右归饮诱导的骨髓间充质干细胞/纤维蛋白胶复合物对膝关节软骨缺损兔软骨修复的影响
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摘要
目的观察右归饮对兔骨髓间充质干细胞(BMSCs)软骨分化的效果及其诱导的BMSCs/纤维蛋白胶复合物对兔膝关节软骨缺损的修复效果。方法分离兔胫骨和股骨骨髓细胞,传代培养得到兔BMSCs,将兔BMSCs分为对照组、诱导组和右归饮低、中、高剂量组,检测BMSCs细胞中Ⅱ型胶原、聚集蛋白多糖mRNA和蛋白表达情况,筛选右归饮后续实验浓度。将膝关节软骨损伤模型兔随机分为5组,每组6只,其中右归饮组将经筛选浓度的右归饮诱导1周的BMSCs/纤维蛋白胶复合物植入兔软骨缺损部位; TGF-β1组将经转化生长因子β1 (TGF-β1)诱导1周的BMSCs/纤维蛋白胶复合物植入兔软骨缺损部位;纤维蛋白胶组将纤维蛋白胶植入兔软骨缺损部分;模型组缺损旷置;另设正常对照组不做任何处理。各组均干预8周后,取膝关节软骨缺损部位,通过大体观察、HE染色及Wakitani评分评估软骨损伤修复效果。结果与对照组比较,右归饮中、高剂量组Ⅱ型胶原蛋白、聚集蛋白多糖mRNA和蛋白水平均明显上调,且以右归饮高剂量组效果最好(P <0. 05),故选右归饮400 mg/ml进行后续实验。正常对照组兔膝关节形态正常,表面光滑,无损伤;模型组兔膝关节软骨缺损处呈暗红色,软骨缺损处组织结构不规则,未见软骨细胞,仅可见少量纤维组织,软骨组织Wakitani评分升高(P <0. 05)。纤维蛋白胶组兔膝关节软骨缺损面积变小,软骨缺损处有修复填充物,Wakitani评分降低(P <0. 05)。与纤维蛋白胶组比较,右归饮组和TGF-β1组兔膝关节软骨缺损基本修复完整,修复部位HE染色与周边正常组织较为接近,可见大量软骨细胞,出现软骨陷窝结构,软骨组织Wakitani评分降低(P <0. 05)。结论右归饮可诱导兔BMSCs的成软骨分化,其所诱导的BMSCs/纤维蛋白胶复合物对兔膝关节软骨缺损修复具有促进作用。
Objective To observe the effects of Yougui Decoction( 右归饮) on cartilage differentiation of rabbit bone marrow mesenchymal stem cells( BMSCs) and the effects of its induced BMSCs/fibrin glue complex on the repair of rabbit knee articular cartilage defects. Methods Rabbit tibia and femur bone marrow cells were isolated and subcultured to obtain rabbit BMSCs. Rabbit BMSCs were divided into a control group,an induction group and Yougui Decoction low,medium and high dose groups. The expression of type Ⅱ collagen and aggrecan mRNA in BMSCs cells and the expression of the protein was detected. The concentration of the subsequent experiment of Yougui Decoction was screened. Rabbits with knee joint cartilage injury were randomly divided into 5 groups,with 6 rabbits in each group. Among them,the Yougui Decoction group was implanted with the right concentration of BMSCs/fibrin glue complex for 1 week into the rabbit cartilage defect site; TGF-β1 group implanted BMSCs/fibrin glue complex induced by transforming growth factor β1( TGF-β1) for 1 week into rabbit cartilage defect site; fibrin glue group implanted fibrin glue into rabbit cartilage defect part; the model group's defect was put aside; another normal control group was set up without any treatment. After 8 weeks of intervention in each group,the cartilage defects of the knee joint were taken,and the cartilage injury repair effect was evaluated by gross observation,HE staining and Wakitani score.Results Compared with the control group,the mRNA and protein levels of type Ⅱ collagen and aggrecan were significantly up-regulated in the middle and high dose groups of Yougui Decoction,and the effect was the best in the high-dose group of Yougui Decoction( P < 0. 05). Therfore,Yougui Decoction 400 mg/ml was selected for subsequent experiments. In the normal control group,the knee joints were normal in shape and smooth,and there was no damage. In the model group,the knee joint articular cartilage defect was dark red,and the cartilage defect was irregular.No chondrocytes were seen,and only a small amount of fibrous tissue and cartilage tissue were observed. The Wakitani score was increased( P < 0. 05). In the fibrin glue group,the knee articular cartilage defect area was smaller,the cartilage defect had repair filler,and the Wakitani score was decreased( P < 0. 05). Compared with the fibrin glue group,the knee joint articular cartilage defects in the Yougui Decoction group and the TGF-β1 group were basically repaired intact. The HE staining at the repair site was close to that of the surrounding normal tissues. A large number of chondrocytes were seen,and the cartilage lacuna structure appeared. The Wakitani score pof cartilage tissue was reduced( P < 0. 05). Conclusion Yougui Decoction can induce chondrogenic differentiation of rabbit BMSCs,and the induced BMSCs/fibrin glue complex can promote the repair of rabbit knee articular cartilage defects.
