[1]夏炳江,沈兴潮,胡松峰,等.大鼠肾虚型椎间盘软骨终板退变病证结合模型的构建与评价[J].中医正骨,2021,33(05):1-10.
　XIA Bingjiang,SHEN Xingchao,HU Songfeng,et al.Construction and evaluation of disease-syndrome combination rat model of intervertebral disc cartilage endplate degeneration due to kidney deficiency[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2021,33(05):1-10.
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大鼠肾虚型椎间盘软骨终板退变病证结合模型的构建与评价()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期数:
2021年05期
页码:
1-10
栏目:
基础研究
出版日期:
2021-05-20

文章信息/Info

Title:
Construction and evaluation of disease-syndrome combination rat model of intervertebral disc cartilage endplate degeneration due to kidney deficiency
作者:
夏炳江; 沈兴潮; 胡松峰; 韦金忠; 许杨
(绍兴市中医院,浙江 绍兴 312000)
Author(s):
XIA Bingjiang; SHEN Xingchao; HU Songfeng; WEI Jinzhong; XU Yang
Shaoxing Hospital of Traditional Chinese Medicine,Shaoxing 312000,Zhejiang,China
关键词:
椎间盘退行性变;  软骨终板退变;  肾虚;  聚集蛋白聚糖;  含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶;  骨小梁数目;  骨小梁分离度;  骨小梁厚度;  大鼠; Sprague-Dawley;  疾病模型; 动物;  动物实验
Keywords:
intervertebral disc degeneration;  cartilage endplate degeneration;  kidney deficiency;  aggrecan;  a disintesrin and metalloprotease with thrombospondin typeⅠmotifs;  trabecular number;  trabecular separation;  trabecular thickness;  rats; Sprague-Dawley;  disease models; animal;  animal experimentation
摘要:
目的:探讨采用切除双侧卵巢结合阻断软骨终板营养供应法构建大鼠肾虚型椎间盘软骨终板(cartilage endplate,CEP)退变病证结合模型的可行性。方法:将108只12周龄SPF级雌性SD大鼠随机分为空白组、CEP退变模型组、病证结合模型组,每组36只。CEP退变模型组大鼠于椎间盘CEP下方骨质处注射无水酒精,病证结合模型组大鼠摘除双侧卵巢并于椎间盘CEP下方骨质处注射无水酒精,空白组大鼠不做任何处理。造模后观察并记录大鼠表观特征,测定肾虚症状体征量化评分。分别于造模后4周、8周,从各组随机抽取6只大鼠,先摄尾椎X线片,测算尾椎骨密度; 再处死大鼠,取出尾椎,行Micro-CT扫描测算骨小梁数目(trabecular number,TN)、骨小梁分离度(trabecular separation,TS)、骨小梁厚度(trabecular thickness,TT); 再从各组随机抽取6只大鼠,切取造模椎间盘,采用HE染色观察各组大鼠椎间盘CEP组织退变情况,采用实时定量聚合酶链反应法检测椎间盘CEP组织聚集蛋白聚糖和含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(a disintesrin and metalloprotease with thrombospondin typeⅠmotifs,ADAMTS)-5基因表达量。结果:①一般情况。在造模过程中,所有大鼠均无意外死亡发生。CEP退变模型组1只大鼠术后3 d出现尾部切口愈合不良、坏死现象,换药后1周左右切口愈合; 其余大鼠尾部切口均愈合良好。②大鼠表观特征。造模后,病证结合模型组大鼠逐渐出现毛发枯槁易脱落、饮食增多、精神倦怠、两眼无神、大便干结变硬、活动减少、反应迟钝等肾虚表现,空白组与CEP退变模型组大鼠无上述表现。③肾虚症状体征量化评分。时间因素和分组因素存在交互效应(F=14.326,P=0.000)。3组大鼠肾虚症状体征量化评分总体比较,差异有统计学意义,即存在分组效应(F=22.317,P=0.000)。造模后不同时间点大鼠肾虚症状体征量化评分的差异有统计学意义,即存在时间效应(F=50.279,P=0.000)。