鹿茸肽促进人脂肪来源间充质干细胞增殖及成软骨分化的研究
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摘要
外伤或慢性骨关节疾病导致的关节软骨损伤和缺损在临床上十分常见,关节软骨由于无血管、淋巴管及神经组织,同时软骨细胞增殖能力有限,不能迁移,因此其自我修复能力非常有限。目前常用的治疗方法均不能真正恢复软骨的组织结构及生物力学特点。组织工程学是应用细胞生物学和工程学的原理,研究开发生物替代物来修复损伤组织形态、恢复损伤组织功能的一门科学。它的提出、建立和发展为治疗组织、器官缺损和功能障碍提供了一种新的方法。
作为组织工程软骨的首要因素,种子细胞的选择和获得对组织的构建至关重要。自体软骨细胞是目前临床唯一得到应用的组织工程软骨种子细胞。但由于其供区有限,分离培养困难,体外增殖能力差,因此应用受到限制。胚胎干细胞、诱导的多潜能干细胞由于伦理、安全性等问题距离临床应用还有很远距离。成体干细胞在体内广泛存在,特别是脂肪来源的间充质干细胞,其来源广泛、获取简单,是理想的组织工程种子细胞。
但如何在体外快速扩增出大量干细胞,并将其更有效的向软骨细胞方向诱导分化依然是目前亟待解决的问题。当前常用的促进干细胞增殖、分化的添加物是各种细胞因子,然而这些细胞因子均十分昂贵,并且单纯应用一种或几种细胞因子也很难模拟体内的多因子环境。由此我们想到了鹿茸。鹿茸是哺乳动物唯一可以完全再生的器官。在其快速生长期,生长速度可达每天1-2cm,并且鹿茸在3个月的时间内将完成生长、软骨化、骨化的全过程。研究表明鹿茸中含有大量多肽类生长因子,是天然的细胞因子库,这种因子的组合是天然的最佳组合。并且许多因子无种属特异性。因此我们选择鹿茸来研究其是否含有能促进脂肪干细胞增殖及分化的有效成分。
首先本实验建立了方便有效的从皮下脂肪组织中分离、纯化hADMSCs的方法：即I型胶原酶消化、贴壁培养法。应用本方法可以从临床废弃的皮下脂肪组织中分离得到大量形态均一、贴壁生长的间充质干细胞。细胞呈典型的漩涡状、鱼群样生长,细胞增殖速度快，群体倍增时间47.8小时。流式细胞术检测所获得的细胞表达CD29、CD105、CD73和CD90抗原,不表达CD45、CD34、CD14、CD79α和HLA-DR。多向分化潜能是鉴定间充质干细胞的重要标志,本实验通过各种不同的诱导剂分别将所获细胞向脂肪细胞、成骨细胞、软骨细胞方向诱导分化,证实其具有多向分化潜能,符合间充质干细胞的鉴定标准。
随后选取新鲜鹿茸,经缓冲溶液及乙醇提取天然鹿茸肽组分,并应用超滤分离、凝胶层析、高效液相色谱等技术对其进行进一步分离纯化。并应用CCK-8实验,对鹿茸肽组分的促脂肪干细胞增殖活性进行研究,结果显示鹿茸肽粗提物能促进hADMSCs的增殖,特别是其寡肽组分,具有更强的促增殖活性。应用质谱法对该寡肽组分进行分子量检测,显示其由分子量位于500-600Da之间的两个寡肽组成。
最后,我们应用细胞微团培养模式,研究鹿茸肽对脂肪间充质干细胞成软骨分化的作用,结果表明单独应用鹿茸肽成软骨诱导分化作用微弱,但其与TGF-β1联合应用可起到协同作用,可以促进细胞内PGC-1α、SOX9、Co12al基因的表达,促进细胞外基质糖胺聚糖和二型胶原的聚集。从而促进脂肪间充质干细胞向软骨细胞分化。其作用呈浓度依赖性,以12.5ug/ml-25.0ug/ml最为适宜。其机制可能与鹿茸肽含多种天然来源的优化组合的细胞因子有关,同时鹿茸肽中的寡肽组分也可能通过促进细胞间及细胞与细胞外基质间相互作用来促进脂肪干细胞向软骨细胞分化。
总之,本实验建立了简单、有效的分离高纯度人脂肪来源间充质干细胞的方法,并从新鲜鹿茸中提取、分离出具有促进hADMSCs增殖活性的组分,证实其为分子量位于500-600Da的两个寡肽成分组成。本实验发现单独应用酸醇法提取的鹿茸肽不能有效使hADMSCs向软骨细胞分化,但其与TGF-β1的成软骨诱导分化作用有明显的协同作用。并且这种作用有一定的浓度依赖性,以12.5ug/ml-25.0ug/ml最为适宜。
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.
作为组织工程软骨的首要因素,种子细胞的选择和获得对组织的构建至关重要。自体软骨细胞是目前临床唯一得到应用的组织工程软骨种子细胞。但由于其供区有限,分离培养困难,体外增殖能力差,因此应用受到限制。胚胎干细胞、诱导的多潜能干细胞由于伦理、安全性等问题距离临床应用还有很远距离。成体干细胞在体内广泛存在,特别是脂肪来源的间充质干细胞,其来源广泛、获取简单,是理想的组织工程种子细胞。
但如何在体外快速扩增出大量干细胞,并将其更有效的向软骨细胞方向诱导分化依然是目前亟待解决的问题。当前常用的促进干细胞增殖、分化的添加物是各种细胞因子,然而这些细胞因子均十分昂贵,并且单纯应用一种或几种细胞因子也很难模拟体内的多因子环境。由此我们想到了鹿茸。鹿茸是哺乳动物唯一可以完全再生的器官。在其快速生长期,生长速度可达每天1-2cm,并且鹿茸在3个月的时间内将完成生长、软骨化、骨化的全过程。研究表明鹿茸中含有大量多肽类生长因子,是天然的细胞因子库,这种因子的组合是天然的最佳组合。并且许多因子无种属特异性。因此我们选择鹿茸来研究其是否含有能促进脂肪干细胞增殖及分化的有效成分。
首先本实验建立了方便有效的从皮下脂肪组织中分离、纯化hADMSCs的方法：即I型胶原酶消化、贴壁培养法。应用本方法可以从临床废弃的皮下脂肪组织中分离得到大量形态均一、贴壁生长的间充质干细胞。细胞呈典型的漩涡状、鱼群样生长,细胞增殖速度快，群体倍增时间47.8小时。流式细胞术检测所获得的细胞表达CD29、CD105、CD73和CD90抗原,不表达CD45、CD34、CD14、CD79α和HLA-DR。多向分化潜能是鉴定间充质干细胞的重要标志,本实验通过各种不同的诱导剂分别将所获细胞向脂肪细胞、成骨细胞、软骨细胞方向诱导分化,证实其具有多向分化潜能,符合间充质干细胞的鉴定标准。
随后选取新鲜鹿茸,经缓冲溶液及乙醇提取天然鹿茸肽组分,并应用超滤分离、凝胶层析、高效液相色谱等技术对其进行进一步分离纯化。并应用CCK-8实验,对鹿茸肽组分的促脂肪干细胞增殖活性进行研究,结果显示鹿茸肽粗提物能促进hADMSCs的增殖,特别是其寡肽组分,具有更强的促增殖活性。应用质谱法对该寡肽组分进行分子量检测,显示其由分子量位于500-600Da之间的两个寡肽组成。
最后,我们应用细胞微团培养模式,研究鹿茸肽对脂肪间充质干细胞成软骨分化的作用,结果表明单独应用鹿茸肽成软骨诱导分化作用微弱,但其与TGF-β1联合应用可起到协同作用,可以促进细胞内PGC-1α、SOX9、Co12al基因的表达,促进细胞外基质糖胺聚糖和二型胶原的聚集。从而促进脂肪间充质干细胞向软骨细胞分化。其作用呈浓度依赖性,以12.5ug/ml-25.0ug/ml最为适宜。其机制可能与鹿茸肽含多种天然来源的优化组合的细胞因子有关,同时鹿茸肽中的寡肽组分也可能通过促进细胞间及细胞与细胞外基质间相互作用来促进脂肪干细胞向软骨细胞分化。
总之,本实验建立了简单、有效的分离高纯度人脂肪来源间充质干细胞的方法,并从新鲜鹿茸中提取、分离出具有促进hADMSCs增殖活性的组分,证实其为分子量位于500-600Da的两个寡肽成分组成。本实验发现单独应用酸醇法提取的鹿茸肽不能有效使hADMSCs向软骨细胞分化,但其与TGF-β1的成软骨诱导分化作用有明显的协同作用。并且这种作用有一定的浓度依赖性,以12.5ug/ml-25.0ug/ml最为适宜。
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.