Objective To observe the effects of Yougui Decoction( 右归饮) on cartilage differentiation of rabbit bone marrow mesenchymal stem cells( BMSCs) and the effects of its induced BMSCs/fibrin glue complex on the repair of rabbit knee articular cartilage defects. Methods Rabbit tibia and femur bone marrow cells were isolated and subcultured to obtain rabbit BMSCs. Rabbit BMSCs were divided into a control group,an induction group and Yougui Decoction low,medium and high dose groups. The expression of type Ⅱ collagen and aggrecan mRNA in BMSCs cells and the expression of the protein was detected. The concentration of the subsequent experiment of Yougui Decoction was screened. Rabbits with knee joint cartilage injury were randomly divided into 5 groups,with 6 rabbits in each group. Among them,the Yougui Decoction group was implanted with the right concentration of BMSCs/fibrin glue complex for 1 week into the rabbit cartilage defect site; TGF-β1 group implanted BMSCs/fibrin glue complex induced by transforming growth factor β1( TGF-β1) for 1 week into rabbit cartilage defect site; fibrin glue group implanted fibrin glue into rabbit cartilage defect part; the model group's defect was put aside; another normal control group was set up without any treatment. After 8 weeks of intervention in each group,the cartilage defects of the knee joint were taken,and the cartilage injury repair effect was evaluated by gross observation,HE staining and Wakitani score.Results Compared with the control group,the mRNA and protein levels of type Ⅱ collagen and aggrecan were significantly up-regulated in the middle and high dose groups of Yougui Decoction,and the effect was the best in the high-dose group of Yougui Decoction( P < 0. 05). Therfore,Yougui Decoction 400 mg/ml was selected for subsequent experiments. In the normal control group,the knee joints were normal in shape and smooth,and there was no damage. In the model group,the knee joint articular cartilage defect was dark red,and the cartilage defect was irregular.No chondrocytes were seen,and only a small amount of fibrous tissue and cartilage tissue were observed. The Wakitani score was increased( P < 0. 05). In the fibrin glue group,the knee articular cartilage defect area was smaller,the cartilage defect had repair filler,and the Wakitani score was decreased( P < 0. 05). Compared with the fibrin glue group,the knee joint articular cartilage defects in the Yougui Decoction group and the TGF-β1 group were basically repaired intact. The HE staining at the repair site was close to that of the surrounding normal tissues. A large number of chondrocytes were seen,and the cartilage lacuna structure appeared. The Wakitani score pof cartilage tissue was reduced( P < 0. 05). Conclusion Yougui Decoction can induce chondrogenic differentiation of rabbit BMSCs,and the induced BMSCs/fibrin glue complex can promote the repair of rabbit knee articular cartilage defects.
