阅读说明：本技术 血小板裂解产物促进脐带干细胞活化并协同干预膝骨性关节炎的方法 (Method for promoting umbilical cord stem cell activation and synergistically intervening knee osteoarthritis by using platelet lysate ) 是由 单乐天 于 2020-07-10 设计创作，主要内容包括：血小板裂解产物促进脐带干细胞活化并协同干预膝骨性关节炎的方法,属于脐带干细胞应用技术领域。本发明提出了血小板裂解产物在制备脐带干细胞辅助剂中的应用和血小板裂解产物协同脐带干细胞在制备治疗膝骨性药物中的应用。本发明通过血小板裂解产物的制备,测定了血小板裂解产物纯度、细胞因子含量,测定脐带干细胞增殖活性、周期进展、细胞周期相关基因表达、划痕修复能力、趋化迁移能力和趋化迁移相关基因表达,大鼠行为学及组织病理学观察试验。验证了血小板裂解产物适合作为脐带干细胞的辅助剂,并且进一步明确了基于生长因子的血小板裂解产物作用于脐带干细胞的机制及其在膝骨性关节炎治疗中的协同应用。(A method for promoting the activation of umbilical cord stem cells and cooperatively intervening knee osteoarthritis by using platelet lysate belongs to the technical field of umbilical cord stem cell application. The invention provides application of a platelet lysate in preparing an umbilical cord stem cell adjuvant and application of the platelet lysate in preparing a medicament for treating knee osteoarthritis in cooperation with umbilical cord stem cells. The invention determines the purity and the cytokine content of the platelet lysate, determines the proliferation activity, the cycle progression, the cell cycle related gene expression, the scratch repair capability, the chemotactic migration capability and the chemotactic migration related gene expression of the umbilical cord stem cells, and performs behavioral and histopathological observation tests on rats. The platelet lysate is proved to be suitable for being used as the auxiliary agent of the umbilical cord stem cells, and the mechanism of the platelet lysate acting on the umbilical cord stem cells based on the growth factors and the synergistic application of the platelet lysate in the treatment of knee osteoarthritis are further defined.)

1. Use of platelet lysate as an adjunct to cord stem cells in promoting and enhancing cell therapy.

2. Use of platelet lysates for the proliferation, chemotaxis and regeneration of umbilical cord stem cells based on growth factors.

3. The use of platelet lysate to improve the viability and biological function of umbilical cord stem cells.

4. The application of the platelet lysate and umbilical cord stem cells in preparing a medicament for treating knee osteoarthritis.

5. The application of the platelet lysate and umbilical cord stem cells in preparing cartilage repair medicines for treating knee osteoarthritis.

Technical Field

The invention belongs to the technical field of umbilical cord stem cell application, and particularly relates to a method for promoting umbilical cord stem cell activation and synergistically intervening knee osteoarthritis by using a platelet lysate.

Background

Mesenchymal Stem Cells (MSCs) have self-renewal capacity, multipotentiality, paracrine effect and immunoregulatory function, can promote the regeneration of damaged tissues, and are important therapeutic tools for cell therapy and regenerative medicine. The sources of mesenchymal stem cells are very wide, such as bone marrow, adipose tissue, menstrual blood and the like. Umbilical cord has long been considered a medical waste, but umbilical cord stem cells (human umbilical cord-derived MSCs, huc-MSCs) have higher plasticity and fewer ethical constraints than stem cells derived from bone marrow and the like, and thus umbilical cord is a very promising source of stem cells. huc-MSCs not only have fast proliferation speed and strong amplification capability, but also have the advantages of no formation of teratoma or tumor, strong immunoregulation capability and the like. More interestingly, huc-MSCs were less immunogenic than other stem cells, making huc-MSCs more suitable candidates for allogeneic transplantation.

Under physiological conditions, platelets activate to release growth factors and play an important role not only in hemostasis, but also in wound healing and tissue regeneration. Platelet Lysate (PL) is obtained after freeze-thaw lysis of platelets. The major factors released from PL include Platelet Derived Growth Factor (PDGF), insulin-like growth factor (IGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), transforming growth factor (TGF- β) and Vascular Endothelial Growth Factor (VEGF). These growth factors can promote cell proliferation, chemotaxis and extracellular matrix production, angiogenesis, etc.

MSCs require a separation and in vitro amplification stage in clinical applications such as cell therapy, tissue engineering, regenerative medicine, etc., to achieve clinically significant cell numbers. In clinical applications based on MSCs cell therapy, there is a major technical hurdle that it is difficult to isolate MSCs from tissues with low levels of MSCs content, and to culture MSCs of sufficient quality and quantity. To achieve successful isolation and rapid expansion of MSCs, the culture medium needs to be supplemented with large amounts of biologically active substances, such as fetal bovine serum. Currently, due to the safety issues associated with immune responses and zoonotic infections using fetal bovine serum, most studies will use serum replacement as a medium supplement or select serum-free medium instead of fetal bovine serum. However, serum replacement or serum-free medium often cannot completely replace the effect of fetal bovine serum, and is expensive and not suitable for large-scale expansion of MSCs. The growth factor-enriched PL has great potential, and can enable MSCs to be amplified in a large scale in the absence of fetal calf serum in one culture period, and meanwhile, the stability of the genome is kept for clinical use. The scientific rationale behind this usage is based on the presence of large amounts of growth factors in platelets that promote stem cell proliferation. However, human blood is generally not a convenient source, even though it is safer than fetal bovine serum. Thus PL is not suitable as a replacement for fetal bovine serum, but still can assist in the culture and application of MSCs.

