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[1]勞楊駿,裘一丹,徐彬,等.軸向負(fù)載誘導(dǎo)小鼠椎間盤退變模型的建立[J].中醫(yī)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.
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軸向負(fù)載誘導(dǎo)小鼠椎間盤退變模型的建立()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第31卷
期數(shù):
2019年05期
頁碼:
1-6
欄目:
基礎(chǔ)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:
勞楊駿1; 裘一丹2; 徐彬1; 盛紅楓1; 沈立鋒1; 劉亦楊1; 余偉波1; 吳承亮2
(1.浙江省立同德醫(yī)院,浙江 杭州 310012; 2.浙江中醫(yī)藥大學(xué),浙江 杭州 310053)
Author(s):
LAO Yangjun1; QIU Yidan2; XU Bin1; SHENG Hongfeng1; SHEN Lifeng1; LIU Yiyang1; YU Weibo1; WU Chengliang2
1.Tongde Hospital of Zhejiang Province,Hangzhou 310012,Zhejiang,China 2.Zhejiang University of Traditional Chinese Medicine,Hangzhou 310053,Zhejiang,China
關(guān)鍵詞:
椎間盤退行性變;  小鼠;  疾病模型; 動(dòng)物;  軸向負(fù)載;  熱板;  動(dòng)物實(shí)驗(yàn)
Keywords:
intervertebral disc degeneration;  mice;  disease models; animal;  axial load;  hot plate;  animal experimentation
摘要:
目的:探討建立軸向負(fù)載誘導(dǎo)小鼠椎間盤退變模型的有效方法。方法:將24只16月齡SPF級小鼠隨機(jī)分為模型組和對照組,每組12只。每日10:00、15:00、19:00將模型組小鼠放入自制熱板鼠籠中,打開熱板并設(shè)定溫度為50 ℃,從小鼠開始跳躍至力竭約10 min左右; 對照組小鼠不做任何處理。分別于造模1個(gè)月和3個(gè)月后從模型組和對照組隨機(jī)抽取6只小鼠,采用頸椎脫臼法處死,完整取下L3～4椎間盤和L3、L4椎體,制作連續(xù)冠狀位切片后,分別行蘇木素-伊紅(hematoxylin-eosin,HE)染色和阿爾新藍(lán)-蘇木素(alcian blue-hematoxylin,ABH)染色,在光學(xué)顯微鏡下觀察腰椎間盤組織形態(tài)學(xué)變化,通過免疫組織化學(xué)染色法在光學(xué)顯微鏡下觀察腰椎間盤組織中Ⅱ型膠原蛋白的表達(dá)情況,并通過繪圖軟件測量椎間盤高度和軟骨終板厚度。結(jié)果:①腰椎間盤組織形態(tài)學(xué)觀察結(jié)果。HE染色顯示造模1個(gè)月及3個(gè)月后,對照組腰椎間盤組織基本正常; 模型組腰椎間盤組織可見退行性改變,其中造模3個(gè)月后較造模1個(gè)月后退變更嚴(yán)重,椎間盤纖維環(huán)出現(xiàn)裂隙,軟骨終板分層結(jié)構(gòu)紊亂、排列不規(guī)則,髓核破壞或皺縮,軟骨終板內(nèi)出現(xiàn)多個(gè)骨化中心。ABH染色結(jié)果顯示造模1個(gè)月和3個(gè)月后,對照組軟骨終板細(xì)胞排列整齊,軟骨終板細(xì)胞數(shù)目較多,軟骨基質(zhì)染色較藍(lán),部分椎間盤軟骨終板內(nèi)存在較少呈紅染的骨化中心,但骨化中心形態(tài)較小; 模型組軟骨終板細(xì)胞排列混亂,細(xì)胞數(shù)目明顯減少,且軟骨基質(zhì)染色變淺,軟骨終板出現(xiàn)多個(gè)骨化中心,且骨化中心形態(tài)較大。②腰椎間盤免疫組織化學(xué)觀察結(jié)果。造模1個(gè)月和3個(gè)月后,對照組Ⅱ型膠原蛋白在髓核、纖維環(huán)內(nèi)層和軟骨終板均有陽性表達(dá),染色較深; 模型組Ⅱ型膠原蛋白的陽性表達(dá)在髓核中央及纖維環(huán)外層較對照組明顯降低,且造模3個(gè)月后Ⅱ型膠原蛋白在髓核與纖維環(huán)外層的表達(dá)較造模1個(gè)月后降低更明顯。③腰椎間盤形態(tài)學(xué)測量結(jié)果。造模1個(gè)月后,模型組椎間盤高度和軟骨終板厚度均小于對照組[(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個(gè)月后,模型組椎間盤高度與對照組比較,差異無統(tǒng)計(jì)學(xué)意義[(0.197±0.052)mm,(0.235±0.053)mm,t=1.478,P=0.163]; 模型組軟骨終板厚度小于對照組[(0.060±0.012)mm,(0.093±0.018)mm,t=4.092,P=0.001]。結(jié)論:用熱板誘導(dǎo)小鼠跳躍,可以有效建立軸向負(fù)載誘導(dǎo)小鼠椎間盤退變模型。
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.

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備注/Memo

備注/Memo:
基金項(xiàng)目:國家自然科學(xué)基金項(xiàng)目(81573994); 浙江省基礎(chǔ)公益研究計(jì)劃項(xiàng)目(LGF19H60007)
更新日期/Last Update: 2019-05-20