造模后,3组大鼠肾虚症状体征量化评分随时间变化趋势不一致,空白组大鼠肾虚症状体征量化评分随时间未见明显变化[(1.00±0.71)分,(1.40±0.55)分,(1.80±0.84)分,(2.00±1.00)分,F=1.573,P=0.235],CEP退变模型组和病证结合模型组大鼠肾虚症状体征量化评分随时间均呈升高趋势[(0.80±0.45)分,(1.20±0.45)分,(1.60±0.55)分,(2.20±0.84)分,F=5.095,P=0.012;(1.40±0.55)分,(3.40±1.14)分,(4.60±0.55)分,(6.80±0.84)分,F=39.256,P=0.000]。造模后2周,3组大鼠肾虚症状体征量化评分比较,差异无统计学意义(F=1.400,P=0.284)。造模后4周、6周、8周,3组大鼠肾虚症状体征量化评分比较,组间差异均有统计学意义(造模后4周:F=12.333,P=0.001; 造模后6周:F=32.462,P=0.000; 造模后8周:F=46.083,P=0.000); 空白组和CEP退变模型组大鼠肾虚症状体征量化评分均低于病证结合模型组(造模后4周:LSD-t=4.082,P=0.002; LSD-t=4.491,P=0.001。造模后6周:LSD-t=6.725,P=0.000; LSD-t=7.206,P=0.000。造模后8周:LSD-t=8.485,P=0.000; LSD-t=8.132,P=0.000); 空白组与CEP退变模型组大鼠肾虚症状体征量化评分比较,差异均无统计学意义(造模后4周:LSD-t=0.408,P=0.690; 造模后6周:LSD-t=0.480,P=0.640; 造模后8周:LSD-t=0.354,P=0.730)。④尾椎骨密度。造模后4周,3组大鼠尾椎骨密度比较,差异有统计学意义[(4.15±0.44)g·cm-3,(4.04±0.38)g·cm-3,(3.30±0.33)g·cm-3,F=7.719,P=0.007]; 空白组和CEP退变模型组大鼠尾椎骨密度均高于病证结合模型组(LSD-t=3.682,P=0.003; LSD-t=3.028,P=0.011); 空白组大鼠尾椎骨密度与CEP退变模型组比较,差异无统计学意义(LSD-t=0.655,P=0.525)。造模后8周,3组大鼠尾椎骨密度比较,差异有统计学意义[(4.12±0.42)g·cm-3,(3.88±0.53)g·cm-3,(2.86±0.63)g·cm-3,F=7.927,P=0.006]; 空白组和CEP退变模型组大鼠尾椎骨密度均高于病证结合模型组(LSD-t=3.749,P=0.003; LSD-t=3.035,P=0.010); 空白组大鼠尾椎骨密度与CEP退变模型组比较,差异无统计学意义(LSD-t=0.714,P=0.489)。⑤骨微结构指标。造模后4周,3组大鼠TN、TS比较,组间差异均具有统计学意义[TN:(5.01±0.12)个·mm-1,(4.81±0.12)个·mm-1,(3.86±0.17)个·mm-1,F=95.936,P=0.000; TS:(0.20±0.03)μm,(0.24±0.02)μm,(0.30±0.04)μm,F=10.807,P=0.002]; 空白组和CEP退变模型组大鼠TN高于病证结合模型组(LSD-t=12.991,P=0.000; LSD-t=10.657,P=0.000),TS低于病证结合模型组(LSD-t=4.623,P=0.001; LSD-t=2.736,P=0.018); 空白组大鼠TN和TS与CEP退变模型组比较,组间差异均无统计学意义(TN:LSD-t=0.432,P=0.924; TS:LSD-t=1.887,P=0.084)。造模后8周,3组大鼠TN、TS比较,组间差异均具有统计学意义[TN:(4.91±0.26)个·mm-1,(4.61±0.55)个·mm-1,(3.05±0.44)个·mm-1,F=27.088,P=0.000; TS:(0.22±0.04)μm,(0.23±0.02)μm,(0.29±0.03)μm,F=7.679,P=0.002]; 空白组和CEP退变模型组大鼠TN高于病证结合模型组(LSD-t=6.854,P=0.000; LSD-t=5.750,P=0.000),TS低于病证结合模型组(LSD-t=3.623,P=0.003; LSD-t=3.106,P=0.009); 空白组大鼠TN和TS与CEP退变模型组比较,组间差异均无统计学意义(TN:LSD-t=1.104,P=0.291; TS:LSD-t=0.518,P=0.614)。造模后4周、8周,3组大鼠TT比较,组间差异均无统计学意义[造模后4周:(0.08±0.02)μm,(0.09±0.01)μm,(0.10±0.01)μm,F=1.462,P=0.304; 造模后8周:(0.09±0.01)μm,(0.09±0.02)μm,(0.08±0.01)μm,F=2.400,P=0.171]。⑥椎间盘CEP组织形态。造模后,CEP退变模型组和病证结合模型组大鼠尾椎CEP均出现退变,表现为软骨细胞排列不规则,软骨细胞簇聚,潮线模糊,染色不均,软骨钙化层增厚等,而病证结合模型组退变更明显。⑦椎间盘CEP组织聚集蛋白聚糖和ADAMTS-5基因的表达。造模后4周、8周,3组大鼠椎间盘CEP组织聚集蛋白聚糖基因表达量比较,组间差异均有统计学意义(造模后4周:1.07±0.19,0.74±0.09,0.51±0.07,F=23.498,P=0.000; 造模后8周:1.03±0.22,0.65±0.08,0.43±0.07,F=23.538,P=0.000); 空白组大鼠聚集蛋白聚糖基因表达量高于CEP退变模型组和病证结合模型组(造模后4周:LSD-t=4.045,P=0.002; LSD-t=6.815,P=0.000。