【作者】 李鹏飞；

【导师】 张郑瑶；

【作者基本信息】 大连理工大学 ， 生物工程（专业学位）， 2019， 硕士

【摘要】 骨肉瘤是青少年恶性骨肿瘤中最常见的一种,具有患者低龄化,致残率高和性别差异等特点,目前能够有效治疗骨肉瘤的药物并不常见,临床上多采用阿霉素、长春新碱、顺铂等化疗药物,然而这些化疗药物具有副作用大,预后差,易复发,易耐药等缺点,因此对于骨肉瘤治疗的创新药物研究十分重要。鹿茸是我国具有悠久历史的名贵药材,是哺乳动物唯一可再生的器官,含有丰富的氨基酸、生长因子、磷脂等有效成分,在治疗骨折、骨质疏松、提高免疫力以及治疗肝肾疾病具有良好的效果,也有研究发现鹿茸对于肿瘤疾病也具有一定的作用。随着技术的发展,研究人员开始对鹿茸的有效成分进行提取,获得了包括鹿茸多肽、鹿茸胶原、鹿茸蛋白水解肽等有效成分,并对其进行了药理活性的研究,取得了众多研究成果,为鹿茸多肽的应用奠定了坚实的基础。本文采用的鹿茸多肽是本课题组前期获得的,其氨基酸序列为VLSATDKTNVLAAWGKVGGNAPAFGA EALERM。利用MTT方法检测鹿茸多肽对骨肉瘤细胞增殖的影响,将浓度为0、0.625、1.25、2.5、5、10、20、40、80μg/ml的鹿茸多肽作用于骨肉瘤细胞MG-63和U2OS,分别作用24 h、48 h和72 h,实验结果表明从2.5μg/ml开始鹿茸多肽能够显著的抑制骨肉瘤细胞MG-63和U2OS的增殖(p<0.01),并随着时间和浓度的升高,作用更加显著。通过0、20、80μg/ml鹿茸多肽作用于细胞划痕,24小时后对划痕进行测量,并与0 h时的初始划痕宽度对比,计算出骨肉瘤细胞的迁移距离和迁移率。结果显示加入鹿茸多肽的实验组细胞迁移距离明显小于空白对照组(p<0.05),实验表明鹿茸多肽能够显著的抑制骨肉瘤细胞的迁移。通过western blot和qPCR检测骨肉瘤细胞的EMT和凋亡的相关蛋白和基因的变化,发现鹿茸多肽能够显著抑制MG-63中TGF-β1的表达,并且抑制MG-63细胞凋亡和EMT的发生,同时鹿茸多肽能够促进U2OS中TGF-β1的表达,同时促进U2OS细胞的凋亡和EMT的发生。对两种细胞进行TGF-β1沉默后发现MG-63和U2OS细胞的TGF-β1的表达均显著下降,并且两种细胞的凋亡和EMT均被抑制,可以得出TGF-β1是鹿茸多肽调控骨肉瘤细胞的关键因子,鹿茸多肽能够通过干预TGF-β信号通路对骨肉瘤细胞的凋亡和EMT过程进行调控。此外鹿茸多肽对两种骨肉瘤细胞TGF-β1表达的影响是不同的,进而导致鹿茸多肽对两种细胞凋亡和EMT的调控作用不同,这可能是由于MG-63和U2OS细胞的细胞类型不同所致,其作用机制需进一步探究。更多还原

【Abstract】 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.更多还原