In the prior art, reports about the amplification culture of umbilical cord stem cells by platelet lysate have been provided, and an animal serum-free and foreign protein-free umbilical cord mesenchymal stem cell culture method is disclosed before, but the technology does not provide a specific cell molecular mechanism related to the proliferation, chemotaxis and regeneration of the platelet lysate to the umbilical cord stem cells, and in addition, in the existing research reports, the auxiliary effect of the platelet lysate to stem cell transplantation treatment is lack of the verification in the aspect of animal experiments. In addition, the platelet lysate and the umbilical cord stem cells are combined to be used for treating knee osteoarthritis, so that the reference basis of the auxiliary effect of the platelet lysate on stem cell transplantation treatment in the aspect of in-vivo animal experiments is provided, and the experimental basis is provided for clinical application of the knee osteoarthritis.

Disclosure of Invention

In view of the above problems in the prior art, it is an object of the present invention to devise a method for providing a platelet lysate to promote the activation of umbilical cord stem cells and to synergistically interfere with osteoarthritis of the knee. The invention proves that the platelet lysate is used as an auxiliary agent for promoting and enhancing umbilical cord stem cells in cell therapy, the platelet lysate is used as an auxiliary supplement for the application of the umbilical cord stem cells, and the platelet lysate and the umbilical cord stem cells are combined to be applied to a cartilage repair action mechanism in the treatment of knee osteoarthritis.

In order to achieve the purpose, the invention adopts the following technical scheme:

use of platelet lysate as an adjunct to cord stem cells in promoting and enhancing cell therapy.

Use of platelet lysates for the proliferation, chemotaxis and regeneration of umbilical cord stem cells based on growth factors.

The use of platelet lysate to improve the viability and biological function of umbilical cord stem cells.

The application of the platelet lysate and umbilical cord stem cells in preparing a medicament for treating knee osteoarthritis.

The application of the platelet lysate and umbilical cord stem cells in preparing cartilage repair medicines for treating knee osteoarthritis.

The invention has the following beneficial effects:

(1) it was verified that platelet lysate is suitable as an adjuvant for umbilical cord stem cells. In addition, the growth factor in the platelet lysate plays an important role in the proliferation, chemotaxis, regeneration, etc. of umbilical cord stem cells.

(2) The mechanism of the platelet lysate acting on the umbilical cord stem cells based on the growth factors and the synergistic application of the platelet lysate in the treatment of knee osteoarthritis are verified, and the viability and the biological function of the umbilical cord stem cells are improved. The active action of the platelet lysate on umbilical cord stem cells is utilized to combine the two so as to achieve the potential of synergistic treatment of knee Osteoarthritis (OA).

Drawings

In FIG. 1, A is a diagram of identifying the purity of human platelet lysate by flow cytometry, B is a diagram of detecting the concentration of growth factor of human platelet lysate by ELISA method, and C is a diagram of determining the influence of human platelet lysate on the activity of umbilical cord stem cells by CCK-8 method;

in FIG. 2, A is a diagram of the results of flow cytometry detection, and B is a diagram of the results of cell cycle-related molecular targets;

FIG. 3 is a graph showing the result of the cell scratch test;

in FIG. 4, A is a graph showing the effect of human platelet lysate on the chemotactic migration of umbilical cord stem cells, and B is a graph showing the effect of human platelet lysate on molecular targets associated with the chemotactic migration of umbilical cord stem cells;

in FIG. 5, A is a graph showing the staining results of safranin-O sections of human platelet lysate, and B is a graph showing the results of mechanical allodynia and thermal pain detection.

Detailed Description

The invention will be further illustrated with reference to specific embodiments and the accompanying drawings.

Given that Platelet Lysates (PL) produce abundant growth factors, PL is assumed to be useful not only in cell culture but also as an adjuvant for promoting and enhancing MSCs in cell therapy. To test this hypothesis, human umbilical cord stem cells were isolated and their effects on MSCs were evaluated at both the cellular and molecular level. Since MSCs have significant efficacy in knee Osteoarthritis (OA), the synergistic effect of PL and MSCs in combination on cartilage damage was further evaluated using a rat OA model, providing evidence for the adjuvant effect of PL in MSCs-based cell therapy. At present, the mechanism of PRP (platelet rich plasma) or PL for the treatment of osteoarthritis has not been elucidated, and the use of PL in combination with MSCs for cartilage regeneration has not been attempted. Therefore, the data of the invention surpass the prior knowledge and provide new information for regenerative medicine, namely PL is used as an auxiliary supplement of the application of MSCs, and the PL is combined with the PL to be used in a cartilage repair action mechanism in OA treatment.