[1]吴捷,张若谷,邱敏,等.骨健口服液联合间充质干细胞来源外泌体治疗小鼠膝骨关节炎的实验研究[J].中医正骨,2024,36(04):28-36.
　WU Jie,ZHANG Ruogu,QIU Min,et al.Gujian(骨健)peroral liquids combined with mesenchymal stem cell-derived exosome against knee osteoarthritis in mice:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(04):28-36.
点击复制

骨健口服液联合间充质干细胞来源外泌体治疗小鼠膝骨关节炎的实验研究()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期数:
2024年04期
页码:
28-36
栏目:
基础研究
出版日期:
2024-04-20

文章信息/Info

Title:
Gujian(骨健)peroral liquids combined with mesenchymal stem cell-derived exosome against knee osteoarthritis in mice:an experimental study
作者:
吴捷1; 张若谷1; 邱敏1; 程良艳1; 徐建波1; 金敏伟2; 金红婷2; 王萍儿2
1.浙江中医药大学第一临床医学院,浙江 杭州 310053; 2.浙江省中医院,浙江 杭州 310006
Author(s):
WU Jie1; ZHANG Ruogu1; QIU Min1; CHENG Liangyan1; XU Jianbo1; JIN Minwei2; JIN Hongting2; WANG Pinger2
1.The First Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 2.Zhejiang Provincial Hospital of Traditional Chinese Medicine,Hangzhou 310006,Zhejiang,China
关键词:
骨关节炎; 膝;  小鼠;  间质干细胞;  外泌体;  骨健口服液;  软骨;  动物实验
Keywords:
osteoarthritis; knee;  mice;  mesenchymal stem cells;  exosomes;  Gujian(骨健)peroral liquids;  cartilage;  animal experimentation
摘要:
目的:探讨骨健口服液联合间充质干细胞来源外泌体(mesenchymal stem cell-derived exosome,MSC-EXO)治疗小鼠膝骨关节炎(knee osteoarthritis,KOA)的效果及作用机制。方法:将25只12周龄雄性无特定病原C57BL/6J小鼠随机分为假手术组、模型组、骨健组、MSC-EXO组、骨健MSC-EXO组,每组5只。假手术组仅切开关节囊而不切断内侧半月板,其余4组均采用内侧半月板失稳法构建KOA模型。骨健组、骨健MSC-EXO组小鼠每日用0.2 mL的骨健口服液灌胃,假手术组、模型组、MSC-EXO组小鼠每日用等量的生理盐水灌胃,均连续8周。MSC-EXO组、骨健MSC-EXO组小鼠每周膝关节腔注射1次10 μL的MSC-EXO,其余各组小鼠每周膝关节腔注射1次等量的磷酸盐缓冲液(phosphate buffer solution,PBS),均连续8周。干预8周后处死小鼠,进行小鼠膝关节Micro-CT分析、膝关节软骨组织病理学观察、膝关节软骨组织中Ⅱ型胶原蛋白α1链(collagenⅡα1,Col2A1)表达水平检测。结果:①小鼠膝关节Micro-CT分析结果。假手术组、骨健组、MSC-EXO组、骨健MSC-EXO组小鼠的骨体积分数均低于模型组(P=0.004,P=0.007,P=0.040,P=0.001),骨健组、MSC-EXO组小鼠的骨体积分数与骨健MSC-EXO组的差异均无统计学意义(P=0.108,P=0.126)。假手术组、骨健组、MSC-EXO组、骨健MSC-EXO组小鼠的骨小梁厚度均大于模型组(P=0.015,P=0.023,P=0.023,P=0.009),骨健组、MSC-EXO组小鼠的骨小梁厚度与骨健MSC-EXO组的差异均无统计学意义(P=0.347,P=0.062)。假手术组和骨健组小鼠的骨小梁数量多于模型组(P=0.016,P=0.021),MSC-EXO组、骨健MSC-EXO组小鼠的骨小梁数量与模型组的差异均无统计学意义(P=0.196,P=0.206)。假手术组、骨健组、MSC-EXO组、骨健MSC-EXO组小鼠的骨小梁分离度均小于模型组(P=0.016,P=0.012,P=0.007,P=0.001),骨健MSC-EXO组小鼠的骨小梁分离度小于骨健组(P=0.016),MSC-EXO组小鼠的骨小梁分离度与骨健MSC-EXO组的差异无统计学意义(P=0.224)。②小鼠膝关节软骨组织病理学观察结果。假手术组小鼠的关节软骨表面光滑,软骨细胞排列有序,提示无明显关节软骨退变。与假手术组相比,模型组小鼠的关节软骨组织磨损明显,软骨细胞数量减少且排列紊乱。与模型组相比,骨健组和MSC-EXO组小鼠仅有部分关节软骨磨损,软骨细胞数量增多且排列有序。与骨健组、MSC-EXO组相比,骨健MSC-EXO组小鼠的关节软骨表面光滑,软骨细胞数量增多且排列更为有序。③小鼠膝关节软骨组织中Col2A1表达水平检测结果。假手术组、骨健组、MSC-EXO组、骨健MSC-EXO组小鼠膝关节软骨组织中Col2A1阳性表达面积比均大于模型组(P=0.000,P=0.000,P=0.000,P=0.000),骨健MSC-EXO组小鼠膝关节软骨组织中Col2A1阳性表达面积比大于骨健组和MSC-EXO组(P=0.000,P=0.000)。结论:骨健口服液联合MSC-EXO可改善KOA小鼠的关节软骨病理状态及软骨下骨的异常微结构,并显示出协同治疗作用,其作用机制可能与上调膝关节软骨组织中Col2A1的表达有关。
Abstract:
Objective:To observe the effects of Gujian(骨健,GJ)peroral liquids combined with mesenchymal stem cell-derived exosome(MSC-EXO)on knee osteoarthritis(KOA)in mice,and to explore its mechanism.Methods:Twenty-five 12-week-old specific pathogen free(SPF)-grade male C57BL/6J mice were randomized into sham-operated group,model group,GJ group,MSC-EXO group,GJ combined MSC-EXO group,5 cases in each group.All mice but the ones in sham-operated group were subjected to destabilized medial meniscus(DMM)method on the right knees for inducing KOA,while the ones in sham-operated group underwent surgery for merely incising the joint capsule,but without transecting medial meniscus.After successful modeling,the mice in GJ group and GJ combined MSC-EXO group