补肾活血方联合人脐带血间充质干细胞修复小鼠膝关节软骨缺损的实验研究  

Bushen Huoxue Fang(补肾活血方)combined with human umbilical cord blood-derived mesenchymal stem cells against knee articular cartilage defects in mice:an experimental study

作　　者：岑芊瑶 刘江源 曾庆贺 温经渊 吴丛姿 王旭 吴震[3] 金红婷[2] 陈佳丽 CEN Qianyao;LIU Jiangyuan;ZENG Qinghe;WEN Jingyuan;WU Congzi;WANG Xu;WU Zhen;JIN Hongting;CHEN Jiali(The Second Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China;Institute of Traumatology and Orthopedics of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China;Tongde Hospital of Zhejiang Province,Hangzhou 310012,Zhejiang,China)

机构地区：[1]浙江中医药大学第二临床医学院,浙江杭州310053 [2]浙江中医药大学骨伤研究所,浙江杭州310053 [3]浙江省立同德医院,浙江杭州310012

出　　处：《中医正骨》2023年第12期5-13,24,共10页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：浙江省中医药科技计划项目(2023ZL037);浙江省基础公益研究计划项目(LGF20H270005)。

摘　　要：目的:探讨补肾活血方联合人脐带血间充质干细胞(human umbilical cord blood-derived mesenchymal stem cells,hUCB-MSCs)修复小鼠膝关节软骨缺损的效果及作用机制。方法:将24只10周龄雌性C57BL/6J小鼠随机分为对照组、模型组、hUCB-MSCs组和补肾活血方联合hUCB-MSCs组,每组6只。除对照组外,其余3组均用针头在小鼠股骨髁滑车关节面上造一个直径0.45 mm、深1 mm的圆柱形缺损。分别于造模后第1周、第2周、第3周,向hUCB-MSCs组、补肾活血方联合hUCB-MSCs组小鼠膝关节腔内注射10μL的hUCB-MSCs悬液(200个细胞·μL^(-1)),向对照组和模型组小鼠膝关节腔内注射10μL的生理盐水;各组小鼠均每周注射1次。造模后第2天,补肾活血方联合hUCB-MSCs组小鼠给予补肾活血方浓缩液(20μL·g-1)灌胃,对照组、模型组和hUCB-MSCs组小鼠每天给予等量生理盐水灌胃;各组小鼠均每天灌胃1次,共4周。灌胃结束后第2天,脱颈处死各组小鼠,切取小鼠右侧膝关节,以Micro-CT观察小鼠膝关节软骨缺损区骨微结构;以阿尔新蓝-苏木素染色观察膝关节软骨缺损区软骨退变情况,并采用国际骨关节炎研究学会(Osteoarthritis Research Society International,OARSI)评分对关节软骨退变情况进行评估;采用免疫组织化学染色测定小鼠膝关节软骨缺损区软骨中Ⅱ型胶原蛋白α1链(collagen typeⅡalpha 1 chain,Col2a1)和基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)的表达量。结果:①小鼠膝关节软骨缺损区骨微结构观察结果。对照组、hUCB-MSCs组和补肾活血方联合hUCB-MSCs组小鼠膝关节软骨缺损区骨松质的骨密度均高于模型组(LSD-t=18.425,P=0.000;LSD-t=-10.186,P=0.000;LSD-t=-7.487,P=0.000),骨体积分数均高于模型组(LSD-t=7.242,P=0.002;LSD-t=-5.243,P=0.006;LSD-t=-5.441,P=0.006),骨小梁厚度均大于模型组(LSD-t=7.575,P=0.002;LSD-t=-10.005,P=0.002;LSD-t=-5.409,P=0.006),骨小梁数量均多于模型组(LSD-t=9.166,P=0.000;LSD-tObjective:To explore the effects and mechanism of Bushen Huoxue Fang(补肾活血方,BSHXF)combined with human umbilical cord blood-derived mesenchymal stem cells(hUCB-MSCs)in repairing knee articular cartilage defects in mice.Methods:Twenty-four 10-week-old female C57BL/6J mice were selected and randomized into control group,model group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group,6 ones in each group.A cylindrical defect(0.45 mm in diameter and 1 mm in depth)was created on the articular surface of trochlea of femoral condyle with a needle in mice except for the ones in control group.At week 1,2 and 3 after the mode-ling,the mice in hUCB-MSCs group and BSHXF combined with hUCB-MSCs group were intervened by knee intra-articular injection of hUCB-MSCs suspension(10μL,200 cells/μL),while the ones in control group and model group with the same dose of normal saline.All mice were intervened once a week.On day 2 after the modeling,the mice in BSHXF combined with hUCB-MSCs group were intragastric administrated with BSHXF concentrate in dosage of 20μL/g,while the ones in control group,model group,and hUCB-MSCs group with the same dose of normal saline.All mice were intervened once a day for consecutive 4 weeks.On day 2 after the end of intragastric administration,all mice were sacrificed by cervical dislocation and their right knees were harvested.The bone microstructure and cartilage degeneration in the knee articular cartilage defect zone were observed by using Micro-CT and alcian blue-hematoxylin(ABH)staining,respectively,and the knee articular cartilage degeneration was evaluated by using Osteoarthritis Research Society International(OARSI)score.Furthermore,the expression levels of collagen typeⅡalpha 1 chain(Col2a1)and matrix metalloproteinase 13(MMP13)in the cartilage of knee articular cartilage defect zone were detected by using immunohistochemical staining.Results:①In the knee articular cartilage defect zone,the control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group exhibited hig

关 键 词：膝关节 软骨 关节 小鼠 软骨缺损 间质干细胞 脐带血 补肾活血方 骨微结构 动物实验 

分 类 号：R684[医药卫生—骨科学]