[1]许勇,薛彬,孟祥超,等.伸筋活血汤对低氧诱导因子-1α缺失小鼠椎间盘退变的影响…[J].中医正骨,2018,30(01):12-17.
　XU Yong,XUE Bin,MENG Xiangchao,et al.Effect of Shenjin Huoxue Tang(伸筋活血汤)on intervertebral disc degeneration in mice with hypoxia-inducible factor-1α deficiency[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(01):12-17.
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伸筋活血汤对低氧诱导因子-1α缺失小鼠椎间盘退变的影响…()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期数:
2018年01期
页码:
12-17
栏目:
基础研究
出版日期:
2018-01-20

文章信息/Info

Title:
Effect of Shenjin Huoxue Tang(伸筋活血汤)on intervertebral disc degeneration in mice with hypoxia-inducible factor-1α deficiency
作者:
许勇; 薛彬; 孟祥超; 李中伟; 奚小冰; 李飞跃
上海交通大学医学院附属瑞金医院,上海 200025
Author(s):
XU Yong; XUE Bin; MENG Xiangchao; LI Zhongwei; XI Xiaobing; LI Feiyue
Ruijin Hospital affiliated to Shanghai Jiaotong University medical college,Shanghai 200025,China
关键词:
椎间盘退行性变;  缺氧诱导因子1;  伸筋活血汤;  小鼠;  动物实验
Keywords:
Keywords intervertebral disc degeneration;  hypoxia-inducible factor-1;  Shenjin Huoxue Tang;  mice;  animal experimentation
摘要:
目的:观察伸筋活血汤对低氧诱导因子(hypoxia-inducible factor,HIF)-1α缺失小鼠椎间盘退变的影响。方法:通过杂交繁殖获得同窝HIF-1α缺失小鼠和正常小鼠。取2、4、8周龄HIF-1α缺失小鼠及正常小鼠,每个时间点2种小鼠各取5只,处死后取L3～L5节段腰椎组织制备切片,分别进行HE染色和番红-固绿染色。取16只4周龄HIF-1α缺失小鼠,随机分为2组,每组8只,分别以伸筋活血汤和生理盐水灌胃。灌胃每次0.12 mL,每天1次,共30 d。处死小鼠取L3～L5节段腰椎组织制备切片,分别进行HE染色、番红-固绿染色及免疫组化染色后观察。结果:HE染色显示,与正常小鼠相比,2周龄HIF-1α缺失小鼠髓核占整个椎间盘组织的比例减少,髓核中的细胞仍以含有大液泡的脊索细胞为主,但已有大量髓核细胞变小,髓核内细胞结构排列稍紊乱; 4周龄HIF-1α缺失小鼠椎间盘内的细胞种类和数量迅速下降,髓核细胞排列紊乱; 8周龄HIF-1α缺失小鼠髓核细胞数量减少十分明显,纤维组织填充于髓核中,细胞外基质增加。番红-固绿染色显示,与正常小鼠相比,2周龄HIF-1α缺失小鼠椎间盘中聚集蛋白聚糖减少,4周龄HIF-1α缺失小鼠椎间盘中聚集蛋白聚糖减少更加明显,8周龄HIF-1α缺失小鼠髓核出现裂痕及钙化,髓核骨化明显。4周龄HIF-1α缺失小鼠经药物干预30 d后,HE染色结果显示,伸筋活血汤组小鼠椎间盘终板无明显破损和骨化,髓核细胞数量较多,排列较整齐; 生理盐水组小鼠椎间盘髓核内细胞呈梭形,体积变小,数量明显减少,排列严重紊乱,细胞外基质增加,髓核中出现裂痕,纤维组织填充于髓核中,甚至出现终板破坏。番红-固绿染色结果显示,伸筋活血汤组小鼠骨和软骨终板染色明显,椎间盘髓核内细胞数量较多,椎间盘骨化现象不明显; 生理盐水组骨和软骨终板染色较浅,出现裂痕及钙化,细胞数量极少。免疫组化染色结果显示,伸筋活血汤组聚集蛋白聚糖阳性染色的细胞数量明显较多,阳性信号多分布在细胞内; 生理盐水组聚集蛋白聚糖阳性染色的细胞数量较少,阳性信号多分布在细胞外。生理盐水组聚集蛋白聚糖阳性染色细胞的灰度值小于伸筋活血汤组(129.889±9.839,154.333±9.622,t=0.200,P=0.000)。结论:HIF-1α缺失可导致小鼠椎间盘退变,伸筋活血汤能够延缓HIF-1α缺失小鼠的椎间盘退变。
Abstract:
ABSTRACT Objective:To observe the effect of Shenjin Huoxue Tang(伸筋活血汤,SJHXT)on intervertebral disc degeneration in mice with hypoxia-inducible factor(HIF)-1α deficiency.Methods:The littermate HIF-1α deficiency mice and normal mice were obtained through crossbreeding.Two-,4- and 8-week-old HIF-1α deficiency mice and normal mice were fetched,and 5 cases were randomly selected from each kind of mice at each time point and were executed,and then their vertebral tissues of L3-L5 were fetched out and were sectioned for HE staining and safranin O-fast green staining.Sixteen 4-week-old HIF-1α deficiency mice were fetched and were randomly divided into 2 groups,8 cases in each group,and they were intragastric administrated with SJHXT(SJHXT group)and normal saline(NS group)respectively,0.12 mL at a time,once a day for consecutive 30 days.Then the mice were executed,and their vertebral tissues of L3-L5 were fetched out and were sectioned for HE staining,safranin O-fast green staining