引文
[1]邱芳萍.马波,王志兵，等.鹿角盘蛋白的分离纯化与活性研究[J].长春工业大学学报：自然科学版.2007,28(3)：144-147.
[2]SJ Suh, KS Kim, A Lee, et al. Prevention of collagen-induced arthritisin mice by Cervus koreanTEMMINCK var. mantchuricus Swinhoe[J]. Environmental Toxicology and Pharmacology,2007,23:147-153.
[3]Kim YK, Kim KS, Chung KH, etal.Inhibitory effects of deer antler aqua-acupuncture, the pilose antler of Cervus Korean TEMMINCK var mantchuricus Swinhoe, on type Ⅱ collagen-induced arthritis in rats[J]. Int Immunopharmacol.2003,3(7):1001-1010.
[4]Kim KS, Choi YH, Kim KH. Protective and anti-arthritic effects of deer antler aqua-acupuncture (DAA), inhibiting dihydroorotate dehydrogenase. on phosphate ions-mediated chondrocyte apoptosis and rat collagen-induced arthritis[J]. Int Immunopharmacol.2004,4(7):963-73.2004,4(7):963-973.
[5]郭颖杰,周秋丽.刘平,等.鹿茸多肤对骨、软骨细胞增殖的实验研究[J],1998，19(2)：74-76
[6]周秋丽,王丽娟.郭颖杰.等.鹿茸多肽对实验性骨折的治疗作用及机理研究[J].白求恩医科大学学报.1999.25(5)：586-588.
[7]李银清.赵雨，唐仁能.等.梅花鹿茸胶原酶解物对去势大鼠骨质疏松症防治作用的实验研究[J].天然产物研究与开发，2010.22(4)：578-581.
[8]段冷昕，马吉胜.翁梁.等.鹿茸总多肽对维A酸致骨质疏松大鼠的防治作用[J].中国药学杂志.2007.42(4)：264-267.
[9]王华，林喆，刘强，等.鹿茸寡肽的制备及其促成骨细胞的增殖作用[J].高等学校化学学报，2008.29(9)：1791-1796.
[10]Li YJ, Kim TH, Kwak HB, etal. Chloroform extract of deer antler inhibits osteoclast differentiation and bone resorption[J]. J Ethnopharmacol.2007, 113(2):191-198.
[11]陈晓东.林建华.鹿茸多肽对体外培养大鼠软骨细胞去分化现象的作用[J].中国骨与关节损伤杂志,2009,24(8)：708-710.
[12]赵文海，赵长伟,闻辉，等.鹿茸多肽对兔骨性关节炎软骨细胞金属蛋白酶mRNA表达的影响[J].中国社区医师，2007.24：18.
[13]侯建平.李丽静,王晓丽，等.纳米鹿茸粗多肽对体外培养的软骨细胞代谢的影响[J].中国中医基础医学杂志.2006,12(7)：555-556.
[14]修忠标.林建华.张春霞.鹿茸多肽对兔软骨细胞体外凋亡的影响[J].福建中医学院学报，2006.16(3)：17-19.
[15]张志平.廖琦.李勇.等.鹿茸多肽对体外正常及凋亡软骨细胞代谢的影响[J].江西中医学院学报.2005.17 (2)：48-49.
[16]李立军.路来金，陈雷.等.鹿茸多肽促进大鼠坐骨神经再生的实验研究[J].辽宁中医杂志.2004,31(4)：343-344.
[17]王克利，路来金.宫旭.等.鹿茸多肽-PLGA复合膜促进大鼠周围神经再生[J].吉林大学学报(医学版).2006，32(2)：199-202.
[18]路来金，王克利.李立军，等.鹿茸多肽对周围神经再生的影响[J].中国修复重建外科杂志，2008，12(12)：1459-1461.
[19]王旭凯.王英，杨有庚,等.鹿茸多肽对p-淀粉样蛋白诱导脊髓神经元细胞凋亡的保护作用[J].浙江中医药大学学报，2009,33(1)：45-47.
[20]李振华.冷向阳.高忠礼.鹿茸多肽对脊髓损伤保护作用的实验研究[J].中国骨伤.2008.21(4)：285-286.
[21]霍玉书,霍虹.鹿茸神经生长因子活性及促分化作用的研究[J].中药新药与临床药理，1997,8(02)：79-81.
[22]严铭铭，曲晓波,钟英杰,等.梅花鹿鹿茸中促进海马神经细胞增殖蛋白的分离纯化[J].中草药.2007,38(8：1163-1167.
[23]徐惠波.王本祥.张洁.等.鹿茸神经节甙酯对小鼠学习记忆功能的影响[J].中国药理学通报.1991.7(5)：385-388.
[24]王晓丽.张大方.鹿茸提取物对东莨宕碱及亚硝酸钠所致小鼠学习记忆障碍的影响[J].长春中医学院学报，2005，21(4)：37-38.
[25]翁梁,周秋丽,池岛桥,等.马鹿茸促进表皮细胞和软骨细胞分裂的新多肽[J].药学学报,2001,36(12)：913-916.
[26]贾晓燕,路来金,宣昭鹏.鹿茸多肽-壳聚糖-蜂蜜混悬剂对猪皮肽褥疮的促愈合作用[J].中国矫形外科杂志，2010,18(6)：498-502.
[27]王丽鸣.鹿茸多肽治疗顽固性鼓膜穿孔30例[J].新中医,2004，36(10)：59-6
[28]曹丽华.韩梅.王丽鸣.鹿茸多肽加速顽固性鼓膜穿孔愈合的疗效观察[J].长春中医学院学报.2006，22(1)：45.
[29]唐巍然，于晓红.阐杰.等.鹿茸多糖对免疫功能低下模型小鼠细胞免疫功能的影响[J].中国中医药科技.2000.7(4)：234.
[30]陈书明，聂向庭.鹿茸醇提物对用环磷酰胺处理的小白鼠红细胞免疫功能的影响[J].经济动物学报,2000,4(11)：23.
