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CN-119752783-B - Mesenchymal stem cell secretion component with high IL-10 expression, preparation method thereof and application thereof in preventing and treating ischemic cerebral apoplexy

CN119752783BCN 119752783 BCN119752783 BCN 119752783BCN-119752783-B

Abstract

A mesenchymal stem cell secretion component with high IL-10 expression, a preparation method thereof and application thereof in preventing and treating ischemic cerebral apoplexy belong to the technical field of cerebral apoplexy treatment. In order to solve the problems of clinical medication and medication method of the current stem cells in ischemic cerebral apoplexy, the invention provides a special treatment mesenchymal stem cell secretion component (hMSC-M), wherein the secretion component is a stem cell secretion component with high IL-10 expression obtained by inducing the expanded human mesenchymal cells by utilizing a basic culture medium containing human IFN-gamma protein and TNF-alpha mimetic peptide, harvesting, culturing and repeatedly freezing and thawing. According to the invention, animal experiments show that hMSC-M has remarkable effects on preventing and treating ischemic cerebral apoplexy, so that a new medicinal application is developed for stem cell application, a foundation is laid for developing related medicaments for efficiently preventing and treating ischemic cerebral apoplexy injury, and a new application scheme and thinking are provided.

Inventors

  • LI HULUN

Assignees

  • 黑龙江优本干细胞研究有限公司

Dates

Publication Date
20260508
Application Date
20241224

Claims (5)

  1. 1. A preparation method of a mesenchymal stem cell secretion component with high IL-10 expression, which is characterized by comprising the following steps: s1, performing amplification culture, namely obtaining a sufficient amount of human mesenchymal stem cells by amplification culture, wherein the sufficient amount of human mesenchymal stem cells are used as first mesenchymal stem cells; S2, performing induction culture, namely placing the first mesenchymal stem cells obtained in the S1 into an induction culture medium, and culturing 12-24 h at the temperature of 2-5% O 2 ,5% CO 2 and 37 ℃ to obtain second mesenchymal stem cells, wherein cytokines are added into the induction culture medium on the basis of a basic culture medium, the cytokines are human IFN-gamma mimetic peptides and TNF-alpha mimetic peptides, the amino acid sequence of the human IFN-gamma mimetic peptides is shown as SEQ ID NO.1, and the amino acid sequence corresponding to the TNF-alpha mimetic peptides is NKHNRKI; S3, harvesting and culturing, namely discarding the culture solution obtained by the culture in S2, washing cells twice by using normal saline, digesting the harvested cells by trypsin, inoculating the harvested cells into an ultralow adsorption 96U bottom plate with an inoculum size of 1X 10 6 /mL, centrifuging, adding alpha-MEM culture medium containing 5-10% human AB serum, and culturing at a temperature of 5% O 2 ,5% CO 2 and 37 ℃ for 12 h; S4, repeatedly freezing and thawing, namely placing the 96-well plate in a-80 refrigerator to freeze 8 h, thawing at room temperature, repeating for 2 times, and collecting cell lysate, namely the mesenchymal stem cell secretion component with high IL-10 expression.
  2. 2. The method according to claim 1, wherein the step of S1 is to resuscitate the human mesenchymal stem cells in a 37 ℃ water bath, then inoculate the resultant cells in a basal medium, and culture the resultant cells at a temperature of 5% O 2 ,5% CO 2 and 37 ℃ until the cell confluence is 70% -90%.
  3. 3. The method according to claim 1, wherein the amount of S2 added of the human IFN- γ mimetic peptide is 200-1000U/mL and the amount of TNF- α mimetic peptide added is 5-10 ng/mL.
  4. 4. The method according to claim 3, wherein the optimal addition amount of the human IFN-gamma mimetic is 600U/mL and the optimal addition amount of the TNF-alpha mimetic is 10 ng/mL.
  5. 5. The method of claim 1, wherein the basal medium of S2 is a-MEM medium containing a diabody and 10-20% FBS or diabody and 10-20% human AB serum.

