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[1]夏炳江,沈興潮,胡松峰,等.大鼠腎虛型椎間盤軟骨終板退變病證結(jié)合模型的構(gòu)建與評價[J].中醫(yī)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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大鼠腎虛型椎間盤軟骨終板退變病證結(jié)合模型的構(gòu)建與評價()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期數(shù):
2021年05期
頁碼:
1-10
欄目:
基礎(chǔ)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; 沈興潮; 胡松峰; 韋金忠; 許楊
(紹興市中醫(yī)院,浙江 紹興 312000)
Author(s):
XIA Bingjiang; SHEN Xingchao; HU Songfeng; WEI Jinzhong; XU Yang
Shaoxing Hospital of Traditional Chinese Medicine,Shaoxing 312000,Zhejiang,China
關(guān)鍵詞:
椎間盤退行性變;  軟骨終板退變;  腎虛;  聚集蛋白聚糖;  含Ⅰ型血小板結(jié)合蛋白基序的解聚蛋白樣金屬蛋白酶;  骨小梁數(shù)目;  骨小梁分離度;  骨小梁厚度;  大鼠; Sprague-Dawley;  疾病模型; 動物;  動物實驗
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:探討采用切除雙側(cè)卵巢結(jié)合阻斷軟骨終板營養(yǎng)供應(yīng)法構(gòu)建大鼠腎虛型椎間盤軟骨終板(cartilage endplate,CEP)退變病證結(jié)合模型的可行性。方法:將108只12周齡SPF級雌性SD大鼠隨機分為空白組、CEP退變模型組、病證結(jié)合模型組,每組36只。CEP退變模型組大鼠于椎間盤CEP下方骨質(zhì)處注射無水酒精,病證結(jié)合模型組大鼠摘除雙側(cè)卵巢并于椎間盤CEP下方骨質(zhì)處注射無水酒精,空白組大鼠不做任何處理。造模后觀察并記錄大鼠表觀特征,測定腎虛癥狀體征量化評分。分別于造模后4周、8周,從各組隨機抽取6只大鼠,先攝尾椎X線片,測算尾椎骨密度; 再處死大鼠,取出尾椎,行Micro-CT掃描測算骨小梁數(shù)目(trabecular number,TN)、骨小梁分離度(trabecular separation,TS)、骨小梁厚度(trabecular thickness,TT); 再從各組隨機抽取6只大鼠,切取造模椎間盤,采用HE染色觀察各組大鼠椎間盤CEP組織退變情況,采用實時定量聚合酶鏈反應(yīng)法檢測椎間盤CEP組織聚集蛋白聚糖和含Ⅰ型血小板結(jié)合蛋白基序的解聚蛋白樣金屬蛋白酶(a disintesrin and metalloprotease with thrombospondin typeⅠmotifs,ADAMTS)-5基因表達量。結(jié)果:①一般情況。在造模過程中,所有大鼠均無意外死亡發(fā)生。CEP退變模型組1只大鼠術(shù)后3 d出現(xiàn)尾部切口愈合不良、壞死現(xiàn)象,換藥后1周左右切口愈合; 其余大鼠尾部切口均愈合良好。②大鼠表觀特征。造模后,病證結(jié)合模型組大鼠逐漸出現(xiàn)毛發(fā)枯槁易脫落、飲食增多、精神倦怠、兩眼無神、大便干結(jié)變硬、活動減少、反應(yīng)遲鈍等腎虛表現(xiàn),空白組與CEP退變模型組大鼠無上述表現(xiàn)。③腎虛癥狀體征量化評分。時間因素和分組因素存在交互效應(yīng)(F=14.326,P=0.000)。3組大鼠腎虛癥狀體征量化評分總體比較,差異有統(tǒng)計學(xué)意義,即存在分組效應(yīng)(F=22.317,P=0.000)。造模后不同時間點大鼠腎虛癥狀體征量化評分的差異有統(tǒng)計學(xué)意義,即存在時間效應(yīng)(F=50.279,P=0.000)。造模后,3組大鼠腎虛癥狀體征量化評分隨時間變化趨勢不一致,空白組大鼠腎虛癥狀體征量化評分隨時間未見明顯變化[(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退變模型組和病證結(jié)合模型組大鼠腎虛癥狀體征量化評分隨時間均呈升高趨勢[(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組大鼠腎虛癥狀體征量化評分比較,差異無統(tǒng)計學(xué)意義(F=1.400,P=0.284)。造模后4周、6周、8周,3組大鼠腎虛癥狀體征量化評分比較,組間差異均有統(tǒng)計學(xué)意義(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); 空白組和CEP退變模型組大鼠腎虛癥狀體征量化評分均低于病證結(jié)合模型組(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); 空白組與CEP退變模型組大鼠腎虛癥狀體征量化評分比較,差異均無統(tǒng)計學(xué)意義(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組大鼠尾椎骨密度比較,差異有統(tǒng)計學(xué)意義[(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]; 空白組和CEP退變模型組大鼠尾椎骨密度均高于病證結(jié)合模型組(LSD-t=3.682,P=0.003; LSD-t=3.028,P=0.011); 空白組大鼠尾椎骨密度與CEP退變模型組比較,差異無統(tǒng)計學(xué)意義(LSD-t=0.655,P=0.525)。