[31]Kang SK, Kim KS, Kim SI, et al. Immunosuppressive activity of deer antler extracts of Cervus korean TEMMINCK var. mantchuricus Swinhoe, on type Ⅱ collagen-induced arthritis[J]. in Vitro Cell Dev Biol Anim,2006, 42(3-4):100-107.
[32]陈晓光.杨明，王本详.等.人工培养鹿茸细胞的药理研究[J].中药药理与临床.1990，6(1)：30-32.
[33]田育璋.贺菊香.王丰梅，等.鹿茸对大鼠学丸影响的形态计量[J].青海医学院学报.1997，18(3)：154.
[34]何刚,王本祥.张伟.等.鹿茸多肽对雄鼠黄体生成素和学丸酮分泌的影响[J].中成药.1994,16(11)：33-34.
[35]林志展，施启贤.李聪明.等.探讨台湾水鹿茸粉对改善肾阳虚大鼠性功能的实验研究[C].第七届海峡两岸心血管研讨会.2009：24.
[36]范玉琳.邢增涛.卫功庆，等.鹿茸蛋白的提取分离及其抗肿瘤活性[J].动物经济学报.1998.2(1)：27-31.
[37]熊和丽.鹿茸活性成分的提取分离及其抗肿瘤作用研究[D].西北农林科技大学.2007.
[38]Kim KW, Kim KS, Park SD, et al. Effect of Cervus korean TEMMINCK var. mantchuricus Swinhoe on protease activities, antioxidant and free radical damages in rheumatis arthritis rats[J]. Toxicol In Vitro,2008,22(1):80-86.
[39]陈书明,聂向庭.鹿茸醇提物抗氧化作用的实验研究[J].2000.17(1)：22-24.
[40]陈晓光,金淑莉.邸琳.等.鹿茸提取物体外抗氧化作用[J].中药材.2003.10：733-734.
[41]周冉.李淑芬.张大成.鹿茸提取物体外抗氧化活性分析[J].食品科学2009.30(9)：33-36.
[42]郑帆.李仁宽.王辉林.等.鹿茸冻干粉的酶解及其酶解产物性质的研究[J].中国中药杂志.2010,35(19：2628-2633.
[43]王华.黄宜兵.高科翔.等.酶解鹿茸肽的制备、纯化及抗氧化活性[J].高等学校化学学报.2010,31(12)：2390-2395.
[44]Suttie JM, Gluckman PD, Butler JH, et al. Insulin-like growth factor 1 (IGF-1) antler-stimulating hormone?[J].Endocrinology,1985,116(2):846-8.
[45]Gu L, Mo E, Yang Z, et al. Expression and localization of insulin-like growth factor-I in four parts of the red deer antler[J]. Growth Factors.2007, 25(4):264-279.
[46]郝林琳,刘松财，夏青娟.等.鹿茸多肽的生物学活性研究[J].吉林农业大学学报.2006,28(3)：285-288.
[47]Ko KM, Yip TT, Tsao SW, et al. Epidermal growth factor from deer (Cervus elaphus) submaxillary gland and velvet antler[J]. Gen Comp Endocrinol. 1986,63(3):431-440.
[48]江润祥.高锦明.叶大同.等.鹿茸表皮生长因子[J].动物学报.1987.33(4)：301-308.
[49]Li C, Stanton JA, Robertson TM, et al. Nerve Growth Factor mRNA Expression in the Regenerating Antler Tip of Red Deer (Cervus elaphus)[J]. PLoS One,2007,10:2(1):e148.
[50]Francis SM, Suttie JM. Detection of growth factors and proto-oncogene mRNA in the growing tip of red deer (Cervus elaphus) antler using reverse-transcriptase polymerase chain reaction (RT-PCR)[J]. J Exp Zool, 1998,281(1):36-42.
[51]Lai AK, Hou WL, Verdon DJ, et al. The distribution of the growth factors FGF-2 and VEGF. and their receptors, in growing red deer antler[J]. Tissue Cell,2007,39(1):35-46.
[52]Jackson DW, Simon TM. Chondrocyte transplantation[J]. Arthroscopy,1996, 12:732-738.
[53]Shapiro F, Kiod S, Gincher G. Cell origin and differentiation in the repair of full thickness defect of articular cartilage[J]. J Bone joint Surg(Am),1996, 74 (4):532.
[54]Takagi M, Umetsu Y, Fujiwara M, et al. High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells[J]. J Biosci Bioeng,2007,103(1):98-100.
[55]Yoo JU, Barthel TS, Nishimura K, et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am,1998,80(12):1745-1757.
[56]Homicz MR, Chia SH, Schumacher BL, et al. Human septal chondrocyte redifferentiation in alginate, polyglycolic acid scaffold, and monolayer culture[J]. Laryngoscope,2003,113(1):25-32.
[57]Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs[J]. J Cell Physiol,2006,207(2):331-339.
[58]D'Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances sternness of human MIAMI cells[J]. Bone,2006,39(3):513-522.
[59]Markway BD, Tan GK, Brooke G, et al. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchvmal stem cells in low oxygen environment micropellet cultures[J]. Cell Transplant,2010, 19(1):29-42.
[60]Robins JC, Akeno N, Mukherjee A, et al. Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9[J]. Bone,2005,37(3):313-322.
[61]Lafont JE, Talma S, Murphy CL. Hypoxia-inducible factor 2 alpha is essential for hypoxic induction of the human articular chondrocyte phenotype[J]. Arthritis Rheum,2007,56(10):3297-3306.
[62]Felka T, Schafer R, Schewe B, et al. Hypoxia reduces the inhibitory effect of IL-1beta on chondrogenic differentiation of FCS-free expanded MSC[J]. Osteoarthritis Cartilage,2009,17(10):1368-1376.
[63]Miyanishi K, Trindade MC, Lindsey DP, et al. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro[J]. Tissue Eng,2006,12(6):1419-1428.
[64]Huang CY, Hagar KL, Frost LE, et al. Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[J]. Stem Cells,2004,22(3):313-323.
[65]Thorpe SD, Buckley CT, Vinardell T, et al. The response of bone marrow-derived mesenchymal stem cells to dynamic compression following TGF-beta3 induced chondrogenic differentiation[J]. Ann Biomed Eng,2010, 38(9):2896-2909.
[66]Terraciano V, Hwang N, Moroni L, et al. Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[J]. Stem Cells,2007,25(11):2730-2738.
[67]Kisiday JD, Frisbie DD, Mcllwraith CW, et al. Dynamic compression stimulates proteoglycan synthesis by mesenchymal stem cells in the absence of chondrogenic cytokines[J]. Tissue Eng Part A,2009,15(10):2817-2824.
[68]Wozney JM. The bone morphogenetic protein family:multifunctional cellular regulators in the embryo and adult[J]. Eur J Oral Sci,1998,106 Suppl 1:160-166.
[69]刘勇.转化生长因子-p与骨、软骨代谢及修复[J].国外医学(创伤与外科基本问题分册)，1999,20(2)：67-70.
[70]Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells[J]. Exp Cell Res,1998. 238(1):265-272.
[71]Hellingman CA. Davidson EN. Koevoet W. et al. Smad Signaling Determines Chondrogenic Differentiation of Bone-Marrow-Derived Mesenchymal Stem Cells:Inhibition of Smad1/5/8P Prevents Terminal Differentiation and Calcification [J]. Tissue Eng Part A,2011. 17(7-8):1157-1167.
[72]Tuli R, Tuli S, Nandi S, et al. Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk[J]. J Biol Chem,2003,17; 278(42):41227-41236.