造模后8周:LSD-t=4.329,P=0.001; LSD-t=6.774,P=0.000),CEP退变模型组大鼠聚集蛋白聚糖基因表达量高于病证结合模型组(造模后4周:LSD-t=2.770,P=0.017。造模后8周:LSD-t=2.445,P=0.031)。造模后4周、8周,3组大鼠椎间盘CEP组织ADAMTS-5基因表达量比较,组间差异均有统计学意义(造模后4周:1.06±0.07,1.30±0.08,1.81±0.15,F=64.344,P=0.000; 造模后8周:1.13±0.09,1.59±0.13,2.14±0.10,F=110.156,P=0.000); 空白组大鼠ADAMTS-5基因表达量低于CEP退变模型组与病证结合模型组大鼠(造模后4周:LSD-t=3.543,P=0.004; LSD-t=11.104,P=0.000。造模后8周:LSD-t=6.702,P=0.000; LSD-t=14.821,P=0.000),CEP退变模型组大鼠ADAMTS-5基因表达量低于病证结合模型组(造模后4周:LSD-t=7.562,P=0.000; 造模后8周:LSD-t=8.119,P=0.000)。结论:切除双侧卵巢结合阻断CEP营养供应法是构建大鼠肾虚型椎间盘CEP退变病证结合模型的一种可行方法。
Abstract:
Objective:To explore the feasibility of building a disease-syndrome(DS)combination rat model of intervertebral disc(IVD)cartilage endplate(CEP)degeneration due to kidney deficiency by removing bilateral ovaries and blocking the supply of nutrients to CEPs.Methods:One hundred and eight 12-week-old SPF-grade female Sprague-Dawley(SD)rats were randomly assigned into blank group,CEP degeneration model group and DS combination model group,36 cases in each group.The rats in CEP degeneration model group were intervened by injection anhydrous ethanol into bone below the CEP of IVD.The ones in DS combination model group were removed bilateral ovaries and followed by the same injection as mentioned above.Those in blank group received no intervention.After modeling,the apparent characteristics of the rats were observed and recorded,followed by the measurement of quantified symptom and sign scores of kidney deficiency.Six rats were randomly selected from each group at the 4th and 8th weeks after the modeling respectively,followed by the examination of bone mineral density(BMD)of the caudal vertebrae by X-ray scanning.The rats were sacrificed and the caudal vertebrae were harvested for measuring the trabecular number(TN),trabecular separation(TS)and trabecular thickness(TT)by Micro-CT scanning.Another six rats randomly selected from each group were sacrificed to isolate the IVDs.The hematoxylin-eosin(HE)staining was performed for observing the degeneration of CEPs,and quantitative real-time polymerase chain reaction(qRT-PCR)was employed for detecting the mRNA expression levels of aggrecan(ACAN)and a disintesrin and metalloprotease with thrombospondin type I motifs(ADAMTS)-5 in CEP tissues of IVDs.Results:During the modeling,no accidental death