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Method for preparing blood vessel cells through transdifferentiation of adipose tissue-derived mesenchymal stem cells, and blood vessel cells and application thereof

A technology of mesenchymal stem cells and vascular cells, applied in the field of transdifferentiation of adipose-derived mesenchymal stem cells to prepare vascular cells, can solve the problems of low efficiency of iPS cells, restriction of iPS cell differentiation, cumbersome operation, etc.

Pending Publication Date: 2019-07-23
深圳市伊思科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the traditional method generally involves reprogramming fibroblasts into iPS cells, and then inducing iPS cells to differentiate into vascular endothelial cells. Differentiation to vascular endothelial cells, which makes the transformation from fibroblasts to vascular cells less efficient, which cannot meet actual needs

Method used

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  • Method for preparing blood vessel cells through transdifferentiation of adipose tissue-derived mesenchymal stem cells, and blood vessel cells and application thereof
  • Method for preparing blood vessel cells through transdifferentiation of adipose tissue-derived mesenchymal stem cells, and blood vessel cells and application thereof
  • Method for preparing blood vessel cells through transdifferentiation of adipose tissue-derived mesenchymal stem cells, and blood vessel cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Construction of Adenovirus Recombinant Plasmid

[0081] Use Stbl3 competent cells to amplify pShuttle-CMV-ETV2 plasmids and pAdEasy-2 plasmids (addgene, Plasmid#16401), and extract pShuttle-CMV-ETV2 plasmids, pAdEasy-2 plasmids, and For the pAdEasy-2 plasmid, the pShuttle-CMV-ETV2 plasmid was digested with PacI from NEB Company, identified by electrophoresis, and the linear pShuttle-CMV-ETV2 plasmid was recovered. Wherein, the pShuttle-CMV-ETV2 plasmid is obtained by inserting the ETV2 transcription factor into the plasmid pShuttle-CMV (addgene, Plasmid #16403).

[0082] The linear pShuttle-CMV-ETV2 plasmid and the pAdEasy-2 plasmid were mixed at a molar ratio of 2:1, and transferred into BJ5183E.coli competent cells for intracellular recombination to obtain a bacterial solution containing the adenovirus recombinant plasmid. Spread the bacterial solution on a kanamycin-resistant LB culture dish and culture it upside down at 37°C for 12 hours, pick white, small-diameter...

Embodiment 2

[0084] Preparation of adenovirus carrying ETV2 transcription factor (ie ADV-ETV2)

[0085] 1) Digest the adenovirus recombinant plasmid with Pac I from NEB Company, then extract the digested adenovirus recombinant plasmid through phenol-chloroform extraction, desalting, and ethanol precipitation to recover the linearized adenovirus recombinant plasmid, and wash it with sterile water Dissolve to obtain a linearized adenovirus recombinant plasmid solution with a concentration not lower than 500ng / uL.

[0086] 2) Inoculate 293A cells into a 35mm culture dish at 37°C, 5% CO 2 Cultured in an incubator to a confluence of 60%. Based on the amount of 5ug linearized adenovirus recombinant plasmid added to each 35mm culture dish, and according to the amount of 3uL transfection agent added per 1ug linearized adenovirus recombinant plasmid, the linearized adenovirus recombinant plasmid and transfection Reagent (FuGene 6 (Promega)) was mixed and acted for 15 minutes to form a transfectio...

Embodiment 3

[0115] Transdifferentiation of adipose-derived mesenchymal stem cells to prepare vascular cells (i.e. endothelial cells)

[0116] 1. Cell inoculation

[0117] Day-1: Inoculate mesenchymal stem cells (ASC cells) into 12 wells of a 12-well plate, 3.5×10 4 cells / well, placed at 37°C, 5.0% CO 2 cultured in an incubator.

[0118] The experiment was divided into two groups, and each group had two parallel controls. The settings of each group are shown in Table 1 below.

[0119] Table 1: Grouping situation

[0120]

[0121]

[0122] Wherein, "negative control" means no virus infection, and "ADV-ETV2" means adenovirus carrying ETV2 transcription factor.

[0123] 2. Virus infection

[0124] 1) Day0: digest the ASC cells in the 12-well plate, and count the number of cells. Put the ASC cell complete medium into a 37°C water bath to heat, add ADV-HR (Shandong Weizhen Biotechnology Co., Ltd.) to obtain ADV-HR complete medium containing 10 μg / mL, add 10 μg / mL The adenovirus car...

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Abstract

The invention relates to a method for preparing blood vessel cells through transdifferentiation of adipose tissue-derived mesenchymal stem cells, and the blood vessel cells and an application thereof.The method for preparing blood vessel cells through transdifferentiation of adipose tissue-derived mesenchymal stem cells comprises the following steps of infecting the adipose tissue-derived mesenchymal stem cells with adenovirus carrying ETV2 transcription factors, and performing culture so that the adipose tissue-derived mesenchymal stem cells are subjected to re-programming; and placing the re-programmed adipose tissue-derived mesenchymal stem cells in blood vessel cell disintegrating culture mediums, and performing disintegration and culture so as to obtain the blood vessel cells. Through the adoption of the method, the adipose tissue-derived mesenchymal stem cells can be directly transdifferentiated into the blood vessel cells, and the transdifferentiation efficiency is high.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing vascular cells through transdifferentiation of adipose-derived mesenchymal stem cells, vascular cells and applications thereof. Background technique [0002] Cardiovascular and cerebrovascular diseases are collectively referred to as cardiovascular and cerebrovascular diseases. disease. Cardiovascular and cerebrovascular diseases seriously threaten human health and are characterized by high prevalence, disability and mortality. Although the reconstruction of the blood transport system by means of traditional medicine and surgery can relieve the clinical symptoms of cardiovascular and cerebrovascular diseases to a certain extent, this treatment method requires long-term medication or the pain of surgery, and cannot fundamentally treat cardiovascular and cerebrovascular diseases. Cerebrovascular disease. [0003] Shinya Yamanaka of Japan disclosed for the firs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N5/10A61P9/00A61L27/50A61L27/38
CPCC12N15/86C12N5/069A61K35/44A61P9/00A61L27/507A61L27/3882A61L27/3804C12N2710/10043C12N2510/00
Inventor 林硕李松秦伟
Owner 深圳市伊思科生物科技有限公司
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