[73]Barry F, Boynton RE, Liu B, et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow:differentiation-dependent gene expression of matrix components[J]. Exp Cell Res,2001,268:189-200.
[74]Sakou T.Bone morphogenetic proteins:from basic studies to clinical approaches[J]. Bone,1998,22(6):591-603.
[75]Bentz H, Nathan RM, Rosen DM, et al. Purification and characterization of a unique osteoinductive factor from bovine bone[J]. J Biol Chem,1989, 5:264(34):20805-20810.
[76]Yokouchi Y, Sakiyama J, Kameda T, et al. BMP-2/-4 mediate programmed cell death in chicken limb buds[J]. Development,1996.122(12):3725-3734.
[77]Onishi T, Ishidou Y, Nagamine T, et al. Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type Ⅱ receptor during fracture healing in rats[J]. Bone,1998, 22(6):605-612.
[78]Lein P, Johnson M, Guo X, et al. Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons[J]. Neuron,1995,15(3):597-605.
[79]Song JJ, Celeste AJ, Kong FM, et al. Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation[J]. Endocrinology,1995, 136(10):4293-4297.
[80]Iwasaki M, Nakahara H, Nakase T, et al. Bone morphogenetic protein 2 stimulates osteogenesis but does not affect chondrogenesis in osteochondrogenic differentiation of periosteum-derived cells[J]. J Bone Miner Res,1994,9(8):1195-1204.
[81]Mehlhorn AT, Niemeyer P, Kaschte K, et al. Differential effects of BMP-2 and TGF-betal on chondrogenic differentiation of adipose derived stem cells[J]. Cell Prolif,2007,40(6):809-823.
[82]Estes BT, Wu AW, Guilak F. Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6[J]. Arthritis Rheum,2006,54(4):1222-1232.
[83]Fujita M, Kinoshita Y, Sato E, et al. Proliferation and differentiation of rat bone marrow stromal cells on poly(glycolic acid)-collagen sponge[J]. Tissue Eng,2005, 11(9-10):1346-1355.
[84]Solchaga LA, Penick K. Porter JD, et al. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells[J]. J Cell Physiol,2005,203(2):398-409.
[85]Chiou M, Xu Y, Longaker MT. Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cells[J]. Biochem Biophys Res Commun,2006,5:343(2):644-652.
[86]Longobardi L, O'Rear L, Aakula S, et al. Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling[J]. J Bone Miner Res,2006,21(4):626-636.
[87]Wang L, Detamore MS. Insulin-like growth factor-Ⅰ improves chondrogenesis of predifferentiated human umbilical cord mesenchymal stromal cells[J]. J Orthop Res,2009,27(8):1109-1115.
[88]Fukumoto T, Sperling JW, Sanyal A, et al. Combined effects of insulin-like growth factor-1 and transforming growth factor-betal on periosteal mesenchymal cells during chondrogenesis in vitro[J]. Osteoarthritis Cartilage. 2003, 11(1):55-64.
[89]Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell.2006. 126(4):663-676.
[90]Friedenstein AJ,Petrakova KV, Kurolesova AI,et al. Heterotopic transplants of bone marrow:analysis of precursor cells for osteogenic and hematopoietic tissues [J]. Transplantation,1968,6:230-247.
[91]Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies[J].Tissue Eng 2001,7:211-228.
[92]Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells[J]. Mol Biol Cell,2002,13(12):4279-4295.
[93]Housman TS, Lawrence N, Mellen BG, et al. The safety of liposuction: results of a national survey. Dermatol Surg,2002,28:971-978.
[94]Aust L, Devlin B, Foster SJ, et al. Yield of human adipose-derived adult stem cells from liposuction aspirates[J]. Cytotherapy,2004,6(1):7-14.
[95]Pittenger MF, Mosca JD, McIntosh KR. Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma[J]. Curr Top Microbiol Immunol,2000,251:3-11.
[96]Sakaguchi Y, Sekiya I, Yagishita K. et al. Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source[J]. Arthritis Rheum,2005,52(8):2521-2529.
[97]Sharpless NE, DePinho RA. How stem cells age and why this makes us grow old[J]. Nat Rev Mol Cell Biol,2007,8:703-713.
[98]Yoshimura K, Shigeura T, Matsumoto D, et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates[J]. J Cell Physiol,2006,208(1):64-76.
[99]Barry FP. Mesenchymal stem cell therapy in joint disease[J]. Novartis Found Symp.2003,249:86-96; discussion 96-102,170-4,239-41.
[100]Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure[J]. Cytotherapy,2006, 8(2):166-177.
[101]Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[J]. Cytotherapy,2006,8(4):315-317.
[102]Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell:a better stem cell than BMSC[J]. Cell Biochem Funct,2008,26(6):664-675.
[103]Puissant B, Barreau C, Bourin P, et al. Immunomodulatory effects of human adipose tissue-derived stem cells:comparison with bone marrow mesenchymal stem cells[J]. Br J Haematol,2005,129:118-129.
[104]国家药典委员会编.中华人民共和国药典(2005年版)一部.
[105]Price J, Allen S, Exploring the mechanisms regulating regeneration of deer antlers[J]. Philos Trans R Soc Lond B Biol Sci,2004,29:359(1445):809-822.
[106]王继峰.生物化学[M].北京.中国中医药出版社.2003,1.35.
[107]Gill I, Lopez-Fandino R, Jorba X, et al. Biologically active peptides and enzymatic approaches to their production[J]. Enzyme Microb Technol,1996, 15:18(3):163-183.
[108]H. Newey, D. H. Smyth. Intracellular hydrolysis of dipeptides during intestinal absorption [J]. J Physiol,1960,152(2):367-380.
[109]Hara H, Funabiki R, Iwata M, et al. Portal absorption of small peptides in rats under unrestrained conditions[J]. J Nutr,1984,114(6):1122-1129.
[110]何瑜璇,刘春生,董栗.天然药物中寡肽的研究进展[C].中华中医药学会第十届中药鉴定学术会议论文集.2010.
[111]鲍加荣,李春义,邢秀梅,等.鹿茸再生及干细胞研究进展[J].中国组织工程研究与临床康复.2008,12(51)：10163-10166.
[112]陶红,梁歧.酶法制备大豆寡肽的研究[J].大豆科学.2004,23(1)：26-29.
[113]周秋丽.刘永强.王颖.等.梅花鹿茸和马鹿茸多肽化学性质及生物活性比较[J].中国中药杂志.2001,26(10)：699-702.
[114]Arden N, Nevitt MC. Osteoarthritis:epidemiology[J]. Best Pract Res Clin Rheumatol, 2006,20(1):3-25.
[115]Jackson DW, Simon TM. Aberman HM. Symptomatic articular cartilage degeneration:the impact in the new millennium[J]. Clin Orthop Relat Res, 2001,(391 Suppl):S14-25.
[116]Kon E, Gobbi A, Filardo G, et al. Arthroscopic second-generation autologous chondrocyte implantation compared with microfracture for chondral lesions of the knee:prospective nonrandomized study at 5 years[J]. Am J Sports Med, 2009,37(1):33-41.
[117]Solchaga LA, Goldberg VM, Caplan AI. Cartilage regeneration using principles of tissue engineering[J].Clin Orthop Relat Res,2001, (391 Suppl):S161-70.
[118]Kim D, Kim CH, Moon JI, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins[J]. Cell Stem Cell, 2009,4(6):472-476.
