[1]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):1-6.
 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):1-6.
点击复制

轴向负载诱导小鼠椎间盘退变模型的建立()
分享到:

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第31卷
期数:
2019年05期
页码:
1-6
栏目:
基础研究
出版日期:
2019-05-20

文章信息/Info

Title:
A mouse model of intervertebral disc degeneration induced by axial loads
作者:
劳杨骏1裘一丹2徐彬1盛红枫1沈立锋1刘亦杨1余伟波1吴承亮2
(1.浙江省立同德医院,浙江 杭州 310012; 2.浙江中医药大学,浙江 杭州 310053)
Author(s):
LAO Yangjun1QIU Yidan2XU Bin1SHENG Hongfeng1SHEN Lifeng1LIU Yiyang1YU Weibo1WU Chengliang2
1.Tongde Hospital of Zhejiang Province,Hangzhou 310012,Zhejiang,China 2.Zhejiang University of Traditional Chinese Medicine,Hangzhou 310053,Zhejiang,China
关键词:
椎间盘退行性变 小鼠 疾病模型动物 轴向负载 热板 动物实验
Keywords:
intervertebral disc degeneration mice disease modelsanimal axial load hot plate animal experimentation
摘要:
目的:探讨建立轴向负载诱导小鼠椎间盘退变模型的有效方法。方法:将24只16月龄SPF级小鼠随机分为模型组和对照组,每组12只。每日10:00、15:00、19:00将模型组小鼠放入自制热板鼠笼中,打开热板并设定温度为50 ℃,从小鼠开始跳跃至力竭约10 min左右; 对照组小鼠不做任何处理。分别于造模1个月和3个月后从模型组和对照组随机抽取6只小鼠,采用颈椎脱臼法处死,完整取下L3~4椎间盘和L3、L4椎体,制作连续冠状位切片后,分别行苏木素-伊红(hematoxylin-eosin,HE)染色和阿尔新蓝-苏木素(alcian blue-hematoxylin,ABH)染色,在光学显微镜下观察腰椎间盘组织形态学变化,通过免疫组织化学染色法在光学显微镜下观察腰椎间盘组织中Ⅱ型胶原蛋白的表达情况,并通过绘图软件测量椎间盘高度和软骨终板厚度。结果:①腰椎间盘组织形态学观察结果。HE染色显示造模1个月及3个月后,对照组腰椎间盘组织基本正常; 模型组腰椎间盘组织可见退行性改变,其中造模3个月后较造模1个月后退变更严重,椎间盘纤维环出现裂隙,软骨终板分层结构紊乱、排列不规则,髓核破坏或皱缩,软骨终板内出现多个骨化中心。ABH染色结果显示造模1个月和3个月后,对照组软骨终板细胞排列整齐,软骨终板细胞数目较多,软骨基质染色较蓝,部分椎间盘软骨终板内存在较少呈红染的骨化中心,但骨化中心形态较小; 模型组软骨终板细胞排列混乱,细胞数目明显减少,且软骨基质染色变浅,软骨终板出现多个骨化中心,且骨化中心形态较大。②腰椎间盘免疫组织化学观察结果。造模1个月和3个月后,对照组Ⅱ型胶原蛋白在髓核、纤维环内层和软骨终板均有阳性表达,染色较深; 模型组Ⅱ型胶原蛋白的阳性表达在髓核中央及纤维环外层较对照组明显降低,且造模3个月后Ⅱ型胶原蛋白在髓核与纤维环外层的表达较造模1个月后降低更明显。③腰椎间盘形态学测量结果。造模1个月后,模型组椎间盘高度和软骨终板厚度均小于对照组[(0.211±0.063)mm,(0.289±0.050)mm,t=2.984,P=0.007;(0.074±0.011)mm,(0.097±0.015)mm,t=2.813,P=0.023]; 造模3个月后,模型组椎间盘高度与对照组比较,差异无统计学意义[(0.197±0.052)mm,(0.235±0.053)mm,t=1.478,P=0.163]; 模型组软骨终板厚度小于对照组[(0.060±0.012)mm,(0.093±0.018)mm,t=4.092,P=0.001]。结论:用热板诱导小鼠跳跃,可以有效建立轴向负载诱导小鼠椎间盘退变模型。
Abstract:
Objective:To explore the effective method for building mouse models of intervertebral disc degeneration induced by axial loads.Methods:Twenty-four 16-month-old SPF-grade mice were randomly divided into model group and control group,12 cases in each group.The mice in model group were put into a self-made heating plate cage at 10:00,15:00 and 19:00 every day.The heating plate was turned on and its temperature was set to 50 ℃.Every time the mice kept jumping for about 10 minutes and were exhausted in the end.The mice in control group were not given any intervention.Six mice were randomly selected out from each group respectively and were executed by using cervical dislocation method after 1- and 3-month modeling respectively.Their L3-4 intervertebral disc and bilateral adjacent vertebral bodies were fetched out completely and were sectioned continuously in