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Method for resisting human mesenchymal stem cell aging and enhancing stem characteristics of human mesenchymal stem cells

A stem cell and aging technology, applied in embryonic cells, animal cells, vertebrate cells, etc., can solve the problems that the regulation of aging stem cells has not been reported, and achieve the effects of improving self-renewal ability, delaying aging, and promoting expression

Active Publication Date: 2020-11-24
AFFILIATED HOSPITAL OF ZUNYI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the regulatory role of immune cells on senescent stem cells has not been reported so far

Method used

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  • Method for resisting human mesenchymal stem cell aging and enhancing stem characteristics of human mesenchymal stem cells
  • Method for resisting human mesenchymal stem cell aging and enhancing stem characteristics of human mesenchymal stem cells
  • Method for resisting human mesenchymal stem cell aging and enhancing stem characteristics of human mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Isolation, culture and identification of human mesenchymal stem cells

[0037] The amniotic tissue was stripped from the fresh placenta of healthy full-term cesarean section, and the D-PBS solution containing 1% double antibody (final concentration of penicillin: 100 IU / mL, streptomycin: 100 IU / mL, freshly prepared before use) was used Rinse off residual blood and mucus repeatedly. Press 1 cm 2 After the amniotic membrane is cut into pieces, the amniotic membrane is divided into 50 mL centrifuge tubes, and about 2 times the volume of the amniotic membrane tissue is added with 0.05% trypsin digestion solution containing 0.02% EDTA-2Na, and it is digested in a constant temperature water bath at 37 °C at 185 rpm. For about 30 min, filter with a 300-mesh stainless steel filter, discard the supernatant, and repeat the above steps once. Wash the digested amniotic tissue once with D-PBS solution containing 1% double antibody, add an equal volume of 0.5 mg / mL type ...

Embodiment 2

[0038] Example 2: Separation of human peripheral blood immune cells

[0039] Peripheral blood was taken from fresh normal physical examination population and diluted with an equal volume of sterile D-PBS. Add an appropriate amount of Histopaque-1077 to a 15 mL centrifuge tube, slowly add an equal volume of blood diluted with sterile D-PBS along the tube wall, centrifuge at 2000 rpm for 20 min; absorb the middle buffy coat layer, add an equal volume of sterile D-PBS , 1500 rpm, and centrifuge for 10 min; wash with sterile D-PBS repeatedly once, discard the supernatant; suspend the cell pellet in low-sugar DMEM medium containing 10% FBS, and obtain PBMC immune cells. Using the property that peripheral blood mononuclear cells (PBM) are easy to adhere to and grow on plastic cell culture plates, PBM and peripheral blood lymphocytes (PBL) are separated from the above-mentioned PBMC.

Embodiment 3

[0040] Example 3: Comparison of the reversal effect of co-culture of PBMCs with different cell ratios and aging MSCs on aging cells

[0041] Human amniotic MSCs continuously expanded to passage 9 in vitro and exhibiting replicative senescence were taken, and 10 4 The density per well was inoculated in a 12-well cell culture plate, and freshly isolated PBMCs were added after 16 h, wherein the density of PBMCs was 10 4 , 10 5 , 10 6 , 2×10 6 , 3×10 6 and 4×10 6 / well, the ratios of PBMC and MSC co-cultured cells were 1:1, 10:1, 100:1, 200:1, 300:1 and 400:1, respectively. After PBMCs and MSCs were co-cultured for 72 h, the number of positive senescent MSCs was detected by β-galactosidase staining. The results show( figure 2 ), the rate of β-galactosidase-positive cells in the control group was (77.07±7.23)% in the aging MSC cultured to the 10th passage (hereinafter referred to as "MSC-10"), when PBMC:MSC-10=1:1 or 10 :1, compared with the aging control group, the positi...

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Abstract

The invention discloses a method for resisting human mesenchymal stem cell aging and enhancing stem characteristics of human mesenchymal stem cells. According to the method, immune cells in human peripheral blood and aged human mesenchymal stem cells are cultured in a cell-to-cell contact mode, the aging characteristics of the aged human mesenchymal stem cells can be remarkably reversed, the expression of cell aging markers such as beta galactosidase, P21 and P16 proteins is reduced, and meanwhile, the cell cycle of the aged human mesenchymal stem cells can be obviously adjusted, specifically,the number of cells in the G1 stage is reduced, and the number of cells in the S stage is increased. More importantly, the method can remarkably enhance the stem characteristics of the aged human mesenchymal stem cells, such as self-renewal capacity, multiplication capacity and multi-directional differentiation potential, and the cells obtained by the culture mode are used for treating disease models and are safe and effective. The method can be directly applied to long-term in-vitro amplification of the human mesenchymal stem cells, the problem of cell aging in the amplification process is solved, the cell activity is recovered, and the clinical application effect is improved.

Description

technical field [0001] The invention belongs to the field of stem cells and regenerative medicine, and relates to a method for anti-aging of human mesenchymal stem cells and enhancing their stemness characteristics in the long-term in vitro expansion process. Background technique [0002] In recent years, mesenchymal stem cells (mesenchymal stem cells, MSCs) have become increasingly prominent in the treatment of human diseases due to their embryonic stem cell-like characteristics and paracrine capabilities, and have become an ideal cell source for stem cells and regenerative medicine (Arch Pharm Res . 2012;35:213-21. ). According to the US Clinical Trials Registry, MSCs have been used to treat more than a hundred diseases, especially for refractory diseases (Stem Cells TranslMed 2019; 9:17-27.). However, the amount of mesenchymal stem cells from human tissue is extremely limited, and it is difficult to reach the required cell number for the lowest clinical therapeutic dose:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/073C12N5/078C12N5/0786
CPCC12N5/0605C12N5/0665C12N5/0668C12N2502/11C12N2502/1157C12N5/0662C12N5/0663C12N5/0667C12N2502/00C12N2502/1107C12N2502/1114C12N2502/1164C12N2502/1192A61P1/02A61K35/28
Inventor 肖建辉罗熠钟建江余昌胤
Owner AFFILIATED HOSPITAL OF ZUNYI UNIV
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