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

Construction and evaluation of disease-syndrome combination rat model of intervertebral disc cartilage end-plate degeneration due to kidney deficiency

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。

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).The variation trends of the quantified symptom and sign scores were inconsistent with each other over time in the three groups,specifically in blank group,the scores did not change significantly over time(1.00±0.71,1.40±0.55,1.80±0.84,2.00±1.00,F=1.573,P=0.235),whereas those in CEP degeneration model group and DS combination model group displayed an upward trend over time(0.80±0.45,1.20±0.45,1.60±0.55,2.20±0.84,F=5.095,P=0.012;1.40±0.55,3.40±1.14,4.60±0.55,6.80±0.84,F=39.256,P=0.000).Two weeks after modeling,there was no statistical difference in the quantified symptom and sign scores among the three groups(F=1.400,P=0.284).However,the differences among the three groups at the 4th,6th and 8th weeks after modeling were statistically significant(4th week after modeling:F=12.333,P=0.001;6th week after modeling:F=32.462,P=0.000:8th week after modeling:F=46.083,P=0.000).The quantified symptom and sign scores of the blank group and CEP degeneration model group were lower than those of the DS combination model group(4th week after modeling:LSD-t=4.082,P=0.002;LSD-t=4.491,P=0.001;6th week after modeling:LSD-t=6.725,P=0.000;LSD-t=7.206,P=0.000;8th week after modeling:LSD-t=8.485,P=0.000;LSD-t=8.132,P=0.000).The scores were not significantly different from each other between blank group and CEP degeneration model group(4th week after modeling:LSD-t=0.408,P=0.690;6th week after modeling:LSD-t=0.480,P=0.640;8th week after modeling:LSD-t=0.354,P=0.730).Four weeks after modeling,there was statistical difference in the BMD of caudal vertebrae among the three groups(4.15±0.44,4.04±0.38,3.30±0.33 g/cm(3),F=7.719,P=0.007).The BMD of caudal vertebrae in blank group and CEP degeneration model group were both higher than that of the DS combination model group(LSD-t=3.682,P=0.003;LSD-t=3.028,P=0.011).Compared with the CEP degeneration model group,the blank group exhibited no statistically significant difference in the BMD of caudal vertebrae(LSD-t=0.655,P=0.525).Eight weeks after modeling,the difference in BMD of caudal vertebrae among the three groups were statistically significant(4.12±0.42,3.88±0.53,2.86±0.63 g/cm(3),F=7.927,P=0.006).The BMD of caudal vertebrae in blank group and CEP degeneration model group both increased compared with that in DS combination model group(LSD-t=3.749,P=0.003;LSD-t=3.035,P=0.010).Compared with the CEP degeneration model group,the blank group exhibited no statistically significant difference in the BMD of caudal vertebrae(LSD-t=0.714,P=0.489).Four weeks after modeling,the TN and TS showed statistically significant differences among the three groups(TN:5.01±0.12,4.81±0.12,3.86±0.17 trabeculae/mm,F=95.936,P=0.000;TS:0.20±0.03,0.24±0.02,0.30±0.04μm,F=10.807,P=0.002).The TN was higher and the TS was lower in blank group and CEP degeneration model group compared to DS combination model group(LSD-t=12.991,P=0.000;LSD-t=10.657,P=0.000;LSD-t=4.623,P=0.001;LSD-t=2.736,P=0.018).There was no statistical difference in TN and TS between blank group and CEP degeneration model group(TN:LSD-t=0.432,P=0.924;TS:LSD-t=1.887,P=0.084).Eight weeks after modeling,the comparison of the TN and TS among the three groups showed statistically significant differences(TN:4.91±0.26,4.61±0.55,3.05±0.44 trabeculae/mm,F=27.088,P=0.000;TS:0.22±0.04,0.23±0.02,0.29±0.03μm,F=7.679,P=0.002).Compared with the DS combination model group,both blank group and CEP degeneration model group exhibited increased TN(LSD-t=6.854,P=0.000;LSD-t=5.750,P=0.000),but decreased TS(LSD-t=3.623,P=0.003;LSD-t=3.106,P=0.009).There was no statistical difference in TN and TS between blank group and CEP degeneration model group(TN:LSD-t=1.104,P=0.291;TS:LSD-t=0.518,P=0.614).At the 4th and 8th week after modeling,no statistically significant difference was observed in TT among the three groups(4th week after modeling:0.08±0.02,0.09±0.01,0.10±0.01μm,F=1.462,P=0.304;8th week after modeling:0.09±0.01,0.09±0.02,0.08±0.01μm,F=2.400,P=0.171).After modeling,the rats in CEP degeneration model group and DS combination model group exhibited CEP degeneration in caudal vertebrae,manifested as irregularly arranged and clustered chondrocyte,blurred tidal line,uneven staining as well as calcified and thickened cartilage,and the degeneration in DS combination model group was more severe.At the 4th and 8th week after modeling,the mRNA expression level of ACAN presented statistically significant differences among the three groups(4th week after modeling:1.07±0.19,0.74±0.09,0.51±0.07,F=23.498,P=0.000;8th week after modeling:1.03±0.22,0.65±0.08,0.43±0.07,F=23.538,P=0.000).The mRNA expression level of ACAN was higher in blank group compared to CEP degeneration model group and DS combination model group(4th week after modeling:LSD-t=4.045,P=0.002;LSD-t=6.815,P=0.000.8th week after modeling:LSD-t=4.329,P=0.001;LSD-t=6.774,P=0.000),and was higher in CEP degeneration model group compared to DS combination model group(4th week after modeling:LSD-t=2.770,P=0.017;8th week after modeling:LSD-t=2.445,P=0.031).At the 4th and 8th week after modeling,there was statistical difference in the mRNA expression level of ADAMTS-5 among the three groups(4th week after modeling:1.06±0.07,1.30±0.08,1.81±0.15,F=64.344,P=0.000;8th week after modeling:1.13±0.09,1.59±0.13,2.14±0.10,F=110.156,P=0.000).The mRNA expression level of ADAMTS-5 in blank group was down-regulated as compared with that in CEP degeneration model group and DS combination model group(4th week after modeling:LSD-t=3.543,P=0.004;LSD-t=11.104,P=0.000;8th week after modeling:LSD-t=6.702,P=0.000;LSD-t=14.821,P=0.000),and the down-regulation revealed in CEP degeneration model group compared to DS combination model group(4th week after modeling:LSD-t=7.562,P=0.000;8th week after modeling:LSD-t=8.119,P=0.000).Conclusion:The removal of bilateral ovaries combined with the blockage of nutrition supply to CEP is a feasible method for inducing the DS combination model of IVD CEP degeneration with kidney deficiency in rats.