[1]蔡宏杰,王旭,徐建波,等.小鼠膝骨关节炎阳虚血瘀证模型的构建与评价[J].中医正骨,2023,35(06):18-23.
　CAI Hongjie,WANG Xu,XU Jianbo,et al.Construction and evaluation of a mouse model of knee osteoarthritis with yang-deficiency and blood-stasis syndrome[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(06):18-23.
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小鼠膝骨关节炎阳虚血瘀证模型的构建与评价()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期数:
2023年06期
页码:
18-23
栏目:
基础研究
出版日期:
2023-06-20

文章信息/Info

Title:
Construction and evaluation of a mouse model of knee osteoarthritis with yang-deficiency and blood-stasis syndrome
作者:
蔡宏杰1; 王旭2; 徐建波1; 林士能2; 童培建1; 金红婷1; 陈佳丽1
(1.浙江省中医院,浙江 杭州 310006; 2.浙江中医药大学第一临床医学院,浙江 杭州 310053)
Author(s):
CAI Hongjie1; WANG Xu2; XU Jianbo1; LIN Shineng2; TONG Peijian1; JIN Hongting1; CHEN Jiali1
1.Zhejiang Provincial Hospital of Traditional Chinese Medicine,Hangzhou 310006,Zhejiang,China 2.The First Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China
关键词:
骨关节炎; 膝;  小鼠;  疾病模型; 动物;  阳虚血瘀;  动物实验
Keywords:
osteoarthritis; knee;  mice;  disease models; animal;  syndrome of yang deficiency and blood stasis;  animal experimentation
摘要:
目的:探讨构建小鼠膝骨关节炎(knee osteoarthritis,KOA)阳虚血瘀证模型的有效方法。方法:将12只10周龄雄性无特定病原C57BL/6J小鼠采用内侧半月板失稳(destabilized medial meniscus,DMM)法构建KOA模型。KOA造模成功后,将12只KOA模型小鼠随机分为KOA阳虚血瘀证组和KOA对照组,每组6只。KOA阳虚血瘀证组小鼠于浸在冰水混合物中的塑料鼠箱中饲养,并以冰水混合物为饮用水; KOA对照组小鼠于22 ℃干燥环境中正常饲养。饲养10周后,评价小鼠阳虚血瘀证表现,分析小鼠步态,处死小鼠后进行小鼠膝关节Micro-CT分析、膝关节组织病理学观察及膝关节软骨Ⅱ型胶原蛋白α1链表达检测。结果:①小鼠阳虚血瘀证评价结果。KOA阳虚血瘀证组小鼠的阳虚证评分和血瘀证评分均高于KOA对照组[(13.33±1.37)分,(3.50±1.23)分,t=13.120,P=0.000;(4.83±0.75)分,(0.83±0.75)分,t=9.200,P=0.000]。②小鼠步态分析结果。2组各收集到5只小鼠完整的步态分析数据。KOA阳虚血瘀证组小鼠右后肢的爪面积和步幅均小于KOA对照组[(0.610±0.094)cm2,(0.758±0.042)cm2,t=-3.211,P=0.012;(1.780±0.045)cm,(2.040±0.152)cm,t=-3.677,P=0.006]; 2组小鼠右后肢的摆动期时间和站立期时间比较,组间差异均无统计学意义[(0.060±0.004)s,(0.060±0.009)s,t=-0.096,P=0.963;(0.062±0.009)s,(0.076±0.014)s,t=-1.810,P=0.137]。③小鼠膝关节Micro-CT分析结果。KOA阳虚血瘀证组小鼠的骨体积分数低于KOA对照组[(55.582±2.810)%,(69.248±1.884)%,t=-6.997,P=0.002],骨小梁分离度大于KOA对照组[(0.114±0.009)mm,(0.077±0.010)mm,t=4.883,P=0.008]; 2组小鼠的骨小梁厚度、骨小梁数量比较,组间差异均无统计学意义[(0.148±0.005)mm,(0.210±0.036)mm,t=-2.939,P=0.094;(3.767±0.100)个·mm-1,(3.368±0.547)个·mm-1,t=1.243,P=0.282]。④小鼠膝关节组织病理学观察结果。2组小鼠膝关节软骨均有缺失,且KOA阳虚血瘀证组小鼠膝关节软骨缺失较KOA对照组更严重。⑤小鼠膝关节软骨Ⅱ型胶原蛋白α1链表达检测结果。KOA阳虚血瘀证组小鼠膝关节软骨Ⅱ型胶原蛋白α1链阳性表达面积比和平均光密度均低于KOA对照组[(0.144±0.003)%,(0.163±0.003)%,t=-6.580,P=0.003;(0.051±0.002)lux,(0.095±0.004)lux,t=-15.878,P=0.001]。结论:DMM法联合低温诱导法是构建小鼠KOA阳虚血瘀证模型的有效方法。
Abstract:
Objective:To explore the effective method for building a yang-deficiency-blood-stasis-type knee osteoarthritis(KOA)mouse model.Methods:Twelve 10-week-old specific pathogen free(SPF)-grade male C57BL/6J mice were selected out and were intervened by destabilized medial meniscus(DMM)method for inducing KOA.After successful modeling,the 12 KOA model mice were randomly divided into yang-deficiency-blood-stasis-type KOA group and KOA control group,6 cases in each group.The mice in yang-deficiency-blood-stasis-type KOA group were bred in