鹿茸多肽对人骨髓间充质干细胞BMP-2和Runx2表达的影响

doi:10.13481/j.1671-587x.20150311

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (03): 491-495.doi: 10.13481/j.1671-587x.20150311

鹿茸多肽对人骨髓间充质干细胞BMP-2和Runx2表达的影响

张洪长¹, 张莹², 刘明昕³, 潘志¹, 律广富¹, 陈港¹, 姜鸿远¹, 郭骏骐⁴

1. 长春中医药大学中医药与生物工程研究开发中心药理研究室, 吉林长春 130117;
2. 吉林大学药学院药理学教研室, 吉林长春 130021;
3. 长春中医药大学图书馆参考咨询部, 吉林长春 130117;
4. 长春中医药大学科技处, 吉林长春 130117

收稿日期:2014-10-09 发布日期:2015-08-01
通讯作者: 郭骏骐,副研究员(Tel:0431-86045421,E-mail:kjcchgk@163.com) E-mail:kjcchgk@163.com
作者简介:张洪长(1961-),男,吉林省长春市人,副研究员,医学博士,主要从事分子生物化学与药理学方面的研究。
基金资助:
国家自然科学基金资助课题(81374025);吉林省科技厅科技发展计划项目资助课题(20140414051GH)

Influence of velvet antler polypeptides in expressions of BMP-2 and Runx2 in human bone marrow mesenchymal stem cells

ZHANG Hongchang¹, ZHANG Ying², LIU Mingxin³, PAN Zhi¹, LYU Guangfu¹, CHEN Gang¹, JIANG Hongyuan¹, GUO Junqi⁴

1. Department of Pharmacology, Center of Traditional Chinese Medicine and Bioengineering, Changchun University of Chinese Medicine, Changchun 130117, China;
2. Department of Pharmacology, School of Pharmacy, Jilin University, Changchun 130021, China;
3. Department of Reference, Library of Changchun University of Chinese Medicine, Changchun 130117, China;
4. Department of Technology, Changchun University of Chinese Medicine, Changchun 130117, China

Received:2014-10-09 Published:2015-08-01

摘要/Abstract

摘要：

目的:探讨鹿茸多肽(VAP)对人骨髓间充质干细胞(hBMSCs)中骨形态发生蛋白2(BMP-2) 和骨特异性转录因子2(Runx2)基因和蛋白表达的影响,阐明其对骨合成及hBMSCs向成骨细胞方向分化的作用机制。方法:体外培养的hBMSCs分为对照组和5、10、15、20 g·L^-1 VAP组,通过检测各组hBMSCs中碱性磷酸酶(ALP)活性,确定体外用药最佳药物浓度;CCK-8法检测对照组和10 g·L^-1 VAP组hBMSCs增殖情况;荧光定量 PCR 法与Western blotting法测定对照组和10 g·L^-1 VAP组hBMSCs中BMP-2和Runx2基因与蛋白的表达。结果:VAP各组细胞中ALP活性均高于对照组,其中以10 g·L^-1 VAP组细胞中ALP活性最高(P<0.01);CCK-8法,10 g·L^-1 VAP组hBMSCs增殖率明显高于对照组(P<0.05),VAP诱导培养第9天hBMSCs增殖率最高,且进入平台期,之后hBMSCs的增殖率开始下降;荧光定量 PCR和Western blotting检测,10 g·L^-1 VAP组hBMSCs中BMP-2和Runx2基因及蛋白表达水平明显高于对照组,差异有统计学意义(P<0.05)。结论:VAP能够提高hBMSCs中ALP活性,促进hBMSCs增殖分化,上调hBMSCs中BMP-2及其下游Runx2基因和蛋白的表达,这可能是VAP对hBMSCs骨形成及骨代谢影响的分子调控机制之一。

关键词: 鹿茸多肽, 人骨髓间充质干细胞, 骨形态发生蛋白2, 骨特异性转录因子2

Abstract:

Objective To investigate the influence of velvet antler polypeptides (VAP) in the expressions of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (Runx2) gene and protein in human bone marrow mesenchymalstem cells (hBMSCs), and to clarify the mechanism of VAP in bone synthesis and bone differentiation direction. Methods The cultured hBMSCs were divided into control group and 5, 10, 15, 20 g ·L^-1 VAP groups.By detecting the alkaline phosphatase (ALP) activities in hBMSCs in various groups, the optimal concentration of the drug in vitro was selected;CCK-8 assay was used to detect the proliferation of hBMSCs in control group and 10 g·L^-1 VAP group;the BMP-2 and Runx2 gene and protein expressions in control group and 10 g·L^-1 VAP group were determined with fluorescence quantitative PCR and Western blotting method. Results The ALP activities in VAP groups were higher than that in control group, and the ALP activity in 10 g ·L^-1 VAP group was the highest (P<0.01).The CCK-8 assay Results showed that the proliferation rate of the hBMSCs in 10 g ·L^-1 VAP group was higher than that in control group (P<0.05), the VAP-induced proliferation of cultured hBMSCs was the highest at the 9th day, then went into the plateau and began to decline;the quantitative PCR and Western blotting Results showed that the expression levels BMP-2 and Runx2 gene and protein in the hBMSCs in 10 g ·L^-1 VAP group were significantly higher than those in control group, and the differences were statistically significant(P<0.05). Conclusion VAP can improve the ALP activity in hBMSCs and promote the proliferation and differentiation of hBMSCs, and increase the expressions of BMP-2 gene and its Runx2 gene and protein, which may be one of the molecular regulation mechanisms of VAP for bone formation and bone metabolism of hBMSCs.