and immunohistochemical staining.Results:The HE staining results showed that,compared to normal mice,(1)the proportion of nucleus pulposus to whole intervertebral disc of 2-week-old HIF-1α deficiency mice decreased,and the cells in nucleus pulposus were still dominated by notochord cells containing large vacuoles,and a large number of nucleus pulposus cells shrinked and their intracellular structure was slightly disorganized;(2)the cell type and number decreased rapidly in intervertebral disc and nucleus pulposus cells were disorganized in 4-week-old HIF-1α deficiency mice;(3)the nucleus pulposus cells decreased obviously and the extracellular matrix increased and the nucleus pulposus was filled with fibrous tissues in 8-week-old HIF-1α deficiency mice.The safranin O-fast green staining results showed that,compared to normal mice,the aggrecan in intervertebral disc decreased in 2-week-old HIF-1α deficiency mice and decreased more obviously in 4-week-old HIF-1α deficiency mice; and cracks,calcifications and obvious ossification appeared in nucleus pulposus in 8-week-old HIF-1α deficiency mice.After 30-day drug intervention on 4-week-old HIF-1α deficiency mice,the HE staining results showed that(1)no obvious damage and ossification were found in intervertebral disc endplate and a large number of nucleus pulposus cells arranged in order in mice of SJHXT group;(2)the cells in intervertebral disc nucleus pulposus of mice in NS group decreased in volume and number and presented with fusiform cells and arranged in great disorder,moreover,the extracellular matrix increased and cracks and a large amount of fibrous tissues were found in nucleus pulposus and the destroyed endplate was also found.The safranin O-fast green staining results showed that(1)the stains in bony and cartilaginous endplates were obvious in mice of SJHXT group,moreover,there were abundant cells in intervertebral disc nucleus pulposus,and the intervertebral disc ossification was not so obvious;(2)the stains presented with light colour in bony and cartilaginous endplates of mice in NS group,moreover,cracks,calcifications and few cells appeared in bony and cartilaginous endplates.The immunohistochemical staining results showed that many cells with positive aggrecan staining were found in SJHXT group and few cells with positive aggrecan staining were found in NS group; and the positive signals mainly distributed inside the cells in SJHXT group and outside the cells in NS group.The gray values of cells with positive aggrecan staining were less in NS group compared to SJHXT group(129.889+/-9.839,154.333+/-9.622,t=0.200,P=0.000).Conclusion:HIF-1α deficiency can lead to intervertebral disc degeneration in mice,and SJHXT can delay intervertebral disc degeneration in mice with HIF-1α deficiency.

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备注/Memo

备注/Memo:
基金项目:上海市中医药传承和创新项目(ZY3-CCCX-1-1014); 上海市名老中医学术经验研究工作室建设项目(SHGZS-2017010); 2017年全国名老中医药专家传承工作室建设项目(MLZJGZS-2017001) 通讯作者:李飞跃 E-mail:sklifeiyue@163.com
更新日期/Last Update: 2018-06-02