[1]邱慈鑫,余添赐,杨树华,等.龟鹿二仙胶不同提取部位对豚鼠软骨细胞活性的影响[J].中医正骨,2014,26(10):7-11.
　Qiu Cixin*,Yu Tianci,Yang Shuhua,et al.Effect of different extract fraction of GUILU ERXIAN gelatin on the chondrocytes activity in Guinea pigs[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2014,26(10):7-11.
点击复制

龟鹿二仙胶不同提取部位对豚鼠软骨细胞活性的影响()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第26卷
期数:
2014年10期
页码:
7-11
栏目:
基础研究
出版日期:
2014-10-30

文章信息/Info

Title:
Effect of different extract fraction of GUILU ERXIAN gelatin on the chondrocytes activity in Guinea pigs
作者:
邱慈鑫; 余添赐; 杨树华; 魏迎辰; 陈泽华; 林海英; 王和鸣; 李楠
福建中医药大学,福建 福州 350122
Author(s):
Qiu Cixin*; Yu Tianci; Yang Shuhua; Wei Yingchen; Chen Zehua; Lin Haiying; Wang Heming; Li Nan.*
Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China
关键词:
龟鹿二仙胶 提取部位 软骨细胞 胶原Ⅱ型 Bcl-2 Bax 基质金属蛋白酶类 动物实验
Keywords:
GUILU ERXIAN gelatin;  Extract fraction;  Chondrocytes;  Collagen TypeⅡ;  Bcl-2;  Bax;  Matrix Metalloproteinases;  Animal experimentation
摘要:
目的:探讨龟鹿二仙胶不同提取部位对豚鼠软骨细胞活性的影响。方法:采用溶剂极性依次递增分离法分别制备龟鹿二仙 胶的三氯甲烷提取物、正丁醇提取物和水提取物。然后分别以三氯甲烷提取物(烷提组)、正丁醇提取物(醇提组)和水提取物(水 提组)及生理盐水(对照组)对4组Wistar大鼠进行灌胃,制备含药血清,再分别以这些含药血清对体外培养的豚鼠软骨细胞进行干 预。干预结束后分别采用免疫组织化学技术、实时荧光定量聚合酶链式反应法及酶联免疫吸附法测定经4种含药血清干预后的豚 鼠软骨细胞中Ⅱ型胶原蛋白、Bcl-2和Bax基因及MMP-3水平。结果:①Ⅱ型胶原蛋白水平。4组细胞Ⅱ型胶原蛋白水平比较,差异 有统计学意义[(0.178±0.002),(0.188±0.003),(0.177±0.003),(0.173±0.001),F=32.905,P=0.000]; 对 照组Ⅱ型胶原蛋白水平低于其余3组(P=0.003,P=0.000,P=0.023); 醇提组高于烷提组和水提组(P=0.000,P=0.000); 烷提组和水 提组比较,差异无统计学意义(P=0.367)。②Bcl-2基因和Bax基因水平。4组细胞Bcl-2基因水平比较,差异有统计学意义 [(0.569±0.052),(1.000±0.000),(0.636±0.072),(0.387±0.061),F=136.767,P=0.000]; 对照组低于其余 3组(P=0.000,P=0.000,P=0.000); 醇提组高于烷提组和水提组(P=0.000,P=0.000); 烷提组低于水提组(P=0.045)。4组细胞Bax 基因水平比较,差异有统计学意义[(1.789±0.240),(1.000±0.000),(1.374±0.226), (2.251±0.196),F=47.445,P=0.000]; 对照组高于其余3组(P=0.000,P=0.000,P=0.000); 醇提组低于烷提组和水提组 (P=0.000,P=0.003); 烷提组高于水提组(P=0.000)。③基质金属蛋白酶3水平。4组细胞基质金属蛋白酶3水平比较,差异有统计 学意义[(11.828±0.363)ng·mL-1,(10.685±0.317)ng·mL-1,(11.397±0.314) ng·mL-1,(13.136±0.379)ng·mL-1,F=71.401,P=0.000]; 对照组高于其余3组 (P=0.000,P=0.000,P=0.000); 醇提组低于烷提组和水提组(P=0.000,P=0.000); 烷提组高于水提组(P=0.019)。结论:龟鹿二仙 胶的三氯甲烷、正丁醇和水3种提取部位均能提高软骨细胞中Ⅱ型胶原和Bcl-2基因水平,降低Bax基因和基质金属蛋白酶3水平, 其中以正丁醇提取部位的作用最强。
Abstract:
Objective:To study the effect of different extract fraction of GUILU ERXIAN gelatin on the chondrocytes activity in Guinea pig.Methods:The trichloromethane extractive,n-butanol extractive and water extractive were made from GUILU ERXIAN gelatin respectively by the separation method of increasing solvent polarity progressively.The Wistar rats in 4 groups were intragastric administrated with trichloromethane extractive,n-butanol extractive,water extractive and normal saline respectively.Then the chondrocytes of Guinea pigs cultured in vitro were intervened by the medicated serums extracted from the Wistar rats respectively.After intervention,the levels of Collagen TypeⅡ,Bcl-2 gene,Bax gene and MMP-3 in the chondrocytes of Guinea pigs were determined by using immunohistochemical technique,real-time fluorescent quantitative polymerase chain reaction and enzyme linked immunosorbent assay respectively.Results:There was statistical difference in the level of Collagen TypeⅡbetween the 4 groups(0.178+/-0.002,0.188+/-0.003,0.177+/- 0.003,0.173+/-0.001,F=32.905,P=0.000).The level of Collagen TypeⅡwas lower in the control group compared with the other 3 groups(P=0.003,P=0.000,P=0.023)and was higher in the n-butanol extractive group compared with trichloromethane extractive group and water extractive group(P=0.000,P=0.000)and there was no statistical difference between n-butanol extractive group and water extractive group(P=0.367).There was statistical difference in the level of Bcl-2 gene between the 4 groups(0.569+/-0.052,1.000+/-0.000,0.636+/-0.072,0.387+/- 0.061,F=136.767,P=0.000).The level of Bcl-2 gene was lower in the control group comk pared with the other 3 groups(P=0.000,P=0.000,P=0.000),and was higher in the n-butanol extractive group compared with trichloromethane extractive group and water extractive group(P=0.000,P=0.000)and was lower in the trichloromethane extractive group compared with water extractive group(P=0.045).There was statistical difference in the level of Bax gene between the 4 groups(1.789+/-0.240,1.000+/-0.000,1.374+/-0.226,2.251+/- 0.196,F=47.445,P=0.000).The level of Bax gene was higher in the control group compared with the other 3 groups (P=0.000,P=0.000,P=0.000)and was lower,in the n-butanol extractive group compared with trichloromethane extractive group and water extractive group(P=0.000,P=0.003)and was higher in the trichloromethane extractive group compared with the water extractive group(P=0.000).There was statistical difference in the level of MMP-3 between the 4 groups(11.828+/-0.363,10.685+/-0.317,11.397+/-0.314,13.136+/-0.379 ng/mL,F=71.401,P=0.000).The level of MMP-3 was higher in the control group compared with the other 3 groups(P=0.000,P=0.000,P=0.000)and was lower in the n-butanol extractive group compared with trichloromethane extractive group and water extract group(P=0.000,P=0.000)and was higher in the trichloromethane extractive group compared with water extractive group(P=0.019).Conclusion:The extract fraction of GUILU ERXIAN gelatin including trichloromethane,n-butanol and water can up-regulate the levels of Collagen TypeⅡand Bcl-2 gene and down-regulate the levels of Bax gene and MMP-3,and the effect of n-butanol is the strongest.