引文
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[6]王伟东,邹阳,沈建国,等.右归饮经过调节SDF-1的表达影响OA模型大鼠关节软骨退变的研究简[J].中华中医药杂志,2016,36(8):3308-3311.
[7]武海军,银和平,李树文,等.密度梯度离心和贴壁筛选法分离培养兔骨髓间充质干细胞的形态学观察[J].中华临床医师杂志(电子版),2012,6(22):7261-7269.
[8]张加廷,张东正,丁一,等.后内侧入路建立兔膝关节软骨缺损动物模型[J].中华实验外科杂志,2016,33(4):1145-1149.
[9]WAKITANI S,GOTO T,YOUNG RG,et al. Repair of large full-thickness aritucular defects with allograft articular chondrocytes embedded inacollagengel[J]. Tissue Eng,1998,4(4):429-444.
[10]DOMINICI M,LE BK,MUELLER I,et al. Minimal criteria for defining multipotent mesenchymal stromal cells.The international society for cellular therapy position statement[J]. Cytotherapy,2006,8(4):315-324.
[11]张鹏,施杞,王拥军.论“肾者,作强之官,伎巧出焉”对中医骨伤科的指导意义[J].中医杂志,2012,53(9):795-796.
[12]KATSARA O,ATTUR M,RUOFF R,et al. Increased activity of the chondrocyte translational apparatus accompanies osteoarthritic changes in human and rodent knee cartilage[J]. Arthritis Rheumatol,2017,69(3):586-597.
[13]陈祁青,金红婷,应俊,等.右归饮含药血清对骨髓间充质干细胞成软骨分化的促进作用及其microRNA表达谱分析[J].中华中医药杂志,2015,30(5):1380-1386.
[14]KUSUYAMA J,KAMISONO A,SEONG C,et al.Spleen tyrosine kinase influences the early stages of multilineage differentiation of bone marrow stromal cell lines by regulating phospholipase C gamma activities[J]. J Cell Physiol,2017,233(3):2549-2559.
[15]NUMMENMAA E,HMLINEN M,MOILANEN T,et al. Effects of FGF-2 and FGF receptor antagonists on MMP enzymes,aggrecan,and typeⅡcollagen in primary human OA chondrocytes[J]. Scand J Rheumatol,2015,44(4):321-330.
[16]BABADAGLI ME,TEZCAN B,YILMAZ ST,et al.Matrilin-3 as a putative effector of C-type natriuretic peptide signaling during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells[J]. Mol Biol Rep,2014,41(9):5549-5555.
[17]YING J,WANG P,ZHANG S,et al. Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells[J]. Life Sci,2018,45(192):84-90.
[18]刘相杰,宋科官.生物支架材料及间充质干细胞在骨组织工程中的研究与应用[J].中国组织工程研究,2018,22(10):148-154.
[19]MORIN KT,TRANQUILLO RT. In vitro models of angiogenesis and vasculogenesis in fibrin gel[J]. Exp Cell Res,2013,319(16):2409-2417.
[20]MURPHY KC,HUGHBANKS ML,BINDER BY,et al.Engineered fibrin gels for parallel stimulation of mesenchymal stem cell proangiogenic and osteogenic potential[J]. Ann Biomed Eng,2015,43(8):2010-2021.
[21]VASEENON T,TOCHIGI Y,HEINER AD,et al.Organ-level histological and biomechanical responses from localized osteoarticular injury in the rabbit knee[J]. J Orthop Res,2011,29(3):340-346.
[2]CHAWLA A,TWYCROSS-LEWIS R,MAFFULLI N. Microfracture produces inferior outcomes to other cartilage repair techniques in chondral injuries in the paediatric knee[J]. Br Med Bull,2015,116(1):93-103.
[3]GARCA-GARCA A,DE CASTILLEJO CL,MNDEZFERRER S. BMSCs and hematopoiesis[J]. Immunol Lett,2015,168(2):129-135.