造模后8周,3組大鼠尾椎骨密度比較,差異有統(tǒng)計學(xué)意義[(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]; 空白組和CEP退變模型組大鼠尾椎骨密度均高于病證結(jié)合模型組(LSD-t=3.749,P=0.003; LSD-t=3.035,P=0.010); 空白組大鼠尾椎骨密度與CEP退變模型組比較,差異無統(tǒng)計學(xué)意義(LSD-t=0.714,P=0.489)。⑤骨微結(jié)構(gòu)指標(biāo)。造模后4周,3組大鼠TN、TS比較,組間差異均具有統(tǒng)計學(xué)意義[TN:(5.01±0.12)個·mm-1,(4.81±0.12)個·mm-1,(3.86±0.17)個·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]; 空白組和CEP退變模型組大鼠TN高于病證結(jié)合模型組(LSD-t=12.991,P=0.000; LSD-t=10.657,P=0.000),TS低于病證結(jié)合模型組(LSD-t=4.623,P=0.001; LSD-t=2.736,P=0.018); 空白組大鼠TN和TS與CEP退變模型組比較,組間差異均無統(tǒng)計學(xué)意義(TN:LSD-t=0.432,P=0.924; TS:LSD-t=1.887,P=0.084)。造模后8周,3組大鼠TN、TS比較,組間差異均具有統(tǒng)計學(xué)意義[TN:(4.91±0.26)個·mm-1,(4.61±0.55)個·mm-1,(3.05±0.44)個·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]; 空白組和CEP退變模型組大鼠TN高于病證結(jié)合模型組(LSD-t=6.854,P=0.000; LSD-t=5.750,P=0.000),TS低于病證結(jié)合模型組(LSD-t=3.623,P=0.003; LSD-t=3.106,P=0.009); 空白組大鼠TN和TS與CEP退變模型組比較,組間差異均無統(tǒng)計學(xué)意義(TN:LSD-t=1.104,P=0.291; TS:LSD-t=0.518,P=0.614)。造模后4周、8周,3組大鼠TT比較,組間差異均無統(tǒng)計學(xué)意義[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椎間盤CEP組織形態(tài)。造模后,CEP退變模型組和病證結(jié)合模型組大鼠尾椎CEP均出現(xiàn)退變,表現(xiàn)為軟骨細胞排列不規(guī)則,軟骨細胞簇聚,潮線模糊,染色不均,軟骨鈣化層增厚等,而病證結(jié)合模型組退變更明顯。⑦椎間盤CEP組織聚集蛋白聚糖和ADAMTS-5基因的表達。造模后4周、8周,3組大鼠椎間盤CEP組織聚集蛋白聚糖基因表達量比較,組間差異均有統(tǒng)計學(xué)意義(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); 空白組大鼠聚集蛋白聚糖基因表達量高于CEP退變模型組和病證結(jié)合模型組(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退變模型組大鼠聚集蛋白聚糖基因表達量高于病證結(jié)合模型組(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組大鼠椎間盤CEP組織ADAMTS-5基因表達量比較,組間差異均有統(tǒng)計學(xué)意義(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); 空白組大鼠ADAMTS-5基因表達量低于CEP退變模型組與病證結(jié)合模型組大鼠(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退變模型組大鼠ADAMTS-5基因表達量低于病證結(jié)合模型組(造模后4周:LSD-t=7.562,P=0.000; 造模后8周:LSD-t=8.119,P=0.000)。結(jié)論:切除雙側(cè)卵巢結(jié)合阻斷CEP營養(yǎng)供應(yīng)法是構(gòu)建大鼠腎虛型椎間盤CEP退變病證結(jié)合模型的一種可行方法。
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)...

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備注/Memo

備注/Memo:
基金項目:浙江省自然科學(xué)基金項目(LQ18H270006)
通訊作者:夏炳江 E-mail:[email protected]
更新日期/Last Update: 1900-01-01