FAM60A modified mesenchymal stem cell as well as preparation method and application thereof

A FAM60A, 1. FAM60A technology, applied in the field of FAM60A-modified mesenchyme, can solve problems such as cell proliferation stagnation, and achieve the effects of delaying or even inhibiting aging, improving therapeutic potential, and resisting replication

Pending Publication Date: 2022-04-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of CRISPR-Cas9 to knock out FAM60A in cells leads to the stagnation of cell proliferation

Method used

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  • FAM60A modified mesenchymal stem cell as well as preparation method and application thereof
  • FAM60A modified mesenchymal stem cell as well as preparation method and application thereof
  • FAM60A modified mesenchymal stem cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1 FAM60A intervention modification can affect the expression level of FAM60A protein in AMSC cells:

[0169] Western Blot method was used to detect the protein expression level of FAM60A in native AMSC, AMSC-Ctrl and AMSC-FAM60A cells. The WB strips were scanned by ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd), and then analyzed by Image J software (Rawak Software, Inc. Germany) for gray ratio analysis. The mRNA expression level of FAM60A in the above cells was detected by PCR method.

[0170] Such as figure 1 As shown in A, the expression level of FAM60A protein in the second-generation AMSC (including mAMSC-FAM60A and hAMSC-FAM60A) modified by FAM60A intervention was significantly lower than that of the control group AMSC-Ctrl and natural AMSC; while the FAM60A mRNA in hAMSC and mAMSC cells After FAM60A intervention modification, the difference was not significant, showing that the modification mainly affects the expression of F...

Embodiment 2

[0172] Example 2 FAM60A intervention modification resists AMSC cell senescence induced by culture and passage:

[0173] Natural AMSC, AMSC-Ctrl and AMSC-FAM60A cells derived from mouse and adult adipose tissue were passed to the 8th passage (mAMSC) and 20th passage (hAMSC), respectively, and the cell replication senescence model induced by culture passage was established.

[0174]The staining of native AMSC, AMSC-Ctrl and AMSC-FAM60A cells was compared by SA-β-gal staining. The staining of 5 visual fields was counted by light microscope, and the statistical analysis was carried out. The results showed that the positive rate of SA-β-gal blue staining of natural hAMSC was 33.5±3.1%, the positive rate of hAMSC-Ctrl was 35.9±4.1%, while the positive rate of blue staining of hAMSC-FAM60A cells was only 8.8±1.6% %. The positive rate of SA-β-gal blue staining in natural mAMSC was 66.5±8.2%, that of mAMSC-Ctrl was 73.5±9.3%, but that of mAMSC-FAM60A cells was only 21.2±4.5%.

[017...

Embodiment 3

[0179] Example 3 FAM60A intervention modification inhibits AMSC aging induced by chemotherapy drugs and oxidative stress:

[0180] Cell senescence was induced by doxorubicin (Dox). Spread AMSC on a 6-well plate, culture overnight, then add 200nM Dox to treat for 48 hours, then change the normal medium and culture for another 6 days, collect cells, and use Western Blot method to detect FAM60A and p21 in natural AMSC, AMSC-Ctrl and AMSC-FAM60A cells and p16 protein expression levels. After the WB bands were scanned by ChemiScope Western Blot imaging system, the gray scale ratio analysis was performed by Image J software.

[0181] Such as image 3 It was shown that FAM60A intervention modification can significantly reduce the increase of FAM60A, p21 and p16 protein expression in AMSC cells induced by chemotherapeutic drugs. In addition, in 5-FU, TNF-α, H 2 o 2 And D-galactose-induced cell senescence model, also has a similar phenomenon. It shows that FAM60A intervention mod...

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Abstract

The invention belongs to the technical field of biology, relates to a method for inhibiting aging of mesenchymal stem cells, and provides FAM60A modified mesenchymal stem cells for inhibiting aging of mesenchymal stem cells as well as a preparation method and application of the FAM60A modified mesenchymal stem cells. The aging of the adipose-derived mesenchymal stem cells is inhibited by intervening and modifying the post-transcription level of the FAM60A gene, and compared with the preparation period of the traditional cell component AMSC, the preparation period of the main cell component AMSC-FAM60A of the biological preparation for treating liver diseases provided by the invention can be shortened by 50% or above; moreover, the adverse effect caused by AMSC aging related cell function damage can be eliminated, so that the application potential of the cell preparation in clinical treatment of liver diseases is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for inhibiting the aging of mesenchymal stem cells, in particular to a method for inhibiting the aging of adipose-derived mesenchymal stem cells through FAM60A modification, thereby improving their proliferative ability and therapeutic potential. FAM60A-modified mesenchymal stem cells aged by mesenchymal stem cells and their preparation method and application. Background technique [0002] Mesenchymal stem cells (Mesenchymal stem cells, MSCs) have been proven to have strong differentiation potential, self-renewal ability, immune regulation and targeted therapy functions. MSC cell transplantation has shown clear efficacy in the treatment of various degenerative diseases and acute and chronic tissue injuries. Studies at the animal level have shown that transplantation of MSCs can relieve liver inflammation and promote liver cell regeneration in mouse acute and chronic liver inj...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N15/87A61K35/28A61P1/16A61P39/06
CPCC12N5/0667C12N15/113C12N15/87A61K35/28A61P39/06A61P1/16C12N2510/00C12N2310/14
Inventor 刘艳宁楼国华岑叶蕾齐津津
Owner ZHEJIANG UNIV
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