[1]吴刚,童培建.补肾活血汤含药血清干预体外培养大鼠骨髓间充质干细胞成软骨分化及补肾活血汤联合骨髓间充质干细胞治疗大鼠膝骨关节炎的实验研究[J].中医正骨,2018,30(01):6-11.
　WU Gang,TONG Peijian.Impact of Bushen Huoxue Tang(补肾活血汤)medicated serum on chondrogenic differentiation?of rat’s bone marrow derived mesenchymal stem cells cultured in vitro[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(01):6-11.
点击复制

补肾活血汤含药血清干预体外培养大鼠骨髓间充质干细胞成软骨分化及补肾活血汤联合骨髓间充质干细胞治疗大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30卷
期数:
2018年01期
页码:
6-11
栏目:
基础研究
出版日期:
2018-01-20

文章信息/Info

Title:
Impact of Bushen Huoxue Tang(补肾活血汤)medicated serum on chondrogenic differentiation?of rat’s bone marrow derived mesenchymal stem cells cultured in vitro
作者:
吴刚1; 童培建2
1.浙江中医药大学,浙江 杭州 310053; 2.浙江省中医院,浙江 杭州 310006
Author(s):
WU Gang1; TONG Peijian2
1.Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China2.Zhejiang Provincial Hospital of Traditional Chinese Medicine,Hangzhou 310006,Zhejiang,China
关键词:
骨关节炎;  骨髓;  间质干细胞;  软骨; 关节;  补肾活血汤;  大鼠;  动物实验
Keywords:
Keywords osteoarthritis;  bone marrow;  mesenchymal stem cells;  cartilage; articular;  Bushen Huoxue Tang;  rats;  animal experimentation
摘要:
目的:观察补肾活血汤含药血清对大鼠骨髓间充质干细胞(bone marrow stem cell,BMSC)成软骨分化的作用,及补肾活血汤联合BMSC治疗大鼠膝骨关节炎(knee osteoarthritis,KOA)的作用。方法:从4周龄SD大鼠股骨中分离培养BMSC,以8周龄SD大鼠制备补肾活血汤含药血清和空白血清。选择体外培养的第3代BMSC,分为3组,分别加入含10%空白血清的DMEM培养液(空白组)、含10%空白血清的DMEM培养液及基础诱导液(诱导组)、含10%补肾活血汤含药血清的DMEM培养液及基础诱导液(含药血清组),干预后分别进行甲苯胺蓝染色、Ⅱ型胶原免疫组化染色,并以Real-time PCR法检测各组细胞中Ⅱ型胶原mRNA、聚集蛋白聚糖mRNA的水平。取18只8周龄SD大鼠,随机分为模型组、BMSC组及联合组,每组6只。通过切断右前交叉韧带对各组大鼠进行KOA造模。造模术后1周,向BMSC组与联合组大鼠右侧膝关节腔内注射5×106个·mL-1BMSC-PBS溶液(每次0.1 mL,每周1次),向模型组大鼠右侧膝关节腔注射等量PBS溶液; 联合组在关节腔注射的基础上每天以补肾活血汤灌胃(每次4 mL,每天1次)。干预8周后处死大鼠,取右侧膝关节软骨及软骨下骨组织,HE染色后在光学显微镜下观察。结果:干预7 d后,含药血清组细胞甲苯胺蓝染色呈阳性,细胞质呈蓝紫色; 空白组和诱导组染色均为阴性。干预14 d 后,含药血清组染色进一步加深,染色面积增大,呈明显的紫蓝色; 空白组染色为阴性,诱导组可见少量蓝紫色染色。干预7 d、14 d后,Ⅱ型胶原免疫组化染色结果显示,空白组细胞均未见明显阳性染色,诱导组及含药血清组细胞胞浆和细胞间质内呈棕黄色或棕褐色染色,其中干预14 d后含药血清组细胞内染色深于诱导组。干预7 d后,3组大鼠BMSC中Ⅱ型胶原mRNA水平、聚集蛋白聚糖mRNA水平比较,组间差异均有统计学意义(1.02±0.23,1.33±0.11,2.11±0.23,F=47.181,P=0.000; 1.03±0.32,1.36±0.16,1.93±0.10,F=26.508,P=0.000)。含药血清组的Ⅱ型胶原mRNA水平、聚集蛋白聚糖mRNA水平均高于空白组和诱导组(P=0.000,P=0.000; P=0.000,P=0.000),诱导组的Ⅱ型胶原mRNA水平、聚集蛋白聚糖mRNA水平均高于空白组(P=0.017; P=0.029)。药物干预8周后,模型组大鼠膝关节软骨表面粗糙,破坏严重,可见明显纤维增生; BMSC组软骨可见部分修复,表面仍较粗糙,细胞纤维化不明显; 联合组关节软骨面光滑,软骨表面磨损不明显,无软骨表面纤维化。结论:补肾活血汤含药血清能有效促进体外培养的大鼠BMSC向软骨细胞分化; 补肾活血汤灌胃联合BMSC膝关节腔内注射能有效防治KOA大鼠膝关节软骨破坏。
Abstract:
ABSTRACT Objective:To observe the effect of Bushen Huoxue Tang(补肾活血汤,BSHXT)medicated serum on chondrogenic differentiation of rat’s bone marrow derived mesenchymal stem cells(BMSCs)and the curative effect of BSHXT and BMSCs on knee osteoarthritis(KOA)in rats.Methods:The BMSCs were isolated from the femurs of 4-week-old SD rats and were cultured,and BSHXT medicated serum and blank serum were prepared by using 8-week-old SD rats.The third-generation BMSCs cultured in vitro were selected and randomly divided into 3 groups,and they were placed into the DMEM culture fluid added with 10% blank serum(blank group),DMEM culture fluid added with 10% blank serum and basic induced fluid(induced group)and DMEM culture fluid added with 10% BSHXT medicated serum and basic induced fluid(medicated serum group)respectively.After the intervention,the BMSCs were received toluidine blue staining and typeⅡcollagen immunohistochemical staining respectively,and the expression level of typeⅡcollagen mRNA and aggrecan mRNA were detected by using Real-time PCR method.Eighteen eight-week-old SD rats were selected and randomly divided into model group,

