Application of adipose-derived stem cells edited by gene editing technology to improvement of differentiation efficiency

A technology of fat stem cells and chondrocytes, applied in the field of fat stem cells edited by gene editing technology, can solve the problems of insufficient research, low efficiency of exogenous growth factors, short half-life, etc., and achieve good induction effect, good induction efficiency and application The effect of shortening the value and induction time

Active Publication Date: 2021-03-02
西部医美生物科技成都有限公司双流医疗分公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the low efficiency and short half-life of exogenous growth factors, some scholars have proposed a long-term stable induction method, that is, using genes to transfect the target genes of the above growth factors to obtain endogenous growth factors, which play a role through the secretory pathway
Although genetic manipulation can control the secretion of specific growth factors, because chondrogenesis is coordinated by multiple growth factors and other signaling molecules, only one gene can be targeted at a time, which limits the application potential of gene transfection. Not enough, there is a lot of room for improvement

Method used

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  • Application of adipose-derived stem cells edited by gene editing technology to improvement of differentiation efficiency
  • Application of adipose-derived stem cells edited by gene editing technology to improvement of differentiation efficiency
  • Application of adipose-derived stem cells edited by gene editing technology to improvement of differentiation efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of adipose stem cells

[0023] Take 10 g of abdominal subcutaneous fat extracted by vacuum liposuction under sterile conditions, rinse it three times with PBS containing 500U / ml double antibody, remove the blood vessels and connective tissue visible to the naked eye, and cut it into pieces with ophthalmic scissors. , and then rinsed repeatedly with PBS to remove red blood cells as much as possible. Add 2 times the volume of 0.2% type I collagenase, shake and mix, and digest in a constant temperature water bath at 37°C for 60 min. Digestion was terminated by adding an equal volume of low-sugar DMEM medium containing 10% FBS. Centrifuge at 1800r / min for 10min. Discard the upper and middle layers, add 2ml complete medium (low sugar DMEM+10%FBS+100U / ml penicillin-streptomycin) to resuspend the cell pellet, filter with a 70μm cell mesh, adjust the cell concentration to 1×10 in the filtered filtrate 8 / L was inoculated into a 60mm cell culture dish at...

Embodiment 2

[0024] Example 2 Gene editing technology targeting miR-221

[0025] According to the precursor sequence of miR221, several sets of gRNA sequences were designed, and the optimal g4-RNA was obtained by screening as the experimental sequence. The specific gRNA sequence is shown in SEQ ID NO: 1, gRNA4: TGTAGATTTCTGTGTTCGTTAGG.

[0026] In Shanghai Sangong Company, the 5' end of the DNA sequence corresponding to gRNA4 was synthesized with the forward nucleotide sequence of CACC, and the 5' end of its complementary strand was added with the reverse nucleotide sequence of AAAC, respectively. The synthesized forward and reverse nucleotide sequences are denatured and annealed to obtain double-stranded DNA fragments. The specific experimental conditions are as follows: 1ul forward nucleotide sequence (100uM); 1ul reverse nucleotide sequence (100uM); 1ul 10×T4Ligation Buffer (NEB); 6.5ul ddH 2O; 0.5ulT4PNK(NEB). The reaction system was placed in a PCR machine, and the reaction conditi...

Embodiment 3

[0028] Example 3 Preparation of monoclonal antibodies targeting RUNX2

[0029] For the conserved region of RUNX2, the inventors screened and obtained antigenic epitopes with high immunogenicity, the specific sequences are as follows:

[0030] DENYSAELRNASAVMKNQVARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIKVTVDGPREPRRHRQKLDDSKPSLFSDRLSDLGRIPHPSMRVGVPPQNPRPSLNSAPSPFNPQGQSQITDPRQAQSSPPWSYDQSYPSYLSQMTSPSIHSTTPLSSTRGTGLPAITDVPRRISGASELGPFSDPRQFPSISSLTESRFSNPRMHYPATFTYTPPVTSGMSLGMSA

[0031] The antigen peptide synthesized with this sequence was used as the immunogen, and it was thoroughly mixed with an equal volume of Freund's complete adjuvant, and BALB / c mice were subcutaneously injected with 50 μg / mice of fusion protein at multiple points. Four weeks after the initial immunization, multiple subcutaneous injections on the back were given to boost the immunization, and 4 weeks later, the immunization was boosted again, with 50 μg of the immunogen per animal each time. Seven to ten days...

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Abstract

The invention relates to application of adipose-derived stem cells edited by a gene editing technology to improvement of differentiation efficiency. After the optimal gRNA sequence is obtained throughscreening and optimization, miR-221 is knocked out to obtain transgenic adipose-derived stem cells, a specific monoclonal antibody is obtained through RUNX2 screening, the monoclonal antibody can inhibit the activity of RUNX2, and after the monoclonal antibody is added into an induction medium, the adipose-derived stem cells can be specifically promoted to be differentiated into chondrocytes, andgood induction efficiency and application values are realized.

Description

technical field [0001] The invention relates to the biological field, in particular to the use of adipose stem cells edited by gene editing technology in improving differentiation efficiency. Background technique [0002] The treatment of articular cartilage lesions, especially knee joint lesions, has always been one of the most challenging clinical problems. Articular cartilage belongs to hyaline cartilage, which has important functions such as buffering mechanical stress and ensuring flexible movement of joints. However, adult articular cartilage lacks blood vessels, nerves, no lymphatic return, and has a single, highly differentiated tissue cell type. The chondrocytes are embedded in a large number of dense matrix, which limits their proliferation and migration ability. As a result, tissue has limited self-healing capacity, and even minor cartilage damage can lead to osteoarthritis, which can develop into progressive inflammation leading to joint degeneration and loss of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/10C07K16/18
CPCC12N5/0655C12N5/0667C07K16/18C12N2501/15C12N2501/155C12N2500/38C12N2501/33C12N2501/60C12N2501/65C12N2510/00C07K2317/56
Inventor 张海涛陈帅
Owner 西部医美生物科技成都有限公司双流医疗分公司
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