[1]岑芊瑶,刘江源,曾庆贺,等.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[J].中医正骨,2023,35(12):5-13，24.
　CEN Qianyao,LIU Jiangyuan,ZENG Qinghe,et al.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[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(12):5-13，24.
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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()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期数:
2023年12期
页码:
5-13，24
栏目:
基础研究
出版日期:
2023-12-20

文章信息/Info

Title:
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1; 刘江源2; 曾庆贺2; 温经渊2; 吴丛姿2; 王旭2; 吴震3; 金红婷2; 陈佳丽2
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
Author(s):
CEN Qianyao1; LIU Jiangyuan2; ZENG Qinghe2; WEN Jingyuan2; WU Congzi2; WANG Xu2; WU Zhen3; JIN Hongting2; CHEN Jiali2
1.The Second Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 2.Institute of Traumatology and Orthopedics of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 3.Tongde Hospital of Zhejiang Province,Hangzhou 310012,Zhejiang,China
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
Keywords:
knee joint;  cartilage; articular;  mice;  cartilage defects;  mesenchymal stem cells;  umbilical cord blood;  Bushen Huoxue Fang;  bone microstructure;  animal experimentation
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10 μL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10 μL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(20 μL·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-t=-10.014,P=0.000; LSD-t=-9.147,P=0.000),骨小梁分离度均小于模型组(LSD-t=-9.120,P=0.000; LSD-t=10.375,P=0.000; LSD-t=7.650,P=0.002); hUCB-MSCs组骨松质骨小梁数量少于对照组(LSD-t=3.027,P=0.039); 其余各组之间两两比较,差异均无统计学意义。②小鼠膝关节软骨缺损区软骨退变情况观察结果。与对照组相比,模型组小鼠关节软骨退变明显; 与模型组相比,hUCB-MSCs组和补肾活血方联合hUCB-MSCs组小鼠关节软骨退变较轻。对照组、hUCB-MSCs组和补肾活血方联合hUCB-MSCs组小鼠OARSI评分均低于模型组(LSD-t=-11.762,P=0.000; LSD-t=10.947,P=0.000; LSD-t=14.779,P=0.000),补肾活血方联合hUCB-MSCs组和对照组小鼠OARSI评分均低于hUCB-MSCs组(LSD-t=-5.635,P=0.005; LSD-t=-3.443,P=0.026),对照组小鼠OARSI评分低于补肾活血方联合hUCB-MSCs组(LSD-t=-5.914,P=0.004)。③小鼠膝关节软骨缺损区软骨Col2a1和MMP13表达量测定结果。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软骨Col2a1阳性表达面积均大于模型组(LSD-t=9.863,P=0.000; LSD-t=45.990,P=0.000; LSD-t=-17.406,P=0.000),补肾活血方联合hUCB-MSCs组小鼠膝关节软骨缺损区软骨Col2a1阳性表达面积大于hUCB-MSCs组(LSD-t=3.623,P=0.022),补肾活血方联合hUCB-MSCs组和hUCB-MSCs组小鼠膝关节软骨缺损区软骨Col2a1阳性表达面积与对照组的差异均无统计学意义(LSD-t=-1.643,P=0.176; LSD-t=0.533,P=0.623)。对照组、hUCB-MSCs组和补肾活血方联合hUCB-MSCs组MMP13阳性细胞数占比均低于模型组(LSD-t=-14.299,P=0.001; LSD-t=9.688,P=0.001; LSD-t=15.638,P=0.000),补肾活血方联合hUCB-MSCs组MMP13阳性细胞数占比低于hUCB-MSCs组和对照组(LSD-t=-11.488,P=0.007; LSD-t=3.578,P=0.023),hUCB-MSCs组MMP13阳性细胞数占比高于对照组(LSD-t=-9.425,P=0.000)。结论:补肾活血方联合hUCB-MSCs能明显改善小鼠膝关节软骨缺损区骨微结构,修复软骨缺损,其作用机制可能与上调Col2a1的表达和抑制MMP13的表达有关。
Abstract:
Objective: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(10 μL,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 20 μL/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 higher bone mineral density and bone volume fraction,thicker trabeculae,more trabeculae,and lower trabecular separation in cancellous bone compared with that of model group(LSD-t=18.425,P=0.000; LSD-t=-10.186,P=0.000; LSD-t=-7.487,P=0.000; LSD-t=7.242,P=0.002; LSD-t=-5.243,P=0.006; LSD-t=-5.441,P=0.006; LSD-t=7.575,P=0.002; LSD-t=-10.005,P=0.002; LSD-t=-5.409,P=0.006; LSD-t=9.166,P=0.000; LSD-t=-10.014,P=0.000; LSD-t=-9.147,P=0.000; LSD-t=-9.120,P=0.000; LSD-t=10.375,P=0.000; LSD-t=7.650,P=0.002).The hUCB-MSCs group displayed less trabeculae compared to control group(LSD-t=3.027,P=0.039).Further pairwise comparison among other groups showed no statistical significance.②Compared to control group,the knee articular cartilage degenerated more obviously in mice of model group; while,compared to model group,the mice in hUCB-MSCs group and BSHXF combined with hUCB-MSCs group exhibited slight knee articular cartilage degeneration.The OARSI score was lower in control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group compared to model group(LSD-t=-11.762,P=0.000; LSD-t=10.947,P=0.000; LSD-t=14.779,P=0.000),and was lower in BSHXF combined with hUCB-MSCs group and control group compared to hUCB-MSCs group(LSD-t=-5.635,P=0.005; LSD-t=-3.443,P=0.026),and was lowest in control group(LSD-t=-5.914,P=0.004).③The positive expression area of Col2a1 in cartilage of knee articular cartilage defect zone was larger in mice of control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group compared to model group(LSD-t=9.863,P=0.000; LSD-t=45.990,P=0.000; LSD-t=-17.406,P=0.000),and was larger in mice of BSHXF combined with hUCB-MSCs group compared to hUCB-MSCs group(LSD-t=3.623,P=0.022); while,the BSHXF combined with hUCB-MSCs group and hUCB-MSCs group didn't differ from control group in the positive expression area of Col2a1 in cartilage of knee articular cartilage defect zone in mice(LSD-t=-1.643,P=0.176; LSD-t=0.533,P=0.623).The proportion of MMP13-positive cells was lower in control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group compared to model group(LSD-t=-14.299,P=0.001; LSD-t=9.688,P=0.001; LSD-t=15.638,P=0.000),and was lower in BSHXF combined with hUCB-MSCs group and control group compared to hUCB-MSCs group,and was lowest in BSHXF combined with hUCB-MSCs group(LSD-t=-11.488,P=0.007; LSD-t=3.578,P=0.023; LSD-t=-9.425,P=0.000).Conclusion:BSHXF combined with hUCB-MSCs can significantly improve the bone microstructure,and repair the cartilage defects in knee articular cartilage defect zone of mice.It may exert the effects by up-regulating the expression of Col2a1 and inhibiting the expression of MMP13.

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备注/Memo

备注/Memo:
基金项目: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)
通讯作者:陈佳丽 E-mail:chenjiali1585@163.com
更新日期/Last Update: 1900-01-01