occurred in all rats.One rat in CEP degeneration model group presented with poor healing and necrosis at the caudal incision at 3 days after the surgery,and the incision healed about one week after external medication.The incisions of the remaining rats healed well.After modeling,the rats in DS combination model group gradually exhibited such kidney deficiency symptoms as withered hair with a tendency to fall out,increased food intake,mental fatigue,dim eyes,dry stool,reduced movement and slow response,which,however,failed to be observed in rats of blank group and CEP degeneration model group.There was interaction between the time factor and the group factor in quantified symptom and sign scores(F=14.326,P=0.000).The overall comparison of the quantified symptom and sign scores among the three groups revealed a statistically significant difference,implying the presence of group effect(F=22.317,P=0.000).After modeling,the difference in symptom and sign scores quantified at different time points was statistically significant,confirming the presence of time effect(F=50.279,P=0.000)...

参考文献/References:

[1] DELUCCA J F,CORTES D H,JACOBS N T,et al.Human cartilage endplate permeability varies with degeneration and intervertebral disc site[J].J Biomech,2016,49(4):550-557.
[2] VERGROESEN P P,KINGMA I,EMANUEL K S,et al.Mechanics and biology in intervertebral disc degeneration:a vicious circle[J].Osteoarthritis Cartilage,2015,23(7):1057-1070.
[3] 李满意,张子扬,娄玉钤.腰痹的源流及相关历史文献复习(下)[J].风湿病与关节炎,2016,5(6):51-59.
[4] 李满意,张子扬,娄玉钤.腰痹的源流及相关历史文献复习(上)[J].风湿病与关节炎,2016,5(5):39-46.
[5] 夏炳江,童培建.适宜中医药研究的椎间盘退变动物模型的构建[J].中医正骨,2016,28(11):71-73.
[6] 黄正团,曹颖颖.基于恐伤肾的阿尔茨海默病肾虚证大鼠模型的建立与评价[J].现代中西医结合杂志,2019,28(21):2286-2291.
[7] 李思汉,李书楠,周福,等.关于中医证候动物模型研究的思考[J].中华中医药杂志,2019,34(8):3357-3361.
[8] 宋亚刚,李艳,崔琳琳,等.中医药病证结合动物模型的现代应用研究及思考[J].中草药,2019,50(16):3971-3978.
[9] 林传权.病证结合动物模型研究的进展及存在问题分析[J].中华中医药杂志,2019,34(8):3601-3604.
[10] 徐德成,马迎民,范吉平.中医“肾精”的现代医学内涵[J].中医杂志,2017,58(22):1891-1897.
[11] 王媛媛,蒙婷,丁慧敏.温经汤治疗肾虚型更年期综合征的效果及对生殖内分泌和免疫功能的影响[J].现代中西医结合杂志,2020,29(17):1847-1850.
[12] 成金枝,张俊龙,郭蕾,等.阿尔茨海默病肾虚证病证结合动物模型的制备及验证[J].中华中医药杂志,2018,33(3):1063-1066.
[13] 王宇航,魏小丽,宋石龙,等.机械推拿对慢性疲劳综合征肾虚型大鼠行为学及血清IL-1β、IL-6的影响[J].时珍国医国药,2018,29(5):1235-1237.
[14] 徐碧红,李茂清,熊思艺,等.补肾调经膏方对肾虚型卵巢早衰患者性激素水平的影响[J].广州中医药大学学报, 2017,34(2):167-172.
[15] 范怀玲,林国清,郑薏,等.电针关元对肾虚不孕大鼠性激素及其受体表达影响的实验研究[J].世界中西医结合杂志,2019,14(5):655-658.
