about 1.0ml of collagenase solution to each tube, and evenly transfer the cut skins into the centrifuge tubes. Cover the centrifuge tube tightly and place it obliquely in a 37°C incubator, start timing, and place the culture solution at room temperature for later use. After 6 hours of enzyme digestion, take out the...

Embodiment 2

[0045] Example 2: Establishment of SQTs patient-specific iPSC strains

[0046] 2 days before transfection, prepare 1 × 10 6 Skin fibroblasts from patients with SQTs are guaranteed to reach a density of 50-80% on the day of transfection. The cells were replaced with culture medium 1 day before transfection. On the day of transfection, the cells were replaced with 1ml culture medium. Take out an aliquot of Sendai virus from the -80 degree refrigerator, melt it on ice, add it to the cells after it is completely dissolved, and mix it repeatedly. After 24 hours of transfection, the virus was removed and the medium was replaced with fresh culture medium. On day 2 of transfection, no treatment was performed. The culture medium was changed every other day until the 6th day of transfection. On the 7th day of transfection, aspirate the culture medium, rinse the cells with DPBS, digest the cells with 0.25ml TrypLE, centrifuge, and then resuspend the cells in the culture medium at 1x...

Embodiment 3

[0047] Example 3: Generation of SQTs patient-specific cardiomyocytes

[0048]The successfully established iPS cell line begins to differentiate into cardiac muscle at about the 20th passage. First, SQTs patient-specific iPSCs were inoculated into 6-well plates at a ratio of 1:8, and the mTeSR medium was replaced every day. When the cell density reached about 80%, the differentiation operation could be performed. On the first day of differentiation, suck up the old medium, wash it once with myocardial differentiation medium (RPMI+B27-Insulin), add 2ml of differentiation medium containing 8μM CHIR to each well, and replace it with myocardial differentiation medium 2 days later, each well On the third day, 2 ml of myocardial differentiation medium containing 5 μM IWR was added to each well, and the culture was continued for 2 days. On day 5, replace the cardiomyocyte differentiation medium. After the 7th day, the culture medium for cardiomyocytes (RPMI+B27+Insulin) was replaced...

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Abstract

The invention discloses a method for establishing a disease model of 'human-derived' short QT syndrome, specifically for the application of induced pluripotent stem cells (iPSCs) technology to establish a 'human-derived' short QT syndrome (SQTs) disease model. Patient skin fibroblasts from individuals with SQTs disease are reprogrammed to iPSCs, and then differentiated into cardiomyocytes specificto patients with SQTs, to establish a stable model of cardiomyocyte disease in vitro, and use it to analyze disease mechanisms and screen therapeutic drugs; and CRISPR / Cas9 genome editing technologywas used to correct the mutation site of the patient's pathogenic gene, and then the iPSCs were differentiated into cardiomyocytes, and the indexes of iPSCs were detected, which provided a basis for the study of the mechanism of disease.

Description

Technical field: [0001] The present invention relates to the application of somatic cell reprogramming technology to establish patient-specific induced pluripotent stem cell lines and the application of myocardial directed differentiation technology to obtain patient-specific cardiomyocytes, establish a "human-derived" short QT syndrome disease model, and CRISPR / Application of Cas9 genome editing technology. Background technique: [0002] In recent years, many inherited cardiovascular diseases have been reported and their mechanisms have been elucidated, including those with structural abnormalities such as hypertrophic cardiomyopathy, and those without obvious structural changes that can lead to sudden cardiogenic Sudden death disease. Long QT syndrome (LQTs) is the most familiar cardiac ion channelopathy, which mainly causes abnormalities of potassium and sodium channels in cardiomyocytes, but it does not produce obvious cardiac structures lesion. Recently, there has b...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N15/90C12N5/10
CPCC12N5/0657C12N5/0696C12N15/86C12N15/907C12N2506/45C12N2510/00C12N2760/18843C12N2800/107C12N2810/10
Inventor 梁平蒋晨阳果冯冯孙雅逊宫庭钰
Owner ZHEJIANG UNIV
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