[119]Kierdorf U, Li Chunyi, Price JS. Improbable appendages:Deer antler renewal as a unique case of mammalian regeneration[J]. Seminars in cell and developmental biology,2008,20(4):535-542.
[120]Li C, Clark DE, Lord EA, et al. sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery [J]. J The Anatomical record,2002,268:125-130.
[121]段冷听.梅花鹿茸多肽的化学结构及其抗肝纤维化作用[D].2007.吉林大学.
[122]Rerat A. Nutritional value of protein hydrolysis products (oligopeptides and free amino acids) as a consequence of absorption and metabolism kinetics[J]. Arch Tierernahr,1995,48(1-2):23-36.
[123]Iwakoshi E, Hisada M, Minakata H. Cardioactive peptides isolated from the brain of a Japanese octopus. Octopus minor[J]. Peptides,2000, 21(5):623-630.
[124]张勇.朱宇旌.刘勇.等.红三叶寡肽对小鼠免疫功能的影响[J]。安徽农业科学.2007.35(35)：11338-11339.11342.
[125]MASUDA O, NAKAMURA Y, TAKANO T. Antihypertensive peptidesarepresentinaortaafteroraladministrationofsourmilkcontaining these peptides to spontaneously hypertensive rats[J]. J Nutr,1996.126:3063-3068.
[126]黄晓明.李祺福.刘用金,等.新生牛牛脑活性肽NBBP-1对人神经母细胞瘤SK-N-SH细胞凋亡的诱导作用[J].厦门大学学报(自然科学版).2009.48(3)：399-405.
[127]Shytle RD, Ehrhart J, Tan J, et al. Oxidative stress of neural, hematopoietic. and stem cells:protection by natural compounds[J]. Rejuvenation Res,2007, 10(2):173-178.
[128]Shytle DR, Tan J, Ehrhart J, et al. Effects of blue-green algae extracts on the proliferation of human adult stem cells in vitro:a preliminary study[J]. Med Sci Monk,2010,16(1):BR1-5.
[129]Tang X, Zhang C, Zeng W, et al. Proliferating effects of the flavonoids daidzein and quercetin on cultured chicken primordial germ cells through antioxidant action[J]. Cell Biol Int,2006,30(5):445-451.
[130]Pervaiz S, Taneja R, Ghaffari S. Oxidative stress regulation of stem and progenitor cells[J]. Antioxid Redox Signal,2009.11(11):2777-2789.
[131]Bickford PC, Tan J, Shytle RD, et al. Nutraceuticals synergistically promote proliferation of human stem cells[J]. Stem Cells Dev.2006,15(1):118-123.
[132]Tacchetti C, Tavella S, Dozin B, et al. Cell condensation in chondrogenic differentiation. Exp Cell Res,1992,200(1):26-33.
[133]Mwale F, Stachura D, Roughley P, et al. Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation[J]. J Orthop Res,2006,24(8):1791-1798.
[134]Ng LJ, Wheatley S, Muscat GE, et al. SOX9 binds DNA. activates transcription, and coexpresses with type Ⅱ collagen during chondrogenesis in the mouse[J].Dev Biol,1997,183(1):108-121.
[135]Kawakami Y, Tsuda M, Takahashi S, et al. Transcriptional coactivator PGC-1alpha regulates chondrogenesis via association with Sox9[J]. Proc Natl Acad Sci U S A,2005,102(7):2414-2419.
[136]郝林琳,刘松财,张明军,等.鲜马鹿茸不同部位多肽的提取及含量分析.吉林农业大学学报.2009,29(4)：378-380.383.
[137]Fukumoto T, Sperling JW, Sanyal A, et al. Combined effects of insulin-like growth factor-1 and transforming growth factor-betal on periosteal mesenchymal cells during chondrogenesis in vitro[J]. Osteoarthritis Cartilage, 2003, 11(1):55-64.
[138]Indrawattana N, Chen G, Tadokoro M, et al. Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun,2004,320(3):914-949.
[139]Kantlehner M, Schaffner P, Finsinger D, et al. Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation[J].Chembiochem,2000, 1(2):107-114.
[140]Au A, Boehm CA, Mayes AM, et al. Formation of osteogenic colonies on well-defined adhesion peptides by freshly isolated human marrow cells[J]. Biomaterials,2007,28(10):1847-1861.
[141]Lin YC, Brayfield CA, Gerlach JC, et al. Peptide modification of polyethersulfone surfaces to improve adipose-derived stem cell adhesion.Acta Biomater[J].2009,5(5):1416-1424.
[2]SJ Suh, KS Kim, A Lee, et al. Prevention of collagen-induced arthritisin mice by Cervus koreanTEMMINCK var. mantchuricus Swinhoe[J]. Environmental Toxicology and Pharmacology,2007,23:147-153.
[3]Kim YK, Kim KS, Chung KH, etal.Inhibitory effects of deer antler aqua-acupuncture, the pilose antler of Cervus Korean TEMMINCK var mantchuricus Swinhoe, on type Ⅱ collagen-induced arthritis in rats[J]. Int Immunopharmacol.2003,3(7):1001-1010.
[4]Kim KS, Choi YH, Kim KH. Protective and anti-arthritic effects of deer antler aqua-acupuncture (DAA), inhibiting dihydroorotate dehydrogenase. on phosphate ions-mediated chondrocyte apoptosis and rat collagen-induced arthritis[J]. Int Immunopharmacol.2004,4(7):963-73.2004,4(7):963-973.
[5]郭颖杰,周秋丽.刘平,等.鹿茸多肤对骨、软骨细胞增殖的实验研究[J],1998，19(2)：74-76
[6]周秋丽,王丽娟.郭颖杰.等.鹿茸多肽对实验性骨折的治疗作用及机理研究[J].白求恩医科大学学报.1999.25(5)：586-588.
[7]李银清.赵雨，唐仁能.等.梅花鹿茸胶原酶解物对去势大鼠骨质疏松症防治作用的实验研究[J].天然产物研究与开发，2010.22(4)：578-581.
[8]段冷昕，马吉胜.翁梁.等.鹿茸总多肽对维A酸致骨质疏松大鼠的防治作用[J].中国药学杂志.2007.42(4)：264-267.
[9]王华，林喆，刘强，等.鹿茸寡肽的制备及其促成骨细胞的增殖作用[J].高等学校化学学报，2008.29(9)：1791-1796.
[10]Li YJ, Kim TH, Kwak HB, etal. Chloroform extract of deer antler inhibits osteoclast differentiation and bone resorption[J]. J Ethnopharmacol.2007, 113(2):191-198.
[11]陈晓东.林建华.鹿茸多肽对体外培养大鼠软骨细胞去分化现象的作用[J].中国骨与关节损伤杂志,2009,24(8)：708-710.
[12]赵文海，赵长伟,闻辉，等.鹿茸多肽对兔骨性关节炎软骨细胞金属蛋白酶mRNA表达的影响[J].中国社区医师，2007.24：18.
[13]侯建平.李丽静,王晓丽，等.纳米鹿茸粗多肽对体外培养的软骨细胞代谢的影响[J].中国中医基础医学杂志.2006,12(7)：555-556.
[14]修忠标.林建华.张春霞.鹿茸多肽对兔软骨细胞体外凋亡的影响[J].福建中医学院学报，2006.16(3)：17-19.
[15]张志平.廖琦.李勇.等.鹿茸多肽对体外正常及凋亡软骨细胞代谢的影响[J].江西中医学院学报.2005.17 (2)：48-49.
