梅花鹿茸多肽对小鼠成骨细胞MC3T3-E1生物学活性的影响
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摘要
目的探讨3种不同提取方法的梅花鹿茸多肽对小鼠成骨细胞MC3T3-E1细胞生物学活性的影响.方法分别采用匀浆法、匀浆裂解法、匀浆超声法在新鲜梅花鹿茸中提取梅花鹿茸多肽(velvet antler polypeptiddes,VAP),用BCA法对梅花鹿茸多肽蛋白质浓度进行分析,培养MC3T3-E1细胞,采用MTT法检测3种提取方法获得的梅花鹿茸多肽对细胞增殖率的影响,利用茜素红染色对MC3T3-E1细胞进行矿化定量测定.结果匀浆法多肽得率9.08%;匀浆裂解法多肽得率15.56%;匀浆超声法多肽得率13.38%;鹿茸多肽能明显促进MC3T3-E1细胞的增殖,并且匀浆超声法的增殖作用最强.钙沉积物的比较表明:与对照组比较,匀浆法、匀浆裂解法和匀浆超声法对MC3T3-E1细胞中的矿化现象呈现递增趋势.结论 3种提取方法综合比较,采用匀浆超声破碎方法提取梅花鹿茸多肽,不仅可以节约时间,而且提高了梅花鹿茸多肽的提取量,是较为理想的鹿茸多肽提取方式,并且能明显地促进MC3T3-E1细胞的增殖和矿化.
Objective To investigate the biological activities of sika velvet antler polypeptides in three osteoblasts MC3 T3-E1 cells by three different extraction methods. Method The velvet antler polypeptides( VAP) were extracted from fresh sika velvet antler by homogenization method,homogenate lysis method and homogenization ultrasonic method. The protein concentration of the extracted sika velvet antler polypeptide was analyzed by BCA method. Biological activity assay MC3 T3-E1 cells were used as a model. The cell proliferation rate of sika deer velvet polypeptide extracted by three extraction methods was detected by MTT assay. The mineralization of MC3 T3-E1 cells was quantitatively determined by alizarin red staining. Results The yield of peptide homogenate was 9. 08%; the yield of homogenate lysing peptide was 15. 56%; the yield of homogenate ultrasonic peptide was 13. 38%; the velvet antler polypeptide could significantly promote the proliferation of MC3 T3-E1 cells,and the proliferation of homogenization ultrasonic method is the strongest.The calcium deposits showed that the homogenization method,homogenate lysis method and homogenization ultrasonic method showed an increasing trend of mineralization in MC3 T3-E1 cells compared with the control group. Conclusion The three extraction methods are comprehensively compared. The extraction of sika velvet antler polypeptide by homogenization ultrasonic smashing method can not only save time,but also improve the extraction amount of sika velvet antler polypeptide. It is an ideal extraction method for velvet antler polypeptide to promote obviously proliferation and mineralization of MC3 T3-E1 cells.
Objective To investigate the biological activities of sika velvet antler polypeptides in three osteoblasts MC3 T3-E1 cells by three different extraction methods. Method The velvet antler polypeptides( VAP) were extracted from fresh sika velvet antler by homogenization method,homogenate lysis method and homogenization ultrasonic method. The protein concentration of the extracted sika velvet antler polypeptide was analyzed by BCA method. Biological activity assay MC3 T3-E1 cells were used as a model. The cell proliferation rate of sika deer velvet polypeptide extracted by three extraction methods was detected by MTT assay. The mineralization of MC3 T3-E1 cells was quantitatively determined by alizarin red staining. Results The yield of peptide homogenate was 9. 08%; the yield of homogenate lysing peptide was 15. 56%; the yield of homogenate ultrasonic peptide was 13. 38%; the velvet antler polypeptide could significantly promote the proliferation of MC3 T3-E1 cells,and the proliferation of homogenization ultrasonic method is the strongest.The calcium deposits showed that the homogenization method,homogenate lysis method and homogenization ultrasonic method showed an increasing trend of mineralization in MC3 T3-E1 cells compared with the control group. Conclusion The three extraction methods are comprehensively compared. The extraction of sika velvet antler polypeptide by homogenization ultrasonic smashing method can not only save time,but also improve the extraction amount of sika velvet antler polypeptide. It is an ideal extraction method for velvet antler polypeptide to promote obviously proliferation and mineralization of MC3 T3-E1 cells.
引文
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[2]袁伟杰,周益.骨重建与能量代谢共调节机制的研究进展[J].中国血液净化,2014,13(3):173-175.
[3] Salari Sharif P,Abdollahi M,Larijani B.Current,new and future treatments of osteoporosis[J].Rheumat Int,2011,13(3):289-300.
[4] Chunhui Y,Wenjun C,Hui W,et al.Pilose antler peptide protects osteoblasts from inflammatory and oxidative injury through EGF/EGFR signaling[J].Int J Biol Macromol,2017,99:15-20.
[5]黄佳.雌二醇通过Wnt/β-catenin信号通路调节成骨细胞增殖的作用机制研究[D].长沙:中南大学,2014.
[6] Lee S R,Jeon B T,Kim S J,et al.Effects of antler development stage on fatty acid,vitamin and GAGs contents of velvet antler in spotted deer[J].Asian-Australasian Journal of Animal Sciences,2007,20(10):1546-1550.
[7]罗海波,陈伟,周静峰,等.茭白总蛋白质双向电泳技术体系的建立[J].食品与生物技术学报,2013,32(6):581-585.
[8]单佳莉,黄帮裕,麦霖霞,等.双水相萃取技术在生物技术上的应用[J].广东化工,2014,41(16):131-132.