| Osteosarcoma is one of the most common malignant bone tumors.It has the characteristics of low age,high disability rate and gender differences.At present,Adriamycin,vincristine and cisplatin are commonly used in clinic because effective drugs for osteosarcoma are not found.These chemotherapeutic drugs have the disadvantages of high side effects,poor prognosis and easy recurrence,so it is very important to research the drugs for osteosarcoma treatment.Antler is a valuable medicinal material with a long history in China.It is the only renewable organ of mammals and contains abundant amino acids,growth factors,phospholipids and other active ingredients.It has been demonstrated that velvet antler can treat fracture,osteoporosis,liver and kidney diseases and improve immunity effectively.It has also been found that velvet antler exerts certain effects on cancer diseases.With the development of technology,researchers began to extract the active ingredients of velvet antler,and obtained the active ingredients including velvet antler polypeptide,velvet antler collagen,velvet antler protein hydrolytic peptide,and carried out the pharmacological activity research,and obtained many research results,which laid a solid foundation for the application of velvet antler polypeptide.The velvet antler polypeptides used in this paper are peptides obtained by ion exchange chromatography,gel filtration chromatography,reverse high-performance liquid chromatography and so on.The amino acid sequence is VLSATDKTNVLAAWGKVGGNAP AFGAEALERM which identified by MALDI-TOF MS.The velvet antler polypeptide of 0,0.625,1.25,2.5,5,10,20,40,80μg/ml was used to treat osteosarcoma cells MG-63 and U2 OS for 24,48 and 72 hours respectively.MTT method was used to detect the effect of antler polypeptide on the proliferation of osteosarcoma cells.It was found that velvet antler polypeptide could significantly inhibit the proliferation of osteosarcoma cells MG-63 and U2 OS from 2.5 μg/ml.With the increase of time and concentration,the effect of velvet antler polypeptide on the proliferation of osteosarcoma cells MG-63 and U2 OS is more significant.The migration distance and migration rate of osteosarcoma cells were calculated by the action of velvet antler polypeptide at 0,20 and 80 μg/ml on cell scratches.The scratches were measured after 24 hours and compared with those at 0 hours.The results showed that the migration distance of cells in the experimental group was significantly shorter than that in the blank control group(p < 0.05).The experiment showed that the migration of osteosarcoma cells could be inhibited significantly by velvet antler polypeptide.Western blot and qPCR were used to detect the changes of proteins and genes related to epithelial-mesenchymal transition(EMT)and apoptosis in osteosarcoma cells.It was found that velvet antler polypeptide could significantly inhibit the expression of TGF-β1 in MG-63 and inhibit the apoptosis and EMT of MG-63 cells.At the same time,velvet antler polypeptide could promote the expression of TGF-β 1 in U2 OS cells,as well as the apoptosis and EMT of U2 OS cells.The expression of TGF-β1 in MG-63 and U2 OS cells decreased significantly after silencing of TGF-β1 at the same time apoptosis and EMT of both cells were inhibited.It was concluded that TGF-β1 was the key factor for velvet antler polypeptide to regulate osteosarcoma cells.Velvet antler polypeptide regulated the apoptosis and EMT process of osteosarcoma cells by regulating TGF-β signaling pathway.In addition,the effect of velvet antler polypeptide on the expression of TGF-β1 in two osteosarcoma cells is different,which leads to the different regulation of velvet antler polypeptide on apoptosis and EMT in two osteosarcoma cells.This may be due to the different cell types of MG-63 and U2 OS cells,and its mechanism needs to be further explored. |
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