[16]李立军.路来金，陈雷.等.鹿茸多肽促进大鼠坐骨神经再生的实验研究[J].辽宁中医杂志.2004,31(4)：343-344.
[17]王克利，路来金.宫旭.等.鹿茸多肽-PLGA复合膜促进大鼠周围神经再生[J].吉林大学学报(医学版).2006，32(2)：199-202.
[18]路来金，王克利.李立军，等.鹿茸多肽对周围神经再生的影响[J].中国修复重建外科杂志，2008，12(12)：1459-1461.
[19]王旭凯.王英，杨有庚,等.鹿茸多肽对p-淀粉样蛋白诱导脊髓神经元细胞凋亡的保护作用[J].浙江中医药大学学报，2009,33(1)：45-47.
[20]李振华.冷向阳.高忠礼.鹿茸多肽对脊髓损伤保护作用的实验研究[J].中国骨伤.2008.21(4)：285-286.
[21]霍玉书,霍虹.鹿茸神经生长因子活性及促分化作用的研究[J].中药新药与临床药理，1997,8(02)：79-81.
[22]严铭铭，曲晓波,钟英杰,等.梅花鹿鹿茸中促进海马神经细胞增殖蛋白的分离纯化[J].中草药.2007,38(8：1163-1167.
[23]徐惠波.王本祥.张洁.等.鹿茸神经节甙酯对小鼠学习记忆功能的影响[J].中国药理学通报.1991.7(5)：385-388.
[24]王晓丽.张大方.鹿茸提取物对东莨宕碱及亚硝酸钠所致小鼠学习记忆障碍的影响[J].长春中医学院学报，2005，21(4)：37-38.
[25]翁梁,周秋丽,池岛桥,等.马鹿茸促进表皮细胞和软骨细胞分裂的新多肽[J].药学学报,2001,36(12)：913-916.
[26]贾晓燕,路来金,宣昭鹏.鹿茸多肽-壳聚糖-蜂蜜混悬剂对猪皮肽褥疮的促愈合作用[J].中国矫形外科杂志，2010,18(6)：498-502.
[27]王丽鸣.鹿茸多肽治疗顽固性鼓膜穿孔30例[J].新中医,2004，36(10)：59-6
[28]曹丽华.韩梅.王丽鸣.鹿茸多肽加速顽固性鼓膜穿孔愈合的疗效观察[J].长春中医学院学报.2006，22(1)：45.
[29]唐巍然，于晓红.阐杰.等.鹿茸多糖对免疫功能低下模型小鼠细胞免疫功能的影响[J].中国中医药科技.2000.7(4)：234.
[30]陈书明，聂向庭.鹿茸醇提物对用环磷酰胺处理的小白鼠红细胞免疫功能的影响[J].经济动物学报,2000,4(11)：23.
[31]Kang SK, Kim KS, Kim SI, et al. Immunosuppressive activity of deer antler extracts of Cervus korean TEMMINCK var. mantchuricus Swinhoe, on type Ⅱ collagen-induced arthritis[J]. in Vitro Cell Dev Biol Anim,2006, 42(3-4):100-107.
[32]陈晓光.杨明，王本详.等.人工培养鹿茸细胞的药理研究[J].中药药理与临床.1990，6(1)：30-32.
[33]田育璋.贺菊香.王丰梅，等.鹿茸对大鼠学丸影响的形态计量[J].青海医学院学报.1997，18(3)：154.
[34]何刚,王本祥.张伟.等.鹿茸多肽对雄鼠黄体生成素和学丸酮分泌的影响[J].中成药.1994,16(11)：33-34.
[35]林志展，施启贤.李聪明.等.探讨台湾水鹿茸粉对改善肾阳虚大鼠性功能的实验研究[C].第七届海峡两岸心血管研讨会.2009：24.
[36]范玉琳.邢增涛.卫功庆，等.鹿茸蛋白的提取分离及其抗肿瘤活性[J].动物经济学报.1998.2(1)：27-31.
[37]熊和丽.鹿茸活性成分的提取分离及其抗肿瘤作用研究[D].西北农林科技大学.2007.
[38]Kim KW, Kim KS, Park SD, et al. Effect of Cervus korean TEMMINCK var. mantchuricus Swinhoe on protease activities, antioxidant and free radical damages in rheumatis arthritis rats[J]. Toxicol In Vitro,2008,22(1):80-86.
[39]陈书明,聂向庭.鹿茸醇提物抗氧化作用的实验研究[J].2000.17(1)：22-24.
[40]陈晓光,金淑莉.邸琳.等.鹿茸提取物体外抗氧化作用[J].中药材.2003.10：733-734.
[41]周冉.李淑芬.张大成.鹿茸提取物体外抗氧化活性分析[J].食品科学2009.30(9)：33-36.
[42]郑帆.李仁宽.王辉林.等.鹿茸冻干粉的酶解及其酶解产物性质的研究[J].中国中药杂志.2010,35(19：2628-2633.
[43]王华.黄宜兵.高科翔.等.酶解鹿茸肽的制备、纯化及抗氧化活性[J].高等学校化学学报.2010,31(12)：2390-2395.
[44]Suttie JM, Gluckman PD, Butler JH, et al. Insulin-like growth factor 1 (IGF-1) antler-stimulating hormone?[J].Endocrinology,1985,116(2):846-8.
[45]Gu L, Mo E, Yang Z, et al. Expression and localization of insulin-like growth factor-I in four parts of the red deer antler[J]. Growth Factors.2007, 25(4):264-279.
[46]郝林琳,刘松财，夏青娟.等.鹿茸多肽的生物学活性研究[J].吉林农业大学学报.2006,28(3)：285-288.
[47]Ko KM, Yip TT, Tsao SW, et al. Epidermal growth factor from deer (Cervus elaphus) submaxillary gland and velvet antler[J]. Gen Comp Endocrinol. 1986,63(3):431-440.
[48]江润祥.高锦明.叶大同.等.鹿茸表皮生长因子[J].动物学报.1987.33(4)：301-308.
[49]Li C, Stanton JA, Robertson TM, et al. Nerve Growth Factor mRNA Expression in the Regenerating Antler Tip of Red Deer (Cervus elaphus)[J]. PLoS One,2007,10:2(1):e148.
[50]Francis SM, Suttie JM. Detection of growth factors and proto-oncogene mRNA in the growing tip of red deer (Cervus elaphus) antler using reverse-transcriptase polymerase chain reaction (RT-PCR)[J]. J Exp Zool, 1998,281(1):36-42.
[51]Lai AK, Hou WL, Verdon DJ, et al. The distribution of the growth factors FGF-2 and VEGF. and their receptors, in growing red deer antler[J]. Tissue Cell,2007,39(1):35-46.
[52]Jackson DW, Simon TM. Chondrocyte transplantation[J]. Arthroscopy,1996, 12:732-738.
[53]Shapiro F, Kiod S, Gincher G. Cell origin and differentiation in the repair of full thickness defect of articular cartilage[J]. J Bone joint Surg(Am),1996, 74 (4):532.
[54]Takagi M, Umetsu Y, Fujiwara M, et al. High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells[J]. J Biosci Bioeng,2007,103(1):98-100.
[55]Yoo JU, Barthel TS, Nishimura K, et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am,1998,80(12):1745-1757.