Key words: velvet antler polypeptides, human bone marrow mesenchymal stem cells, bone morphogenetic protein-2, runt-related transcription factor 2

中图分类号:

R961

张洪长, 张莹, 刘明昕, 潘志, 律广富, 陈港, 姜鸿远, 郭骏骐. 鹿茸多肽对人骨髓间充质干细胞BMP-2和Runx2表达的影响[J]. 吉林大学学报(医学版), 2015, 41(03): 491-495.

ZHANG Hongchang, ZHANG Ying, LIU Mingxin, PAN Zhi, LYU Guangfu, CHEN Gang, JIANG Hongyuan, GUO Junqi. Influence of velvet antler polypeptides in expressions of BMP-2 and Runx2 in human bone marrow mesenchymal stem cells[J]. Journal of Jilin University Medicine Edition, 2015, 41(03): 491-495.

参考文献

[1] Xiu ZB, Lin JH, Wu ZY, et al.Effect of pilose antler polypeptides on chondrogenic phenotype differentiation of bone marrowderived mesenchymal stem cells in vitro[J].J Clin Rehabilit Tissue Engin Res, 2011, 15(19):3563-3566.

[2] 陈东, 孟晓婷, 刘佳梅, 等.鹿茸多肽对胎大鼠脑神经干细胞体外诱导分化的实验研究[J].解剖学报, 2004, 35(3):240-243.

[3] 牛琼, 杨欣建, 刘黎军.鹿茸多肽促进皮肤创面愈合的研究[J].内蒙古中医药, 2011(23):72-73.

[4] Zha E, Li X, Li D, et al.Immunomodulatory effects of a 3.2 kDa polypeptide from velvet antler of Cervus nippon Temminck[J].Int Immunopharmacol, 2013, 16(2):210-213.

[5] Sui Z, Zhang L, Huo Y, et al.Bioactive components of velvet antlers and their pharmacological properties[J].J Pharm Biomed Anal, 2014, 87:229-240.

[6] Boden SD.The ABCs of BMPs[J].Orthop Nurs, 2005, 24(1):49-54.

[7] Wozney J, Seeherman H.Protein-based tissue engineering in bone and cartilage repair[J].Curr Opin Biotechnol, 2004, 15(5):392-398.

[8] Service RF.Tissue engineers build new bone[J].Science, 2000, 289(5484):1498-1500.

[9] Bosnakovski D, MizunoM, Kim G, et al.Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells[J].Cell Tissue Res, 2005, 319(2):243-253.

[10] 王艳双, 罗速, 张大方, 等.梅花鹿鹿茸I型胶原诱导骨髓基质干细胞成骨分化及其分子机制的研究[J].时珍国医国药, 2013, 24(12):3056-3059.

[11] Rutherford RB, Moalli M, Franceschi RT, et al.Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo[J].Tissue Eng, 2002, 8(3):441-452.

[12] Ripamonti U.Soluble osteogenic molecular signals and the induction of bone formation[J].Biomaterials, 2006, 27(6):807-822.

[13] Yoon BS, Lyons KM.Multiple functions of BMPs in chondrogenesis[J].J Cell Biochem, 2004, 93(1):93-103.

[14] Huang Z, Ren PG, Ma T, et al.Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability[J].Cytokine, 2010, 51(3):305-310.

[15] Noel D, Gazit D, Bouquet C, et al.Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells[J].Stem Cells, 2004, 22(1):74-85.

[16] Ryoo HM, Lee MH, Kim YJ.Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells[J].Gene, 2006, 366(1):51-57.

[17] Shimizu K, Ito A, Honda H.Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering[J].J Biosci Bioeng, 2007, 104(3):171-177.

[18] Karsenty G.Role of Cbfa1 in osteoblast differentiation and function[J].Semin Cell Dev Biol, 2000, 11(5):343-346.

[19] Satija NK, Gurudutta GU, Sharma S, et al.Mesenchymal stem cells:molecular targets for tissue engineering[J].Stem Cells Dev, 2007, 16(1):7-23.

