Stem cell-derived exosome preparation with function of delaying senescence

An exosome and stem cell technology, applied in embryonic cells, germ cells, animal cells, etc., can solve problems such as restricting applications, and achieve the effect of improving cell morphology, reducing DNA damage, and improving therapeutic effects.

Active Publication Date: 2019-01-04
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because embryonic stem cells are restricted by immune rejection and ethics in clinical application, their clinical application is limited. Therefore, scientists are looking for some other ways to replace embryonic stem cells to exert relevant therapeutic effects

Method used

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  • Stem cell-derived exosome preparation with function of delaying senescence
  • Stem cell-derived exosome preparation with function of delaying senescence
  • Stem cell-derived exosome preparation with function of delaying senescence

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1, the present invention provides a method for extracting and identifying exosomes derived from stem cells.

[0015] Reagents such as cell culture medium, double antibody, and trypsin were purchased from Gibco Company; cell culture consumables were purchased from Corning Company.

[0016] The serum required for cell culture is fetal bovine serum that removes exosomes, which was purchased from BI Company. The processing steps are as follows: put the fetal bovine serum in an ultracentrifuge tube, centrifuge at 120,000 g at 4°C for 2 h, and take it out in an ultra-clean bench. Clear, filter with a 0.22 μm needle filter, and store in a -80°C refrigerator for later use.

[0017] See "Animal Cell Culture (Sixth Edition)" for details on the operation steps of cell biology experiments such as cell subculture, cryopreservation and recovery.

[0018] Collect the conditioned medium of hP-MSCs containing exosomes: When the hP-MSCs cultured in a 75 cm2 cell culture flask ar...

Embodiment 2

[0027] Example 2, the present invention provides a method for establishing a senescent cell model.

[0028] During the process of in vitro culture and expansion of mesenchymal stem cells, the cell morphology becomes flat and the proliferation rate slows down. At this time, the expression of cell cycle-related proteins increases, inhibiting cell proliferation, and the activity of β-gal increases, indicating that the cells are in the senescence stage. state, so this replicatively senescent cell was used as a model of cellular senescence.

Embodiment 3

[0029] Example 3, the present invention provides a technique for using exosomes to alleviate the aging of mesenchymal stem cells

[0030] The aged mesenchymal stem cells were treated with exosomes derived from the isolated embryonic stem cells, and the changes in cell proliferation were detected after 12h, 24h, and 48h of treatment. After 48 hours of treatment, extract protein and RNA, detect the expression of related genes and proteins, and detect the DNA damage site γ-H2AX. The detection results of various aging-related indicators show that the exosomes derived from embryonic stem cells can Alleviate the aging of mesenchymal stem cells.

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Abstract

The invention provides a technological means for delaying mesenchymal stem cell senescence and enhancing treatment effect of the mesenchymal stem cells by exosomes secreted by embryonic stem cells. The embryonic stem cells have strong potential of self-renewing and multi-directional differentiation, but cannot be widely applied to medical science due to limitation of immunological rejection and moral principles. The exosomes are vesicles which contain multiple kinds of RNA and protein and are secreted by cells. In recent years, the mesenchymal stem cells are widely applied to clinic treatment,however, with passage amplification in vitro, the mesenchymal stem cells can be senile gradually, and the treatment effect of the mesenchymal stem cells is weakened. The senile mesenchymal stem cellsare treated by the exosomes secreted by the embryonic stem cells, so that the treatment effect of the mesenchymal stem cells is further improved.

Description

technical field [0001] The invention relates to a technical means for delaying the aging of mesenchymal stem cells and enhancing their therapeutic effects by using exosomes secreted by embryonic stem cells, and belongs to the technical field of tissue engineering and new medicine. Background technique [0002] Embryonic stem cells are highly undifferentiated cells that can proliferate indefinitely when cultured in vitro, and have a high potential for self-renewal and multilineage differentiation. Embryonic stem cells can be differentiated into all types of cells and are the most ideal target cells for gene therapy. Due to these excellent characteristics, embryonic stem cells are called "seed cells", which provide a large amount of raw materials for clinical tissue and organ transplantation. However, due to the constraints of immune rejection and ethics in clinical application of embryonic stem cells, their clinical application is limited. Therefore, scientists are looking f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735A61K35/545A61P17/02A61P9/10A61P13/12A61K35/28
CPCA61P9/10A61P13/12A61P17/02A61K35/28A61K35/545C12N5/0606A61K2300/00
Inventor 刘娜张岩李宗金
Owner NANKAI UNIV
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