[56]Homicz MR, Chia SH, Schumacher BL, et al. Human septal chondrocyte redifferentiation in alginate, polyglycolic acid scaffold, and monolayer culture[J]. Laryngoscope,2003,113(1):25-32.
[57]Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs[J]. J Cell Physiol,2006,207(2):331-339.
[58]D'Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances sternness of human MIAMI cells[J]. Bone,2006,39(3):513-522.
[59]Markway BD, Tan GK, Brooke G, et al. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchvmal stem cells in low oxygen environment micropellet cultures[J]. Cell Transplant,2010, 19(1):29-42.
[60]Robins JC, Akeno N, Mukherjee A, et al. Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9[J]. Bone,2005,37(3):313-322.
[61]Lafont JE, Talma S, Murphy CL. Hypoxia-inducible factor 2 alpha is essential for hypoxic induction of the human articular chondrocyte phenotype[J]. Arthritis Rheum,2007,56(10):3297-3306.
[62]Felka T, Schafer R, Schewe B, et al. Hypoxia reduces the inhibitory effect of IL-1beta on chondrogenic differentiation of FCS-free expanded MSC[J]. Osteoarthritis Cartilage,2009,17(10):1368-1376.
[63]Miyanishi K, Trindade MC, Lindsey DP, et al. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro[J]. Tissue Eng,2006,12(6):1419-1428.
[64]Huang CY, Hagar KL, Frost LE, et al. Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[J]. Stem Cells,2004,22(3):313-323.
[65]Thorpe SD, Buckley CT, Vinardell T, et al. The response of bone marrow-derived mesenchymal stem cells to dynamic compression following TGF-beta3 induced chondrogenic differentiation[J]. Ann Biomed Eng,2010, 38(9):2896-2909.
[66]Terraciano V, Hwang N, Moroni L, et al. Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[J]. Stem Cells,2007,25(11):2730-2738.
[67]Kisiday JD, Frisbie DD, Mcllwraith CW, et al. Dynamic compression stimulates proteoglycan synthesis by mesenchymal stem cells in the absence of chondrogenic cytokines[J]. Tissue Eng Part A,2009,15(10):2817-2824.
[68]Wozney JM. The bone morphogenetic protein family:multifunctional cellular regulators in the embryo and adult[J]. Eur J Oral Sci,1998,106 Suppl 1:160-166.
[69]刘勇.转化生长因子-p与骨、软骨代谢及修复[J].国外医学(创伤与外科基本问题分册)，1999,20(2)：67-70.
[70]Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells[J]. Exp Cell Res,1998. 238(1):265-272.
[71]Hellingman CA. Davidson EN. Koevoet W. et al. Smad Signaling Determines Chondrogenic Differentiation of Bone-Marrow-Derived Mesenchymal Stem Cells:Inhibition of Smad1/5/8P Prevents Terminal Differentiation and Calcification [J]. Tissue Eng Part A,2011. 17(7-8):1157-1167.
[72]Tuli R, Tuli S, Nandi S, et al. Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk[J]. J Biol Chem,2003,17; 278(42):41227-41236.
[73]Barry F, Boynton RE, Liu B, et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow:differentiation-dependent gene expression of matrix components[J]. Exp Cell Res,2001,268:189-200.
[74]Sakou T.Bone morphogenetic proteins:from basic studies to clinical approaches[J]. Bone,1998,22(6):591-603.
[75]Bentz H, Nathan RM, Rosen DM, et al. Purification and characterization of a unique osteoinductive factor from bovine bone[J]. J Biol Chem,1989, 5:264(34):20805-20810.
[76]Yokouchi Y, Sakiyama J, Kameda T, et al. BMP-2/-4 mediate programmed cell death in chicken limb buds[J]. Development,1996.122(12):3725-3734.
[77]Onishi T, Ishidou Y, Nagamine T, et al. Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type Ⅱ receptor during fracture healing in rats[J]. Bone,1998, 22(6):605-612.
[78]Lein P, Johnson M, Guo X, et al. Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons[J]. Neuron,1995,15(3):597-605.
[79]Song JJ, Celeste AJ, Kong FM, et al. Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation[J]. Endocrinology,1995, 136(10):4293-4297.
[80]Iwasaki M, Nakahara H, Nakase T, et al. Bone morphogenetic protein 2 stimulates osteogenesis but does not affect chondrogenesis in osteochondrogenic differentiation of periosteum-derived cells[J]. J Bone Miner Res,1994,9(8):1195-1204.
[81]Mehlhorn AT, Niemeyer P, Kaschte K, et al. Differential effects of BMP-2 and TGF-betal on chondrogenic differentiation of adipose derived stem cells[J]. Cell Prolif,2007,40(6):809-823.
[82]Estes BT, Wu AW, Guilak F. Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6[J]. Arthritis Rheum,2006,54(4):1222-1232.
[83]Fujita M, Kinoshita Y, Sato E, et al. Proliferation and differentiation of rat bone marrow stromal cells on poly(glycolic acid)-collagen sponge[J]. Tissue Eng,2005, 11(9-10):1346-1355.
[84]Solchaga LA, Penick K. Porter JD, et al. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells[J]. J Cell Physiol,2005,203(2):398-409.
[85]Chiou M, Xu Y, Longaker MT. Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cells[J]. Biochem Biophys Res Commun,2006,5:343(2):644-652.
[86]Longobardi L, O'Rear L, Aakula S, et al. Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling[J]. J Bone Miner Res,2006,21(4):626-636.
[87]Wang L, Detamore MS. Insulin-like growth factor-Ⅰ improves chondrogenesis of predifferentiated human umbilical cord mesenchymal stromal cells[J]. J Orthop Res,2009,27(8):1109-1115.
[88]Fukumoto T, Sperling JW, Sanyal A, et al. Combined effects of insulin-like growth factor-1 and transforming growth factor-betal on periosteal mesenchymal cells during chondrogenesis in vitro[J]. Osteoarthritis Cartilage. 2003, 11(1):55-64.
[89]Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell.2006. 126(4):663-676.
[90]Friedenstein AJ,Petrakova KV, Kurolesova AI,et al. Heterotopic transplants of bone marrow:analysis of precursor cells for osteogenic and hematopoietic tissues [J]. Transplantation,1968,6:230-247.
[91]Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies[J].Tissue Eng 2001,7:211-228.
[92]Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells[J]. Mol Biol Cell,2002,13(12):4279-4295.
[93]Housman TS, Lawrence N, Mellen BG, et al. The safety of liposuction: results of a national survey. Dermatol Surg,2002,28:971-978.
[94]Aust L, Devlin B, Foster SJ, et al. Yield of human adipose-derived adult stem cells from liposuction aspirates[J]. Cytotherapy,2004,6(1):7-14.
[95]Pittenger MF, Mosca JD, McIntosh KR. Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma[J]. Curr Top Microbiol Immunol,2000,251:3-11.
[96]Sakaguchi Y, Sekiya I, Yagishita K. et al. Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source[J]. Arthritis Rheum,2005,52(8):2521-2529.
[97]Sharpless NE, DePinho RA. How stem cells age and why this makes us grow old[J]. Nat Rev Mol Cell Biol,2007,8:703-713.
[98]Yoshimura K, Shigeura T, Matsumoto D, et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates[J]. J Cell Physiol,2006,208(1):64-76.
[99]Barry FP. Mesenchymal stem cell therapy in joint disease[J]. Novartis Found Symp.2003,249:86-96; discussion 96-102,170-4,239-41.