参考文献/References:

[1] PROCKOP DJ.Marrow stromal cells as stem cells for continual renewal of nonhematopoietic tissues and as potential vectors for gene therapy[J].J Cell Biochem Suppl,1998,30-31:284-285.
[2] YAMASAKI S,MERA H,ITOKAZU M.Cartilage Repair With Autologous Bone Marrow Mesenchymal Stem Cell Transplantation:Review of Preclinical and Clinical Studies[J].Cartilage,2014,5(4):196-202.
[3] LEVILLAIN A,BOULOCHER C,KADERLI S,et al.Meniscal biomechanical alterations in an ACLT rabbit model of early osteoarthritis[J].Osteoarthritis Cartilage,2015,23(7):1186-1193.
[4] SNYDER TN,MADHAVAN K,INTRATOR MA,et al.A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair[J].J Biol Eng,2014,8:10.
[5] SHIRAISHI K,KAMEI N,TAKEUCHI SA,et al.Quality evaluation of human bone marrow mesenchymal stem cells for cartilage repair[J/OL].Stem Cells Int,2017
[2017-11-27].https://www.ncbi.nlm.nih.gov/pubmed/28835756.
[6] BHOSALE AM,RICHARDSON JB.Articular cartilage:structure,injuries and review of management[J].Br Med Bull,2008,87(1):77-95.
[7] DA CUNHA CAVALCANTI FM,DOCA D,COHEN M.Updating on diagnosis and treatment of chondral lesion of the knee[J].Rev Bras Ortop,2012,47(1):12-20.
[8] BECK A,MURPHY DJ,CAREY-SMITH R,et al.Treatment of Articular Cartilage Defects With Microfracture and Autologous Matrix-Induced Chondrogenesis Leads to Extensive Subchondral Bone Cyst Formation in a Sheep Model[J].Am J Sports Med,2016,44(10):2629-2643.
[9] FOLDAGER CB,FARR J,GOMOLL AH.Patients Scheduled for Chondrocyte Implantation Treatment with MACI Have Larger Defects than Those Enrolled in Clinical Trials[J].Cartilage,2016,7(2):140-148.
[10] DENNIS JE,MERRIAM A,AWADALLAH A,et al.A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse[J].J Bone Miner Res,1999,14(5):700-709.
[11] AMIN HD,BRADY MA,ST-PIERRE J,et al.Stimulation of chondrogenic differentiation of adult human bone Marrow-Derived stromal cells by a Moderate-Strength static magnetic field[J].Tissue Eng Part A,2014,20(11/12):1612-1620.
[12] 李西海,刘献祥.骨关节炎的核心病机——本痿标痹[J].中医杂志,2014,55(14):1248-1249.
[13] 李佳,谢晚晴,郑洪新.论关节软骨与肾的联系[J].中华中医药学刊,2015,33(6):1344-1346.
[14] 季卫锋,施伟峰,陈林,等.补肾活血法防治大鼠膝骨性关节炎的实验研究[J].中国骨伤,2012,25(3):246-250.
[15] 李念虎.补肾活血中药含药血清对滑膜细胞分泌TNF-α、IL-1β水平的影响[J].中国骨伤,2007,20(5):302-303.
[16] 李念虎,夏雷,乙胜,等.补肾活血方对骨关节炎模型大鼠关节滑液IL-1β及关节软骨DDR2水平的影响[J].中国中医骨伤科杂志,2013,21(8):1-4.
[17] TANG JL,CUI WD,SONG FL,et al.Effects of mesenchymal stem cells on interleukin-1 beta-treated chondrocytes and cartilage in a rat osteoarthritic model[J].Mol Med Rep,2015,12(2):1753-1760.
[18] DIEKMAN BO.Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells[J].Proc Natl Acad Sci U S A,2012,109(47):19172-19177.

相似文献/References:

[1]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.
[2]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.
[3]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.
[4]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(01):9.
[5]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.
[6]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.
[7]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.
[8]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(01):6.
[9]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(01):15.
[10]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(01):27.

备注/Memo

备注/Memo:
通讯作者:吴刚 E-mail:280784683@qq.com
更新日期/Last Update: 2018-06-02