参考文献/References:

[1] 吴文博.中药复方提取部位的功能与母方整体效应关系之思考[J].医学与哲学,2008,29(7):85.
[2] 郑本端,罗自文.龟鹿二仙膏的药理学研究[J].中成药,2000,22(12):860-861.
[3] 王和鸣,余丹丹,汤亭亭,等.龟鹿二仙胶汤及其拆方对关节软骨细胞增殖的作用[J].中国中医骨伤科杂志,2007,15(7):45- 49.
[4] 李楠,黄远鹏,杜江,等.龟鹿二仙胶及其拆方对体外培养豚鼠软骨细胞活性的影响[J].福建中医药大学学报,2012,22(5):18 -21.
[5] 李楠,尹玉彪,雒焕生,等.龟鹿二仙胶及其拆方对豚鼠软骨细胞Ⅱ型胶原蛋白多糖合成的影响[J].中华中医药杂志,2011,26 (11):2524-2528.
[6] 李楠,雒焕生,赵诣,等.龟鹿二仙胶汤及其拆方对大鼠软骨细胞凋亡基因表达的影响[J].中国中医骨伤科杂志,2011,19 (7):1-3.
[7] 杜江,黄远鹏,李奕修,等.龟鹿二仙胶及其拆方对去势骨关节炎大鼠血清雌二醇浓度及膝关节软骨细胞Ⅱ型胶原表达的影响 [J].中医正骨,2013,25(3):11-20.
[8] 曹胜男,包海鹰.鹿角的化学成分及药理活性研究进展[J].经济动物学报,2011,15(4):230-233.
[9] 李长泉.龟甲药理作用及临床应用的现代研究[J].长春中医学院学报,2003,19(4):55-56.
[10] 唐斌,程绪菊,刘江,等.人参皂苷药理作用研究进展[J].西南军医,2005,7(3):45-47.
[11] 魏永祥,商希礼,张杰,等.枸杞天然活性成分提取的研究进展[J].山东化工,2009,38(7):18-20.
[12] 张业勇,程文丹,陈哲峰,等.人参皂苷Rg1对软骨细胞Ⅱ型胶原表达的影响[J].中国组织工程研究,2013,17(11):1917-1924.
[13] 邓茂,钟桂书,雷启蓉.人参皂甙Rd对培养光老化真皮成纤维细胞影响的实验研究[J].皮肤性病诊疗学杂志,2010,17 (6):403-406.
[14] 杨朝鲜,李雷激,高小青,等.大鼠局灶性脑缺血时人参皂甙Rbl抑制细胞凋亡和调控凋亡基因的表达[J].中风与神经疾病杂 志,2008,25(1):12-16.
[15] 何勇川,李平华.人参皂甙Rg1对氧化损伤兔晶状体上皮细胞凋亡相关基因Bcl-2和Bax表达影响[J].重庆医科大学学 报,2006,31(5):662-665.
[16] 李佳佳,马健.中医衰老理论与抗衰老的实验研究进展[J].中医学报,2013,28(2):213-216.
[17] 彭勇,张荣华,蔡宇,等.补肾活血法延缓衰老的机理探讨[J].陕西中医,2004,25(2):142-144.

备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(30772814),福建省教育厅重点项目(JA13153)
通讯作者:李楠 E-mail:mr.linan@126.com
更新日期/Last Update: 2014-10-30