[100]Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure[J]. Cytotherapy,2006, 8(2):166-177.
[101]Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[J]. Cytotherapy,2006,8(4):315-317.
[102]Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell:a better stem cell than BMSC[J]. Cell Biochem Funct,2008,26(6):664-675.
[103]Puissant B, Barreau C, Bourin P, et al. Immunomodulatory effects of human adipose tissue-derived stem cells:comparison with bone marrow mesenchymal stem cells[J]. Br J Haematol,2005,129:118-129.
[104]国家药典委员会编.中华人民共和国药典(2005年版)一部.
[105]Price J, Allen S, Exploring the mechanisms regulating regeneration of deer antlers[J]. Philos Trans R Soc Lond B Biol Sci,2004,29:359(1445):809-822.
[106]王继峰.生物化学[M].北京.中国中医药出版社.2003,1.35.
[107]Gill I, Lopez-Fandino R, Jorba X, et al. Biologically active peptides and enzymatic approaches to their production[J]. Enzyme Microb Technol,1996, 15:18(3):163-183.
[108]H. Newey, D. H. Smyth. Intracellular hydrolysis of dipeptides during intestinal absorption [J]. J Physiol,1960,152(2):367-380.
[109]Hara H, Funabiki R, Iwata M, et al. Portal absorption of small peptides in rats under unrestrained conditions[J]. J Nutr,1984,114(6):1122-1129.
[110]何瑜璇,刘春生,董栗.天然药物中寡肽的研究进展[C].中华中医药学会第十届中药鉴定学术会议论文集.2010.
[111]鲍加荣,李春义,邢秀梅,等.鹿茸再生及干细胞研究进展[J].中国组织工程研究与临床康复.2008,12(51)：10163-10166.
[112]陶红,梁歧.酶法制备大豆寡肽的研究[J].大豆科学.2004,23(1)：26-29.
[113]周秋丽.刘永强.王颖.等.梅花鹿茸和马鹿茸多肽化学性质及生物活性比较[J].中国中药杂志.2001,26(10)：699-702.
[114]Arden N, Nevitt MC. Osteoarthritis:epidemiology[J]. Best Pract Res Clin Rheumatol, 2006,20(1):3-25.
[115]Jackson DW, Simon TM. Aberman HM. Symptomatic articular cartilage degeneration:the impact in the new millennium[J]. Clin Orthop Relat Res, 2001,(391 Suppl):S14-25.
[116]Kon E, Gobbi A, Filardo G, et al. Arthroscopic second-generation autologous chondrocyte implantation compared with microfracture for chondral lesions of the knee:prospective nonrandomized study at 5 years[J]. Am J Sports Med, 2009,37(1):33-41.
[117]Solchaga LA, Goldberg VM, Caplan AI. Cartilage regeneration using principles of tissue engineering[J].Clin Orthop Relat Res,2001, (391 Suppl):S161-70.
[118]Kim D, Kim CH, Moon JI, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins[J]. Cell Stem Cell, 2009,4(6):472-476.
[119]Kierdorf U, Li Chunyi, Price JS. Improbable appendages:Deer antler renewal as a unique case of mammalian regeneration[J]. Seminars in cell and developmental biology,2008,20(4):535-542.
[120]Li C, Clark DE, Lord EA, et al. sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery [J]. J The Anatomical record,2002,268:125-130.
[121]段冷听.梅花鹿茸多肽的化学结构及其抗肝纤维化作用[D].2007.吉林大学.
[122]Rerat A. Nutritional value of protein hydrolysis products (oligopeptides and free amino acids) as a consequence of absorption and metabolism kinetics[J]. Arch Tierernahr,1995,48(1-2):23-36.
[123]Iwakoshi E, Hisada M, Minakata H. Cardioactive peptides isolated from the brain of a Japanese octopus. Octopus minor[J]. Peptides,2000, 21(5):623-630.
[124]张勇.朱宇旌.刘勇.等.红三叶寡肽对小鼠免疫功能的影响[J]。安徽农业科学.2007.35(35)：11338-11339.11342.
[125]MASUDA O, NAKAMURA Y, TAKANO T. Antihypertensive peptidesarepresentinaortaafteroraladministrationofsourmilkcontaining these peptides to spontaneously hypertensive rats[J]. J Nutr,1996.126:3063-3068.
[126]黄晓明.李祺福.刘用金,等.新生牛牛脑活性肽NBBP-1对人神经母细胞瘤SK-N-SH细胞凋亡的诱导作用[J].厦门大学学报(自然科学版).2009.48(3)：399-405.
[127]Shytle RD, Ehrhart J, Tan J, et al. Oxidative stress of neural, hematopoietic. and stem cells:protection by natural compounds[J]. Rejuvenation Res,2007, 10(2):173-178.
[128]Shytle DR, Tan J, Ehrhart J, et al. Effects of blue-green algae extracts on the proliferation of human adult stem cells in vitro:a preliminary study[J]. Med Sci Monk,2010,16(1):BR1-5.
[129]Tang X, Zhang C, Zeng W, et al. Proliferating effects of the flavonoids daidzein and quercetin on cultured chicken primordial germ cells through antioxidant action[J]. Cell Biol Int,2006,30(5):445-451.
[130]Pervaiz S, Taneja R, Ghaffari S. Oxidative stress regulation of stem and progenitor cells[J]. Antioxid Redox Signal,2009.11(11):2777-2789.
[131]Bickford PC, Tan J, Shytle RD, et al. Nutraceuticals synergistically promote proliferation of human stem cells[J]. Stem Cells Dev.2006,15(1):118-123.
[132]Tacchetti C, Tavella S, Dozin B, et al. Cell condensation in chondrogenic differentiation. Exp Cell Res,1992,200(1):26-33.
[133]Mwale F, Stachura D, Roughley P, et al. Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation[J]. J Orthop Res,2006,24(8):1791-1798.
[134]Ng LJ, Wheatley S, Muscat GE, et al. SOX9 binds DNA. activates transcription, and coexpresses with type Ⅱ collagen during chondrogenesis in the mouse[J].Dev Biol,1997,183(1):108-121.
[135]Kawakami Y, Tsuda M, Takahashi S, et al. Transcriptional coactivator PGC-1alpha regulates chondrogenesis via association with Sox9[J]. Proc Natl Acad Sci U S A,2005,102(7):2414-2419.
[136]郝林琳,刘松财,张明军,等.鲜马鹿茸不同部位多肽的提取及含量分析.吉林农业大学学报.2009,29(4)：378-380.383.
[137]Fukumoto T, Sperling JW, Sanyal A, et al. Combined effects of insulin-like growth factor-1 and transforming growth factor-betal on periosteal mesenchymal cells during chondrogenesis in vitro[J]. Osteoarthritis Cartilage, 2003, 11(1):55-64.
[138]Indrawattana N, Chen G, Tadokoro M, et al. Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun,2004,320(3):914-949.
[139]Kantlehner M, Schaffner P, Finsinger D, et al. Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation[J].Chembiochem,2000, 1(2):107-114.
[140]Au A, Boehm CA, Mayes AM, et al. Formation of osteogenic colonies on well-defined adhesion peptides by freshly isolated human marrow cells[J]. Biomaterials,2007,28(10):1847-1861.
[141]Lin YC, Brayfield CA, Gerlach JC, et al. Peptide modification of polyethersulfone surfaces to improve adipose-derived stem cell adhesion.Acta Biomater[J].2009,5(5):1416-1424.