plastic box submerged in ice-water mixture,and they were fed with ice-water mixture,while the ones in KOA control group were bred normally in a dry environment of 22 ℃.After 10-week feeding,the yang-deficiency and blood-stasis syndrome in KOA model mice were observed and evaluated,and the gaits were analyzed.After that,the mice were executed by using cervical dislocation method,and the knee samples were harvested from their right hind limbs.The knee samples were scanned and analyzed by using micro-CT and CTAn1.10 software respectively,followed by observation on histopathological changes of knee tissues and determination of the expression levels of typeⅡcollagen α1 chain(Col2A1)in knee cartilage.Results:①The scores of yang-deficiency syndrome and blood-stasis syndrome were higher in yang-deficiency-blood-stasis-type KOA group compared to KOA control group(13.33±1.37 vs 3.50±1.23 points,t=13.120,P=0.000; 4.83±0.75 vs 0.83±0.75 points,t=9.200,P=0.000).②The complete gait analysis data was collected from 5 mice in each group.The paw area and stride length of the right hind limbs were smaller in yang-deficiency-blood-stasis-type KOA group compared to KOA control group(0.610±0.094 vs 0.758±0.042 cm(2),t=-3.211,P=0.012; 1.780±0.045 vs 2.040±0.152 cm,t=-3.677,P=0.006).There was no statistical difference in the time of swing phase and stance phase of the right hind limbs between the 2 groups(0.060±0.004 vs 0.060±0.009 seconds,t=-0.096,P=0.963; 0.062±0.009 vs 0.076±0.014 seconds,t=-1.810,P=0.137).③The bone volume fraction was lower and the trabecular separation was greater in yang-deficiency-blood-stasis-type KOA group compared to KOA control group(55.582±2.810 vs 69.248±1.884%,t=-6.997,P=0.002; 0.114±0.009 vs 0.077±0.010 mm,t=4.883,P=0.008); while there was no statistical difference in trabecular thickness and trabecular number between the 2 groups(0.148±0.005 vs 0.210±0.036 mm,t=-2.939,P=0.094; 3.767±0.100 vs 3.368±0.547 trabeculae/mm,t=1.243,P=0.282).④The knee cartilage loss was observed in the model mice of the 2 groups,and it was more severe in yang-deficiency-blood-stasis-type KOA group compared to KOA control group.⑤The positive expression area ratio and average optical density of Col2A1 in knee cartilage were lower in yang-deficiency-blood-stasis-type KOA group compared to KOA control group(0.144±0.003 vs 0.163±0.003%,t=-6.580,P=0.003; 0.051±0.002 vs 0.095±0.004 lux,t=-15.878,P=0.001).Conclusion:Combination of DMM with low temperature induction is an effective method for constructing a mouse model of KOA and yang-deficiency and blood-stasis syndrome.

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中医正骨2023年6月第35卷第6期 J Trad Chin Orthop Trauma,2023,Vol.35,No.6(总423)
(总424)中医正骨2023年6月第35卷第6期 J Trad Chin Orthop Trauma,2023,Vol.35,No.6
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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(81873324); 浙江中医药大学基本科研能力提升项目(2021JKJNTZ014A) 通讯作者:陈佳丽 E-mail:chenjiali1585@163.com
更新日期/Last Update: 1900-01-01