The Promotion Effects Of Velvet Antler Peptides On Proliferation And Chondrogenesis Differentiation Of Human Adipose Derived Mesenchymal Stem Cells

Articular cartilage injury and defect caused by trauma and chronic osteoarthritis are common in clinic. The capacity of intrinsic repair of cartilage is limited due to its avascularity and the limited proliferation and migration ability of chondrocyte. The treatment methods commonly used today can not really restore the organizational structure and biomechanical property of cartilage. Tissue engineering is the development of the alternates to replace or support the function of defective or injured body parts using the principle of cell biology and engineering.It provides a new method to deal with tissue defect and functional disability.As the primary factor in cartilage tissue engineering, the selection and acquisition of seed cells is most important. Autologous chondrocyte is the only seed cell which has been used in clinic, but its application is limited because of the donor site morbility and difficulty in isolation and cultivation. Embryonic stem cells. induced pluripotent stem cells are far from clinical application because of ethic and security problems. Adult stem cells are widespread in the body.especially the adipose derived stem cells. It is an ideal seed cell for tissue engineering because of its accessibility and abundant resource.How to acquire a large number of stem cells in vitro in a short time,and promote their chondrogenesis differentiate is still problems to be solved. Various cytokines have been used to promote stem cell proliferation and differentiation now. However, they are extremely expensive, and a single cytokine or several cytokines can not minic the in vivo multi-factor environment. So we remind the deer velvet antler which is the only organ that can regenerate every year. In the rapid growth period, the growth rate can be 1-2cm per day. and during the 3 months, the velvet antler will reach a hight of 1 meter, and complete the process of growth, chondrogenesis and ossification. Some studies have revealed that velvet contains a large number of polypeptide growth factor. It is a natural repository of cytokines. The combination of these cytokines is a natural status. Some study revealed many factors have no species specificity. So we choose the velvet antler as an object to explore its effects on proliferation and differentiation of human adipose derived mesenchymal stem cells.First, this study established a convenient and effective method to isolate hADMSCs from subcutaneous adipose tissue:collagenaseⅠdigestion, and adherent culture. We can obtain a large number of uniform hADMSCs from the discarded subcutaneous adipose tissue. Cells showed the typical swirling, fish-like growth. The cells can proliferate at a high speed,the population doubling time is 47.8 hours. Flow cytometry examination reveals that the surface antigen CD105, CD73 and CD90 are positive, and the CD45, CD34, CD14, CD79αand HLA-DR are negetive. Multipotent differentiation is a important feature of mesenchymal stem cells. In this experiment, we differentiate the acquired cells to adipocytes, osteoblasts. and chondrocytes using different inducers to confirm their multipotent ability. The cells used in this experiment are coincidence with the minimal criteria for defining multipotent mesenchymal stromal cells proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Celluar Therapy.Then we extract the natural peptides from new velvet antler using buffer solution and ethanol. isolate and purify its component using ultrafiltration. gel chromatography. high performance liquid chromatography technology. We test the component's activity of promoting adipose stem cells proliferation using cell counting kit-8. We discover that the velvet antler peptides can promote adipose stem cells proliferation, and the oligopeptides have a more better activiey. Mass spectrometry examination reveals its molecular weight is between 500-600Da.Finally, we explore the effect of velvet antler peptides in chondrogenesis of adipose derived mesenchymal stem cells. The velvet antler peptides has little effect in chondrogenesis when used lonely, but it has a synergistic effect with TGF-β1. It can promote the expression of PGC-1α,SOX9,Col2a1 genes, and accumulation of glycosaminoglycans and collagen typeⅡ. So it can promote chondrogenesis of TGF-β1, and the effect is dose-dependent, the suitable concentration is 25ug/ml-50ug/ml. The possible mechanism of this effect may be related to the natural combination of cytokines from velvet antler, and the oligopeptides may be effective by promoting the interaction between cells and extracellular matrix.In short, we establish a simple and effective method to isolate adipose derived mesenchymal stem cells with high homogenous, and we extract and purified a component of oligopeptides from velvet antler, and confirm its promotion effect of proliferation in adipose derived mesenchymal stem cells. In this experiment, we find that the velvet antler peptides extracted by acid and ethanol can not induce chondrogenesis. but it has a synergistic effect with TGF-pM. This effect is dose-dependent in a limited concentration, the suitable concentration is 12.5ug/ml-25.0ug/ml.