coronal position for hematoxylin-eosin(HE)staining and alcian blue-hematoxylin(ABH)staining.The histological and morphological changes of lumbar intervertebral disc tissues were observed under the optical microscope,and the protein expression levels of typeⅡcollagen in lumbar intervertebral disc tissues were detected by using immunohistochemical staining,and the intervertebral disc height and cartilage endplate thickness were measured by using drawing software.Results:HE staining results demonstrated that lumbar intervertebral disc tissues of mice were basically normal in control group after 1- and 3-month modeling respectively,while degenerative changes were found in lumbar intervertebral disc tissues of mice in modelgroup and degenerative changes were more visible after 3-month modeling.The degeneration presented with(1)cracks in intervertebral disc annulus fibrosus,(2)disordered and irregularly arranged layered structure of cartilage endplate,(3)damaged or wizened nucleus pulposus,and(4)multiple ossification centers in cartilage endplate.ABH staining results showed that after 1- and 3-month modeling,abundant and regularly arranged cartilage endplate cells were found in control group,and the cartilage matrix presented with blue staining,and a few littlish red stained ossification centers were found in cartilage endplate of some intervertebral discs; while cartilage endplate cells arranged disorderly and decreased significantly in model group,and cartilage matrix presented with light-coloured staining,and multiple largish ossification centers appeared in cartilage endplate.The immunohistochemical staining results of lumbar intervertebral disc tissues showed that positive expressions of typeⅡcollagen were found in nucleus pulposus,inner layer of fibrous rings and cartilage endplate in control group after 1- and 3-month modeling,and the cells were dark stained; while the positive expressions of typeⅡcollagen were obviously lower in center of nucleus pulposus and outer layer of fibrous rings in model group compared to control group,and more obvious decrease was found after 3-month modeling.The intervertebral disc height and cartilage endplate thickness were smaller in model group compared to control group after 1-month modeling(0.211+/-0.063 vs 0.289+/-0.050 mm,t=2.984,P=0.007; 0.074+/-0.011 vs 0.097+/-0.015 mm,t=2.813,P=0.023).There was no statistical difference in intervertebral disc height between model group and control group after 3–month modeling(0.197+/-0.052 vs 0.235+/-0.053 mm,t=1.478,P=0.163).The cartilage endplate thickness was smaller in model group compared to control group(0.060+/-0.012 vs 0.093+/-0.018 mm,t=4.092,P=0.001).Conclusion:The mouse model of intervertebral disc degeneration induced by axial loads can be built effectively by using heating plate for forcing mouse to jump.