were intervened by intragastric administration with 0.2 mL GJ peroral liquids,while the ones in sham-operated group,model group and MSC-EXO group with the same dose of normal saline,once a day for consecutive 8 weeks; moreover,the mice in MSC-EXO group and GJ combined MSC-EXO group were further intervened by knee intra-articular injection of 10 μL MSC-EXO,while the ones in sham-operated group,model group and GJ group by the same dose of phosphate buffer solution(PBS),once a week for consecutive 8 weeks.After the end of intervention,the mice were sacrificed by cervical dislocation method,and their right knees were harvested for scanning and analysing by using Micro-CT,followed by observation on histopathological changes of knee tissues and determination of the expression level of collagenⅡα1(Col2A1)in knee cartilage tissues.Results:①The bone volume fraction was lower in sham-operated group,GJ group,MSC-EXO group and GJ combined MSC-EXO group compared to model group(P=0.004,P=0.007,P=0.040,P=0.001),while,there was no statistical difference between GJ group and GJ combined MSC-EXO group as well as between MSC-EXO group and GJ combined MSC-EXO group(P=0.108,P=0.126).The trabecular thickness was greater in sham-operated group,GJ group,MSC-EXO group and GJ combined MSC-EXO group compared to model group(P=0.015,P=0.023,P=0.023,P=0.009),while,there was no statistical difference between GJ group and GJ combined MSC-EXO group as well as between MSC-EXO group and GJ combined MSC-EXO group(P=0.347,P=0.062).The trabecular number was more in sham-operated group and GJ group compared to model group(P=0.016,P=0.021),while,there was no statistical difference between MSC-EXO group and model group as well as between GJ combined MSC-EXO group and model group(P=0.196,P=0.206).The trabecular separation was lower in sham-operated group,GJ group,MSC-EXO group and GJ combined MSC-EXO group compared to model group(P=0.016,P=0.012,P=0.007,P=0.001),and was lower in GJ combined MSC-EXO group compared to GJ group(P=0.016),while,there was no statistical difference between MSC-EXO group and GJ combined MSC-EXO group(P=0.224).②The mice in sham-operated group presented with smooth articular cartilage surface and neatly arranged chondrocytes,which suggesting no obvious degeneration in articular cartilage; compared with that of sham-operated group,the changes,manifesting as obviously worn articular cartilage tissues,decreased and disordered arranged chondrocytes,were observed in mice of model group; compared with that of model grouop,the mice in GJ group and MSC-EXO group presented with partial worn articular cartilage with increased and neatly arranged chondrocytes; the improvement was better in GJ combined MSC-EXO group in contrast to GJ group and MSC-EXO group.③The positive expression area ratio of Col2A1 in knee cartilage tissues was greater in mice of sham-operated group,GJ group,MSC-EXO group and GJ combined MSC-EXO group compared to model group(P=0.000,P=0.000,P=0.000,P=0.000),and was greater in GJ combined MSC-EXO group compared to GJ group and MSC-EXO group(P=0.000,P=0.000).Conclusion:GJ peroral liquids combined with MSC-EXO can improve the pathological state of articular cartilage and the abnormal microstructure of subchondral bone in KOA mice,and it behaves a synergistic therapeutic action.It may exert the effects by up-regulating the expression of Col2A1 in knee cartilage tissues.