[4]LI X,LI Y,ZUO Y,et al. Osteogenesis and chondrogenesis of biomimetic integrated porous PVA/gel/V-n-HA/pa6scaffolds and BMSCs construct in repair of articular osteochondral defect[J]. J Biomed Mater Res,2015,103(10):3226-3236.
[5]KIM IG,HWANG MP,DU P,et al. Bioactive cellderived matrices combined with polymer mesh scaffold for osteogenesis and bone healing[J]. Biomaterials,2015,50(1):75-86.
[6]王伟东,邹阳,沈建国,等.右归饮经过调节SDF-1的表达影响OA模型大鼠关节软骨退变的研究简[J].中华中医药杂志,2016,36(8):3308-3311.
[7]武海军,银和平,李树文,等.密度梯度离心和贴壁筛选法分离培养兔骨髓间充质干细胞的形态学观察[J].中华临床医师杂志(电子版),2012,6(22):7261-7269.
[8]张加廷,张东正,丁一,等.后内侧入路建立兔膝关节软骨缺损动物模型[J].中华实验外科杂志,2016,33(4):1145-1149.
[9]WAKITANI S,GOTO T,YOUNG RG,et al. Repair of large full-thickness aritucular defects with allograft articular chondrocytes embedded inacollagengel[J]. Tissue Eng,1998,4(4):429-444.
[10]DOMINICI M,LE BK,MUELLER I,et al. Minimal criteria for defining multipotent mesenchymal stromal cells.The international society for cellular therapy position statement[J]. Cytotherapy,2006,8(4):315-324.
[11]张鹏,施杞,王拥军.论“肾者,作强之官,伎巧出焉”对中医骨伤科的指导意义[J].中医杂志,2012,53(9):795-796.
[12]KATSARA O,ATTUR M,RUOFF R,et al. Increased activity of the chondrocyte translational apparatus accompanies osteoarthritic changes in human and rodent knee cartilage[J]. Arthritis Rheumatol,2017,69(3):586-597.
[13]陈祁青,金红婷,应俊,等.右归饮含药血清对骨髓间充质干细胞成软骨分化的促进作用及其microRNA表达谱分析[J].中华中医药杂志,2015,30(5):1380-1386.
[14]KUSUYAMA J,KAMISONO A,SEONG C,et al.Spleen tyrosine kinase influences the early stages of multilineage differentiation of bone marrow stromal cell lines by regulating phospholipase C gamma activities[J]. J Cell Physiol,2017,233(3):2549-2559.
[15]NUMMENMAA E,HMLINEN M,MOILANEN T,et al. Effects of FGF-2 and FGF receptor antagonists on MMP enzymes,aggrecan,and typeⅡcollagen in primary human OA chondrocytes[J]. Scand J Rheumatol,2015,44(4):321-330.
[16]BABADAGLI ME,TEZCAN B,YILMAZ ST,et al.Matrilin-3 as a putative effector of C-type natriuretic peptide signaling during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells[J]. Mol Biol Rep,2014,41(9):5549-5555.
[17]YING J,WANG P,ZHANG S,et al. Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells[J]. Life Sci,2018,45(192):84-90.
[18]刘相杰,宋科官.生物支架材料及间充质干细胞在骨组织工程中的研究与应用[J].中国组织工程研究,2018,22(10):148-154.
[19]MORIN KT,TRANQUILLO RT. In vitro models of angiogenesis and vasculogenesis in fibrin gel[J]. Exp Cell Res,2013,319(16):2409-2417.
[20]MURPHY KC,HUGHBANKS ML,BINDER BY,et al.Engineered fibrin gels for parallel stimulation of mesenchymal stem cell proangiogenic and osteogenic potential[J]. Ann Biomed Eng,2015,43(8):2010-2021.
[21]VASEENON T,TOCHIGI Y,HEINER AD,et al.Organ-level histological and biomechanical responses from localized osteoarticular injury in the rabbit knee[J]. J Orthop Res,2011,29(3):340-346.