参考文献/References:

[1] SEGAR A H,FAIRBANK J C T,URBAN J.Leptin and the intervertebral disc:a biochemical link exists between obesity,intervertebral disc degeneration and low back pain-an in vitro study in a bovine model[J].Eur Spine J,2019,28(2):214-223. [2] 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(7):32-35. [3] 劳杨骏,徐涛涛,杨笑奇,等.小鼠椎间盘退变模型的构建策略[J].中国中医急症,2015,24(10):1783-1786. [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):71-73. [5] BARCZEWSKA M,JEZIERSKA-WOZNIAK K,HABICH A,et al.Evaluation of regenerative processes in the pig model of intervertebral disc degeneration after transplantation of bone marrow-derived mesenchymal stem cells[J].Folia Neuropathol,2018,56(2):124-132. [6] STEFFEN F,BERTOLO A,AFFENTRANGER R,et al.Treatment of naturally degenerated canine lumbosacral intervertebral discs with autologous mesenchymal stromal cells and collagen microcarriers:a prospective clinical study[J].Cell Transplant,2019,28(2):201-211. [7] PENNICOOKE B,HUSSAIN I,BERLIN C,et al.Annulus fibrosus repair using high-density collagen gel:an in vivo ovine model[J].Spine(Phila Pa 1976),2018,43(4):E208-E215. [8] GELALIS I D,CHRISTOFOROU G,CHARCHANTI A,et al.Autologous platelet-rich plasma(PRP)effect on intervertebral disc restoration:an experimental rabbit model[J].Eur J Orthop Surg Traumatol, 2019,29(3):545-551. [9] SUN Q, LIU F,GAO M,et al.Therapeutic evaluation of acupoint stimulation with needle-scapelon on rat model of degenerative cervical intervertebral discs[J]. Biomed Pharmacother,2019,110:677-684. [10] O'CONNELL G D,VRESILOVIC E J,ELLIOT[11] SHOWALTER B L,BECKSTEIN J C,MARTIN J T,et al.Comparison of animal discs used in disc research to human lumbar disc torsion mechanics and collagen content[J].Spine(Phila Pa 1976),2012,37(15): E900-907. [12] ZHANG J,WANG X,LIU H,et al.TNF-ɑ enhances apoptosis by promoting chop expression in nucleus pulposus cells:role of the MAPK and NF-кB pathways[J].J Orthop Res,2019,37(3):697-705 [13] YANG B,O'CONNELL G D.GAG content,fiber stiffness,and fiber angle affect swelling-based residual stress in the intact annulus fibrosus[J].Biomech Model Mechanobiol,2018 Dec 10.doi:10.1007/s10237-018-1105-9. [Epub ahead of print] [14] CHE Y J,GUO J B,LIANG T,et al.Controlled immobilization-traction based on intervertebral stability is conducive to the regeneration or repair of the degenerative disc:An in vivo study on the rat coccygeal model[J].Spine J,2018 Nov 3.pii:S1529-9430(18)31204-X.doi:10.1016/j.spinee.2018.10.018.[Epub ahead of print] [15] SAKAI D,NISHIMURA K,TANAKA M A,et al.Migration of bone marrow-derived cells for endogenous repair in a new tail-looping disc degeneration model in the mouse:a pilot study[J].Spine J,2015,15(6):1356-1365. [16] PAPUGA M O,KWOK E,YOU Z,et al.TNF is required for the induction but not the maintenance of compression-induced BME signals in murine tail vertebrae:limitations of anti-TNF therapy for degenerative disc disease[J].J Orthop Res,2011,29(9):1367-1374. [17] HSIEH A H,LOTZ J C.Prolonged spinal loading induces matrix metalloproteinase-2 activation in intervertebral discs[J].Spine(Phila Pa),2003,28(16):1781-1788. [18] COURT C,COLLIOU O K,CHIN J R,et al.The effect of static in vivo bending on the murine intervertebral disc[J].Spine J,2001,1(4):239-245. [19] ZHOU H,SHEN J,HU Z,et al.Leukemia inhibitory factor promotes extracellular matrix synthesis in degenerative nucleus pulposus cells via MAPK-ERK1/2 signaling pathway[J].Biochem Biophys Res Commun,2018,507(1-4):253-259. [20] MARRAS W S,LAVENDER S A,LEURGANS S E,et al.Biomechanical risk factors for occupationally related low back disorders[J].Ergonomics,1995,38(2):377-410. [21] DU S,SUN Y,ZHAO B.Interleukin-6 serum levels are elevated in individuals with degenerative cervical myelopathy and are correlated with symptom severity[J].Med Sci Monit,2018,24:7405-7413. [22] TAM W K,CHEUNG K M,LEUNG V Y.Intervertebral disc engineering through exploiting mesenchymal stem cells:progress and perspective[J].Curr Stem Cell Res Ther,2016,11(6):505-512. [23] ALLON A A,AUROUER N,YOO B B,et al.Structured coculture of stem cells and disc cells prevent disc degeneration in a rat model[J].Spine J,2010,10(12):1089-1097. [24] CHAN D D,KHAN S N,YE X,et al.Mechanical deformation and glycosaminoglycan content changes in a rabbit annular puncture disc degeneration model[J].Spine(Phila Pa 1976),2011,36(18):1438-1445. [25] DALY C D,GHOSH P,BADAL T,et al.A comparison of two ovine lumbar intervertebral disc injury models for the evaluation and development of novel regenerative therapies[J].Global Spine J,2018,8(8):847-859. [26] LAO Y J,XU T T,JIN H T,et al.Accumulated spinal axial biomechanical loading induces degeneration in intervertebral disc of mice lumbar spine[J].Orthop Surg,2018,10(1):56-63. [27] 黄秀芳,原向伟.Sox9和Ⅱ型胶原蛋白在腰椎间盘退变中的表达及意义[J].中国医药科学,2014,4(3):18-21.T D M.Comparison of animals used in disc research to human lumbar disc geometry[J].Spine,2007,32(3):328-333.

相似文献/References:

[1]张莉,秦丹霞,张细姣.腹针治疗椎间盘源性腰痛[J].中医正骨,2015,27(10):38.
[2]吴青坡,孙国绍,王林杰.后路椎管减压联合腰椎椎弓根钉动态稳定装置内固定 治疗单节段腰椎退行性疾病[J].中医正骨,2015,27(10):42.
[3]王乐,徐无忌.六味地黄丸对兔椎间盘退变模型椎间盘组织中Ⅰ、Ⅱ型胶原表达的影响[J].中医正骨,2016,28(08):1.
 WANG Le,XU Wuji.Effect of Liuwei Dihuang Wan(六味地黄丸)on typeⅠandⅡcollagen expression in intervertebral disc of rabbit model of intervertebral disc degeneration[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(05):1.
[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]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.
[7]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.
[8]陶振宇,张月雷,陈华,等.细胞自噬在酒精性股骨头坏死中的作用及相关机制研究[J].中医正骨,2018,30(10):4.
 TAO Zhenyu,ZHANG Yuelei,CHEN Hua,et al.Roles of cell autophagy in alcohol-induced osteonecrosis of the femoral head and its related mechanisms of action:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(05):4.
[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]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.

备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(81573994); 浙江省基础公益研究计划项目(LGF19H60007)
更新日期/Last Update: 2019-05-20