参考文献/References:

[1] VAN DEN BOSCH M H J.Osteoarthritis year in review 2020:biology[J].Osteoarthritis Cartilage,2021,29(2):143-150.
[2] MURATOVIC D,FINDLAY D M,CICUTTINI F M,et al.Bone marrow lesions in knee osteoarthritis:regional differences in tibial subchondral bone microstructure and their association with cartilage degeneration[J].Osteoarthritis Cartilage,2019,27(11):1653-1662.
[3] KUYINU E L,NARAYANAN G,NAIR L S,et al.Animal models of osteoarthritis:classification,update,and measurement of outcomes[J].J Orthop Surg Res,2016,11:19.
[4] STODDART J C,DANDRIDGE O,GARNER A,et al.The compartmental distribution of knee osteoarthritis-a systematic review and meta-analysis[J].Osteoarthritis Cartilage,2021,29(4):445-455.
[5] REICHENBACH S,FELSON D T,HINCAPIÉ C A,et al.Effect of biomechanical footwear on knee pain in people with knee osteoarthritis:the BIOTOK randomized clinical trial[J].JAMA,2020,323(18):1802-1812.
[6] SHAO H,IM H,CASTRO C M,et al.New technologies for analysis of extracellular vesicles[J].Chem Rev,2018,118(4):1917-1950.
[7] 伏玉龙,郭珈宜,李峰,等.间充质干细胞来源外泌体修复膝骨关节炎软骨损伤作用机制的研究进展[J].中医正骨,2023,35(11):76-79.
[8] VONK L A,VAN DOOREMALEN S F J,LIV N,et al.Mesenchymal stromal/stem cell-derived extracellular vesicles promote human cartilage regeneration in vitro[J].Theranostics,2018,8(4):906-920.
[9] YAN L,LIU G,WU X.Exosomes derived from umbilical cord mesenchymal stem cells in mechanical environment show improved osteochondral activity via upregulation of LncRNA H19[J].J Orthop Translat,2020,26:111-120.
[10] 胡雪琴,金红婷,施振宇,等.髓病理论及其在骨病学中的应用[J].中医正骨,2018,30(11):39-42.
[11] 胡雪琴,金红婷,肖鲁伟,等.“浙派中医”骨伤学家肖鲁伟辨治“髓系骨病”的学术经验[J].浙江中医药大学学报,2019,43(10):1071-1073.
[12] 汪小健,王瑞,顾勇,等.骨健口服液治疗原发性骨质疏松症的临床疗效分析[J].时珍国医国药,2022,33(10):2424-2427.
[13] WU C,GE Q,SHI Z,et al.Elucidation of the underlying mechanism of Gujian oral liquid acting on osteoarthritis through network pharmacology,molecular docking,and experiment[J/OL].Biomed Res Int,2022,2022:9230784[2023-11-02].https://pubmed.ncbi.nlm.nih.gov/35937393/.
[14] HUNTER D J,BIERMA-ZEINSTRA S.Osteoarthritis[J].Lancet,2019,393(10182):1745-1759.
[15] SPROUSE R A,HARRIS G D,SPROUSE G D E.A practical approach to knee OA[J].J Fam Pract,2020,69(7):327-334.
