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Method for promoting reprogramming of mouse fibroblasts into myocardial cells under low oxygen conditions

A technology of fibroblasts and cardiomyocytes, applied in epidermal cells/skin cells, animal cells, vertebrate cells, etc.

Inactive Publication Date: 2016-06-01
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There is no method for hypoxia to promote the reprogramming of mouse fibroblasts into cardiomyocytes in the prior art

Method used

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  • Method for promoting reprogramming of mouse fibroblasts into myocardial cells under low oxygen conditions
  • Method for promoting reprogramming of mouse fibroblasts into myocardial cells under low oxygen conditions
  • Method for promoting reprogramming of mouse fibroblasts into myocardial cells under low oxygen conditions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Isolation and cultivation of mouse fibroblasts and cardiomyocytes

[0035] C57BL / 6J mice and Kunming white pups were taken from 1 to 3 days after birth, and the primary fibroblasts were cultured. It was observed that the primary fibroblasts began to swim out and adhere to the wall 3 days after the back skin tissue block adhered to the wall. figure 1 A), the digested cells are in suspension, and they are round transparent balls under the light microscope. The 3rd to 5th passage fibroblasts were selected for induction, the cells at this time have high purity and the best growth state.

[0036] Cardiomyocytes were inoculated in a culture dish by trypsin digestion, and the cells floated in a single spherical shape in the culture medium. After 12 hours of culture, they began to adhere to the wall and grow. They were round and then became spindle-shaped. figure 1 B).

Embodiment 2

[0037] Example 2 Establishment of Mouse Fibroblasts Directly Reprogrammed into Cardiomyocyte Model

[0038] Molecular cloning and lentivirus infection of fibroblasts

[0039] Construction of lentiviral vector

[0040] Primers were designed according to the sequences of Gata4, Mef2c and Tbx5 to amplify Gata4, Mef2c and Tbx5 genes respectively; high-quality recombinant plasmids were extracted and purified, and lentiviral plasmid expression vectors carrying the target genes were constructed. Assisted by GeneCopoeia.

[0041] Virus fluid production

[0042] 293T is a lentivirus packaging cell, which is an anchorage-dependent epithelioid cell, and its growth medium is DMEM (containing 10% FBS). Adherent cells grow and proliferate in culture to form a monolayer of cells. Viruses were packaged in 293T cells that were in good condition and had fewer passages. The 293T cell supernatant 48 hours after transfection was collected, centrifuged and filtered to obtain the virus liquid. ...

Embodiment 3

[0060] Example 3 Effect of hypoxia on the efficiency of direct reprogramming of fibroblasts into cardiomyocytes

[0061] Direct reprogramming of fibroblasts into cardiomyocytes in a hypoxic microenvironment

[0062] During the reprogramming process, cells were cultured in normoxia (control group) and hypoxia (experimental group with 2% O 2 +5%CO 2 +93%N 2 ), choose 6h and 12h hypoxic pretreatment. Mouse skin fibroblasts were infected with lentivirus, and the cell morphology was observed every 24 hours during the reprogramming process. The cell fluorescence appeared on the 3rd day after transfection. At this time, the cell fluorescence was weak and the number was small. The number of fluorescent cells in the hypoxic 12h group was more than that in the hypoxic 12h group, and the number of fluorescent cells in the hypoxic pretreatment group for 12h was more than that in the normoxia group. The fluorescence intensity of the cells at 1, 2, 3, and 4 weeks after transduction grad...

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Abstract

The invention discloses a method for promoting reprogramming of mouse fibroblasts into myocardial cells under low oxygen conditions. A reprogramming factor is utilized to directly reprogram mouse fibroblasts into efficiently differentiated myocardial cells; on such basis, the efficiency of low oxygen on direct reprogramming to generate myocardial cells and the variation of the HIF-1 expression level in the reprogramming process from fibroblasts to myocardial cells are detected, and the effect of low oxygen on the cell direct reprogramming is systematically explained. The expression conditions of the multi-potential transcription factor and epigenetic regulation factor under low oxygen stimulation are detected. The individualized myocardial cells obtained by directly reprogramming body cells are used for repairing myocardial infarction, thereby laying a foundation for enhancing the clinical cellular transplantation efficiency and safety. The research of the relationship between the low oxygen microenvironment and reprogramming provides a new idea for researching the reprogramming mechanism under low oxygen conditions.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for promoting the reprogramming of mouse fibroblasts into cardiomyocytes by hypoxia. Background technique [0002] Cardiovascular disease is a major disease that threatens human health, especially myocardial infarction has become one of the main causes of human death. Many myocardial regeneration strategies are aimed at addressing the shortage of transplanted organs. In myocardial replacement therapy, cardiomyocytes induced by adult cells are a good choice for cell replacement therapy in the treatment of myocardial ischemic diseases. How to safely and efficiently obtain induced cardiomyocytes is an urgent problem to be solved in the treatment of myocardial infarction and heart failure with personalized cell therapy. Direct reprogramming of fibroblasts into cardiomyocytes will provide a new cell source for cardiomyocyte transplantation. Based on previous studies, it is shown...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N5/0657C12N2500/02C12N2501/998C12N2506/09
Inventor 刘慧雯王艳艳师淑君赵丹刘东华王舒瑶陈曦许玮蔚汪思媛
Owner HARBIN MEDICAL UNIVERSITY
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