[20] Matsubara T, Kida K, Yamaguchi A, et al.BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation[J].J Biol Chem, 2008, 283(43):29119-29125.

[21] 王翠艳, 魏芳晶, 阴淑莹, 等.大鼠骨髓间充质干细胞的分离、培养和鉴定[J].中国老年学杂志, 2013, 33(5):1086-1088.

[22] 王婷婷, 周雪颖, 刘黎青, 等.骨髓间充质干细胞诱导分化为心肌细胞的研究进展[J].中国老年学杂志, 2013, 34(4):1144-1145.

鹿茸多肽对人骨髓间充质干细胞BMP-2和Runx2表达的影响

Influence of velvet antler polypeptides in expressions of BMP-2 and Runx2 in human bone marrow mesenchymal stem cells

RichHTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	张鹏程, 冷向阳, 路博丞, 张远超, 王英, 王旭凯. 鹿茸多肽联合GDNF基因修饰的雪旺细胞对Aβ_25-35诱导脊髓神经元凋亡的保护作用[J]. 吉林大学学报(医学版), 2018, 44(06): 1190-1193.
[2]	龚庆, 宋秋莹, 邱莉晶, 黄晓巍, 赵文海. 聚丙交酯-乙交酯微球搭载鹿茸多肽联合骨髓间充质干细胞对大鼠坐骨神经损伤的修复作用及其机制[J]. 吉林大学学报(医学版), 2018, 44(06): 1174-1178.
[3]	黄晓巍, 徐岩, 韩冬, 林贺, 律广富, 李晓华, 曲晓波, 张冰. 鹿茸多肽介导心肌干细胞分化对终末心肌分化基因ANP和MLC-2v表达的影响[J]. 吉林大学学报(医学版), 2018, 44(02): 249-253.
[4]	路博丞, 冷向阳, 张鹏程, 王英, 王旭凯. 鹿茸多肽联合GDNF基因修饰的雪旺细胞对人骨髓间质干细胞体外增殖的影响[J]. 吉林大学学报(医学版), 2017, 43(06): 1125-1129.
[5]	朱文赫, 钟秀宏, 张巍, 徐俊杰, 李妍, 沈楠, 张红, 吕士杰. 鹿茸多肽-胶原蛋白/壳聚糖复合材料对家兔下颌骨缺损愈合的促进作用及其机制[J]. 吉林大学学报(医学版), 2017, 43(03): 527-531.
[6]	黄晓巍, 王艳玲, 李哲, 陶贵斌, 张天柱, 李朝政, 徐岩, 刘玥欣, 张丹丹, 曲晓波. 鹿茸多肽对冈田酸致大鼠海马神经元损伤时Tau、Bcl-2和Caspase-3表达的影响[J]. 吉林大学学报(医学版), 2017, 43(01): 26-31.
[7]	吴乃蓬, 王宇, 宋佳, 武振旭, 高田林, 冯祥汝, 付川, 王宗良, 王春艳. 重组人胶原绑定骨形态发生蛋白2在大肠杆菌中的表达、纯化与复性[J]. 吉林大学学报(医学版), 2016, 42(02): 226-230.
[8]	齐鹏鹏, 孟粼, 吴梓萁, 杨涛源, 于士洋, 王景云. 骨形态发生蛋白2在牙槽骨改建中作用的研究进展[J]. 吉林大学学报(医学版), 2015, 41(03): 671-674.
[9]	. 雷公藤多苷对强直性脊柱炎患者成纤维细胞中BMP-2表达的影响[J]. 吉林大学学报(医学版), 2014, 40(06): 1187-1191.
[10]	李智，郎晓讴，宦大为，孙虓. 纳米粒子介导的BMP-2基因转染对移植血管内膜增生的抑制作用[J]. 吉林大学学报(医学版), 2013, 39(1): 82-86.
[11]	徐晓华, 谭岩, 盛传伦, 王冠军. 低剂量辐射诱导人骨髓间充质干细胞分子在P38MAPK通路的信号机制[J]. J4, 2009, 35(5): 779-784.
[12]	邓方阁，张秀英，李艳茹，潘力，李玉林. 人骨髓间充质干细胞体外定向诱导分化为心肌样细胞的超微结构[J]. J4, 2009, 35(1): 13-16.
[13]	邓方阁，张秀英，王心蕊，马英智，李玉林. 体外模拟心肌微环境中人骨髓间充质干细胞向心肌细胞的分化[J]. J4, 2007, 33(2): 257-259.
[14]	姜涛，李叔强，刘建国. 重组PCA13-hBMP2质粒构建Ad5-hBMP2腺病毒载体[J]. J4, 2007, 33(2): 362-365.
[15]	王克利，路来金，宫旭，李相军. 鹿茸多肽-PLGA复合膜促进大鼠周围神经再生[J]. J4, 2006, 32(2): 199-202.