[16] SCHNITZER T J,EASTON R,PANG S,et al.Effect of tanezumab on joint pain,physical function,and patient global assessment of osteoarthritis among patients with osteoarthritis of the hip or knee:a randomized clinical trial[J].JAMA,2019,322(1):37-48.
[17] UTAMAWATIN K,PHRUETTHIPHAT O A,APINYANKUL R,et al.The efficacy of intra-articular triamcinolone acetonide 10 mg vs.40 mg in patients with knee osteoarthritis:a non-inferiority,randomized,controlled,double-blind,multicenter study[J].BMC Musculoskelet Disord,2023,24(1):92.
[18] 陈军,凌仕勇,吴卓一,等.膝关节镜手术术后静脉血栓形成危险因素分析[J].血栓与止血学,2021,27(1):130-132.
[19] 胡琪,袁建涛,宋薇,等.行全膝关节置换术患者术后发生下肢深静脉血栓危险因素的研究进展[J].中外医学研究,2021,19(31):193-196.
[20] 王刘玉,万全会,陈军.黄芪多糖对MC-3T3-E1成骨细胞增殖的影响及作用机制研究[J].中医正骨,2023,35(8):1-7.
[21] 张睿,倪敏,周春根,等.从中医“精气转化”探讨黄芪对间充质干细胞增殖及分化的影响[J].中华中医药学刊,2020,38(10):146-149.
[22] SONG J,ZHANG Y,ZHU Y,et al.Structural characterization and anti-osteoporosis effects of polysaccharide purified from Eucommia ulmoides Oliver cortex based on its modulation on bone metabolism[J/OL].Carbohydr Polym,2023,306:120601[2023-11-02].https://pubmed.ncbi.nlm.nih.gov/36746570/.
[23] 黄进,张进,徐志伟.菟丝子含药血清促进骨髓间充质干细胞增殖的效应及机制[J].中华中医药杂志,2011,26(4):818-822.
[24] QI S,ZHENG H,CHEN C,et al.Du-zhong(eucommia ulmoides oliv.)cortex extract alleviates lead acetate-induced bone loss in rats[J].Biol Trace Elem Res,2019,187(1):172-180.
[25] ALTANEROVA U,JAKUBECHOVA J,REPISKA V,et al.Exosomes of human mesenchymal stem/stromal/medicinal signaling cells[J].Neoplasma,2017,64(6):809-815.
[26] REZNIKOFF C A,BRANKOW D W,HEIDELBERGER C.Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division[J].Cancer Res,1973,33(12):3231-3238.
[27] MUKAI T,KUSUDO T.Bidirectional effect of vitamin D on brown adipogenesis of C3H10T1/2 fibroblast-like cells[J/OL].Peer J,2023,11:e14785[2023-11-02].https://pubmed.ncbi.nlm.nih.gov/36815991/.
[28] MENG X,CUI J,WANG Y,et al.Heterogeneous nuclear ribonucleoprotein A1 interacts with microRNA-34a to promote chondrogenic differentiation of mesenchymal stem cells[J].Am J Transl Res,2017,9(4):1774-1782.
[29] ALONSO-PÉREZ A,GUILLÁN-FRESCO M,FRANCO-TREPAT E,et al.Improved protocol to study osteoblast and adipocyte differentiation balance[J].Biomedicines,2022,11(1):31.
[30] TANG Q Q,OTTO T C,LANE M D.Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte line-age[J].Proc Natl Acad Sci U S A,2004,101(26):9607-9611.
[31] 梁彦.间充质干细胞来源的外泌体对人滋养细胞系HTR8/Svneo迁移的影响的相关研究[D].广州:南方医科大学,2019.

相似文献/References:

