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Aging cell model and preparation method thereof

A cell model and stem cell technology, applied in the biological field, can solve the problems of clinical translational research limitations, inability to understand human aging, and large differences.

Active Publication Date: 2018-03-30
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Model organisms such as nematodes, fruit flies, and mice are very different from humans. The use of model organisms cannot better understand the process of human aging at the theoretical level, and it also greatly limits clinical translation research. Therefore, the new research on human aging The model needs to be generated

Method used

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  • Aging cell model and preparation method thereof
  • Aging cell model and preparation method thereof
  • Aging cell model and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Example 1. Establishment and identification of ATF6-deficient embryonic stem cell lines

[0072] This embodiment relates to the targeted inactivation of the human ATF6 gene in human embryonic stem cells (the genome sequence is GenBank: NC_000001.11 at No. 161,766,244-161,964,070, updated on PRI 12-JUL-2017; the cDNA sequence is GenBank: NM_007348 .3, updated on PRI 10-JUL-2017), to disable ATF6 function, and obtain human embryonic stem cells with ATF6 function loss. Since the biological function of ATF6β is rarely reported, ATF6 is directly used to represent ATF6α, that is, ATF6 in this paper is ATF6α.

[0073] The present invention firstly designs and obtains the homology arm sequence targeting at both sides of the No. 1 exon of the ATF6 gene in the human genome by molecular cloning methods, and then constructs it into the pCR2. Gift from Professor Belmonte, recorded in "Duan, S., Yuan, G., Liu, X., Ren, R., Li, J., Zhang, W., Wu, J., Xu, X., Fu, L ., Li, Y., et al. ...

Embodiment 2

[0129] Example 2, Preparation of ATF6- / -MSC by Directed Differentiation of ATF6- / -ESC in vitro

[0130] In the present invention, the ATF6- / -ESCs obtained in Example 1 were further differentiated into mesenchymal stem cells (ATF6- / -MSCs) in vitro, and it was found that ATF6- / -MSCs could exhibit typical cell senescence symptoms.

[0131] The specific method is as follows:

[0132] 1. Directed differentiation of ATF6- / - mesenchymal stem cells

[0133] ATF6- / -ESCs were differentiated into embryoid bodies (EBs) for 14 days, and EBs were seeded in matrigel (Invitrogen)-coated 6-well plates for culture, and cultured for 2 weeks until fibrous cells appeared . After another passaging, the cell populations in which CD73, CD90 and CD105 were all positive were sorted by flow cytometry ( figure 2 Middle A) are ATF6-deficient (ATF6- / -) mesenchymal stem cells (denoted as ATF6- / -MSC). In the present invention, the H9 cell line is directedly induced to differentiate into mesenchymal stem...

Embodiment 3

[0168] Example 3. Detecting the cell phenotype caused by the imbalance of protein homeostasis produced by ATF6- / - MSC cells induced by stimulators.

[0169] In the present invention, during the process of culturing the aforementioned ATF6- / -MSCs derived from ATF6- / -ESCs, abnormalities in protein homeostasis of cells are detected. details as follows:

[0170] When intracellular protein homeostasis is out of balance, intracellular misfolded proteins increase, and abnormal proteins cannot be degraded, resulting in a significant increase in intracellular protein aggregates. After ATF6+ / + MSC (wild type) and ATF6- / - MSC cells were cultured normally to the late passage (10th passage), the cells were digested and collected, and the probes for labeling protein aggregates (ENZO, ENZ-51035-K100PROTEOSTAT Agresome detection kit ) for staining of wild-type and ATF6- / - MSCs. The specific analysis method is as follows:

[0171] 1) Digest and collect cells, centrifuge at 1000 rpm for 5 mi...

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Abstract

The invention discloses an aging cell model and a preparation method thereof. The preparation method of the aging cell model comprises the following steps of reducing the content or activity of multipotential stem cell ATF6, so as to obtain ATF6 deficiency multipotential stem cell; inducing the ATF6 dificiency multipotential stem cell, so as to obtain ATF6 deficiency mesenchymal stem cell, whereinthe ATF6 deficiency mesenchymal stem cell is the aging cell model; the multipotential stem cell is embryo stem cell or induced multipotential stem cell. The aging cell model has the advantages that the cell propagation ability is decreased, the recession speed is accelerated, the survival ability is decreased, the integration ability is decreased, and the protein homeostasis is unbalanced in thecell; the increasing of protein aggregate in the cell can be used for establishing an individualized medicine screening platform which is used for screening candidate (natural) compounds for regulating and maintaining adult stem cell protein homeostasis, and retarding cell ageing.

Description

technical field [0001] The invention relates to an aging cell model and a preparation method thereof in the field of biotechnology. Background technique [0002] Population aging is an increasingly serious social problem facing the world, especially Chinese society, which brings heavy economic and spiritual burdens to society and families, and will affect the sustainable development of society. Therefore, how to delay aging, make the elderly live a long and healthy life, and achieve "healthy aging" is a necessary condition for ensuring long-term social stability and sustainable economic development. Therefore, aging and aging-related diseases have become hotspots and difficulties that urgently need to be solved and breakthroughs in the global scientific research field. Model organisms such as nematodes, fruit flies, and mice are very different from humans. The use of model organisms cannot better understand the process of human aging at the theoretical level, and it also gr...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85C12N5/10
CPCC07K14/4702C12N5/0606C12N5/0662C12N15/85C12N15/907C12N2510/00C12N2800/107C12N2810/10
Inventor 刘光慧曲静王思任若通
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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