[16] 袁普卫,杨威,康武林,等.豚鼠绝经后肾虚型原发性骨关节炎模型的建立[J].中华中医药杂志,2018,33(4):1625-1630.
[17] 陶黎,刘梅洁,薛欣,等.益肾蠲痹丸对肾虚胶原诱导性关节炎大鼠踝关节骨质破坏的影响[J].中医杂志,2018,59(5):420-426.
[18] 柳源,刁永帅,冯奇,等.“肾主骨”理论的研究进展[J].辽宁中医杂志,2019,46(7):1558-1561.
[19] 黄文庭.肾虚证与骨质疏松症的相关性研究[J].广西中医药,2017,40(5):1-2.
[20] 梁文娜,李西海,李灿东.血清microRNA与绝经后骨质疏松症肾虚证的关系[J].中医正骨, 2017,29(10):53-56.
[21] 袁维,周小小,蔡攀,等.终板下椎体缺血诱导兔椎间盘退行性变模型的建立及终板中内皮素1的表达[J].脊柱外科杂志,2020,18(2):118-124.
[22] 张勇,杜世新,白新明,等.绝经期骨质疏松症与腰椎间盘退变的相关性分析[J].颈腰痛杂志,2019,40(1):125-127.
[23] 徐浩伟,王善金,张树宝,等.骨质疏松与腰椎间盘退变相关性的研究进展[J].中国骨质疏松杂志,2020,26(3):445-448.
[24] VERMA P,DALAL K.ADAMTS-4 and ADAMTS-5:key enzymes in osteoarthritis[J].J Cell Biochem,2011,112(12):3507-3514.
[25] GROMA G,XIN W,GRSKOVIC I,et al.Abnormal bone quality in cartilage oligomeric matrix protein and matrilin 3 double-deficient mice caused by increased tissue inhibitor of metalloproteinases 3 deposition and delayed aggrecan degradation[J].Arthritis Rheum,2012,64(8):2644-2654.
[26] VAN MEURS J B.Osteoarthritis year in review 2016:genetics,genomics and epigenetics[J].Osteoarthritis Cartilage,2017,25(2):181-189.
[27] STANTON H,ROGERSON M,EAST C J,et al.ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro[J].Nature,2005,434(7033):648-652.

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[4]夏炳江,童培建.适宜中医药研究的椎间盘退变动物模型的构建[J].中医正骨,2016,28(11):71.
[5]赵赫,高誉珊,胡振国,等.补肾壮筋汤对腰椎间盘退变大鼠椎间盘及行为学特征影响的实验研究[J].中医正骨,2017,29(07):12.
　ZHAO He,GAO Yushan,HU Zhenguo,et al.Effect of Bushen Zhuangjin Tang(补肾壮筋汤)on intervertebral disc and behavioral characteristics of rats with lumbar intervertebral disc degeneration[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(05):12.
[6]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.
　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(05):12.
[7]夏炳江,侯明生,张磊,等.五福饮对大鼠尾部退变椎间盘的影响及其作用机制…[J].中医正骨,2018,30(01):18.
　XIA Bingjiang,HOU Mingsheng,ZHANG Lei,et al.Effects of Wufu Yin(五福饮)on caudal degenerative intervertebral disc in rats and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(05):18.
[8]刁志君,姜宏,刘锦涛,等.炎症因子在椎间盘退变中的作用[J].中医正骨,2018,30(07):32.
[9]刘汝专,张磊,潘汉升,等.中药复方干预椎间盘退变的实验研究进展[J].中医正骨,2019,31(04):36.
[10]劳杨骏,裘一丹,徐彬,等.轴向负载诱导小鼠椎间盘退变模型的建立[J].中医正骨,2019,31(05):1.
　LAO Yangjun,QIU Yidan,XU Bin,et al.A mouse model of intervertebral disc degeneration induced by axial loads[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(05):1.

备注/Memo

备注/Memo:
基金项目:浙江省自然科学基金项目(LQ18H270006)
通讯作者:夏炳江 E-mail:xiabj2006@163.com
更新日期/Last Update: 1900-01-01