[1]樊庆阳,任凯晶.定制3D打印切模辅助全膝关节置换术治疗 膝骨关节炎合并股骨干骨折畸形愈合[J].中医正骨,2015,27(11):37.
[2]刘晓雅,孙永强,刘国杰.主动快速康复锻炼对全膝关节置换术后关节活动度的影响[J].中医正骨,2015,27(09):73.
[3]郑春松,叶蕻芝,李西海,等.透骨消痛胶囊中补肾柔肝药和活血祛风药治疗 骨关节炎作用方式的计算机模拟比较[J].中医正骨,2015,27(07):6.
　ZHENG Chunsong,YE Hongzhi,LI Xihai,et al.Comparison of the mode of action of Bushen Rougan(补肾柔肝)drugs versus Huoxue Qufeng(活血祛风)drugs contained in Tougu Xiaotong Jiaonang(透骨消痛胶囊)for the treatment of osteoarthritis:A computer simulation study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(04):6.
[4]宋兵华,孙俊英,倪增良,等.全膝关节置换术前CT测量股骨后髁角的临床意义[J].中医正骨,2015,27(07):38.
[5]郑春松,叶蕻芝,李西海,等.独活寄生汤含药血清对白细胞介素1β诱导的 退变关节软骨细胞中基质金属蛋白酶 和环氧化酶2表达的影响[J].中医正骨,2015,27(12):1.
　ZHENG Chunsong,YE Hongzhi,LI Xihai,et al.Impact of Duhuo Jisheng Tang(独活寄生汤)medicated serum on expression of matrix metalloproteinase and cyclooxygenase 2 in degenerative articular chondrocytes induced by interleukin-1 beta[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(04):1.
[6]王金良,孙京涛,李玲,等.骨水泥联合螺钉修复全膝关节置换术中 胫骨平台内侧骨缺损[J].中医正骨,2015,27(12):55.
[7]冯荣,王平,李炳奇,等.铍针刺络拔罐结合中药口服治疗膝骨关节炎合并 原发性血小板增多症1例[J].中医正骨,2015,27(12):73.
[8]蔡云仙.围手术期耳穴按压联合平衡针疗法 在全膝关节置换术后镇痛中的应用[J].中医正骨,2015,27(06):41.
[9]张荣,王健.人工全膝关节置换术的围手术期心理护理[J].中医正骨,2015,27(05):77.
[10]喻长纯,杨明路,王战朝.不同手术方式治疗胫骨平台骨折畸形愈合的体会[J].中医正骨,2015,27(03):37.
[11]孟维娜,明立功,王新德,等.关节镜下清理联合腓骨近1/3段截骨治疗膝骨关节炎[J].中医正骨,2015,27(11):40.
[12]明立功,孟维娜,王新德,等.腓骨近端截骨治疗内侧间室膝骨关节炎的近期疗效观察[J].中医正骨,2015,27(10):25.
[13]张杰,王人彦,张玉柱.膝骨关节炎的治疗进展[J].中医正骨,2015,27(10):68.
[14]梁朝,蔡静怡,闫立,等.针刀疗法改善膝骨关节炎早期疼痛症状的疗效评价[J].中医正骨,2015,27(09):9.
　LIANG Zhao,CAI Jingyi,YAN Li,et al.Evaluation of the curative effect of needle-knife therapy for relieving knee pain in patients with early knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(04):9.
[15]王建武,党建军,李强,等.四联疗法治疗膝骨关节炎[J].中医正骨,2015,27(08):44.
[16]刘红娟,郭会利,郭树农.云克联合中药治疗膝骨关节炎的护理[J].中医正骨,2015,27(08):75.
[17]陈卫衡.探索建立系统的膝骨关节炎中医临床科研范式 和理论体系[J].中医正骨,2015,27(07):1.
[18]帅波,沈霖,杨艳萍,等.加味青娥丸治疗膝骨关节炎的作用机制研究[J].中医正骨,2015,27(07):15.
　SHUAI Bo,SHEN Lin,YANG Yanping,et al.Study on the mechanism of action of Jiawei Qing'e Wan(加味青娥丸)for the treatment of knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(04):15.
[19]梅其杰,袁长深,段戡,等.壮药骨痹方烫熨联合运动疗法治疗膝骨关节炎的临床研究[J].中医正骨,2015,27(07):27.
　MEI Qijie,YUAN Changshen,DUAN Kan,et al.Clinical study of the curative effect of hot compressing and rubbing with packet of Gubi Fang(骨痹方)combined with exercise therapy in the treatment of knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(04):27.
[20]王丹辉,张燕,刘丽娟,等.重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白 关节腔注射联合中药薰洗治疗膝骨关节炎的临床研究[J].中医正骨,2015,27(07):31.
　WANG Danhui,ZHANG Yan,LIU Lijuan,et al.Clinical study on intra-articular injection of TypeⅡrecombinant human tumor necrosis factor receptor-Fc fusion protein combined with Chinese herbal steaming and washing therapy for treatment of knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(04):31.

备注/Memo

备注/Memo:
基金项目:浙江省中医药科技计划项目(2022ZB116) 通讯作者:王萍儿 E-mail:pingerwang@zcmu.edu.cn
更新日期/Last Update: 1900-01-01