Description

Mesenchymal stem cell secretion component with high IL-10 expression, preparation method thereof and application thereof in preventing and treating ischemic cerebral apoplexy Technical Field The invention belongs to the technical field of cerebral apoplexy treatment, and particularly relates to a mesenchymal stem cell secretion component with high IL-10 expression, a preparation method thereof and application thereof in preventing and treating ischemic cerebral apoplexy. Background Cerebral stroke is the second leading disease to death and disability worldwide. Over time, the morbidity and mortality of such diseases continue to rise, bringing great burden to the patient's home and society. According to the latest epidemiological study, ischemic cerebral apoplexy accounts for up to 69.6% in all cerebral apoplexy cases in China. Ischemic stroke has various pathological types and complex pathogenesis, and relates to inflammatory mediator release, inflammatory cell permeation, damage to blood brain barrier and generation of inflammatory factors. Although the therapeutic window of intravascular mechanical thrombolysis or thrombolysis has been expanded in the treatment of ischemic stroke, the time to treat its stenosis and the bleeding complications from surgery remain significant challenges for the clinician. The development of effective prophylactic and therapeutic measures is therefore essential for reducing the incidence of cerebral hemorrhage and improving the prognosis of patients. Stem cell transplantation therapies are considered to be novel therapeutic approaches to promote brain repair following stroke. Among them, human umbilical cord mesenchymal stem cells are widely paid attention because they are easily available and have fewer ethical problems. But the homing ability and the activity of the transplanted stem cells after homing are poor, and the therapeutic effect is reduced. Although derivatives of stem cells, exosomes and vesicles, promote neurogenesis and reduce inflammatory responses, to some extent, they can circumvent the above problems, but they still require more research as cell-free therapies for clinical use, and intravenous injection is the most widespread route of administration in exosome administration, but intravenous injection may present a risk of stagnation of the pulmonary capillary network leading to infusion problems. How to effectively avoid the above-mentioned dilemma is a key problem to be solved when stem cells are used for treating ischemic stroke. Disclosure of Invention Aiming at the defects of the current stem cells in clinical medication and medication methods of cerebral arterial thrombosis, the invention provides a special treatment mesenchymal stem cell secretion component (hMSC-M), wherein the secretion component is a stem cell secretion component with high IL-10 expression obtained by inducing the expanded human mesenchymal cells by utilizing a basic culture medium containing human IFN-gamma mimetic peptide and TNF-alpha mimetic peptide, harvesting, culturing and repeatedly freezing and thawing. The invention utilizes hMSC-M to pretreat mice, finds that the pre-administration of hMSC-M can increase the proportion and the quantity of B cells in peripheral immune organ lymph nodes, and proves that the pre-use of stem cell secretion components can obviously reduce cerebral ischemia volume and improve spleen weight reduction caused by cerebral ischemia through a cerebral ischemia model, and in addition, the hMSC-M has more obvious treatment effect on cerebral ischemia compared with mesenchymal stem cell secretion supernatant (hMSC-L), can improve pathological damage and blood brain barrier leakage of cerebral ischemia, inhibit the expression of inflammatory factors, and promote the expression of downstream SOCS1/STAT6/p-STAT6 channels through activating nuclear translocation of PPAR-gamma, thereby having important clinical application value. In order to solve the technical problems and realize the corresponding technical effects, the invention provides the following technical scheme: the first object of the present invention is to provide a method for preparing a mesenchymal stem cell secretion component with high expression of IL-10, comprising the steps of: s1, performing amplification culture, namely obtaining a sufficient amount of human mesenchymal stem cells by amplification culture, wherein the sufficient amount of human mesenchymal stem cells are used as first mesenchymal stem cells; S2, performing induction culture, namely placing the first mesenchymal stem cells obtained in the S1 into an induction culture medium, and culturing for 12-24 hours at the temperature of 2-5% O 2,5％CO2 and the temperature of 37 ℃ to obtain second mesenchymal stem cells, wherein cytokines are added into the induction culture medium on the basis of a basic culture medium, the cytokines are human IFN-gamma mimetic peptides and TNF-alpha mimetic peptides, the amino acid sequence of th