骨髓间充质干细胞移植联合川芎嗪膝关节腔注射治疗兔膝关节骨关节炎
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摘要
背景:单纯应用骨髓间充质干细胞或单纯应用川芎嗪治疗骨关节炎疗效有限。骨髓间充质干细胞移植复合川芎嗪是否能起到更好的效果未见报道。目的:了解川芎嗪对骨髓间充质干细胞成软骨分化及软骨再生的作用。方法:分离出新西兰白兔(1周龄,广西医科大学动物实验中心提供)骨髓间充质干细胞,培养传至第3代。将健康新西兰大白兔(五六个月龄,广西医科大学动物实验中心提供)40只随机分成4组进行实验:除空白对照组外,其余3组应用伸直位石膏固定法建立兔膝关节骨关节炎模型,所有建模部位均为右侧后肢。造模成功后第2天,空白对照组兔膝关节腔注射生理盐水0.3 mL,每周1次,共4次;单纯川芎嗪组兔膝关节腔注射川芎嗪0.3 mL,每周1次,共4次;单纯干细胞移植组兔膝关节腔内一次性注射骨髓间充质干细胞0.3 mL(细胞浓度为3×108 L-1),同时膝关节腔内注射生理盐水0.3 mL,每周1次;干细胞移植联合川芎嗪组兔膝关节腔内一次性注射骨髓间充质干细胞0.3 mL(细胞浓度为3×10~8L~(-1)),同时膝关节腔内注射川芎嗪0.3 mL,每周1次,共4次;干预后第6,12周采集各组兔膝关节液。结果与结论:(1)空白对照组膝关节软骨无明显磨损,周围滑膜无明显增生及炎性改变;单纯川芎嗪组膝关节磨损较为严重,软骨破坏达到3级,但并未暴露软骨下骨,周围滑膜增生明显;单纯干细胞移植组、干细胞移植联合川芎嗪组膝关节轻度磨损,1级或2级,周围滑膜增生及炎性改变不明显,两组在大体观下无法辨别磨损严重程度的差异;(2)单纯川芎嗪组、干细胞移植联合川芎嗪组兔关节液中白细胞介素1β及白细胞介素6水平明显低于单纯干细胞移植组(P <0.05)。在第6周时,也就是兔膝关节骨关节炎初期,软骨破坏不明显的时候,单纯川芎嗪组兔膝关节液炎症因子水平最低;在第12周时,干细胞移植联合川芎嗪组兔关节液炎症因子水平最低;(3)结果表明,川芎嗪对骨髓间充质干细胞成软骨分化及软骨再生的促进作用不明显,但是川芎嗪能明显降低膝关节骨关节炎的炎症水平,可以用来缓解膝关节骨关节炎患者的临床症状。
BACKGROUND:Mesenchymal stem cells or ligustrazine alone has limited efficacy in the treatment of osteoarthritis. Whether bone marrow mesenchymal stem cell transplantation combined with ligustrazine can achieve better results has yet to be reported.OBJECTIVE:To define whether ligustrazine has the function to promote the chondrogenic differentiation and regeneration of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were isolated from New Zealand white rabbits(1 week old, provided by the Animal Experimental Center of Guangxi Medical University in China) and cultured to the third generation. Forty healthy New Zealand white rabbits(5-6 months old, provided by the Animal Experimental Center of Guangxi Medical University) were randomly divided into four groups(n=10 per group). Except for the blank control group, models of knee osteoarthritis were made in the right hind limbs of rabbits in the extension position using the plaster fixation method in the other three groups. On the 2~(nd) day after the successful modeling, the rabbits in the blank control group were injected with 0.3 mL of normal saline once a week for 4 weeks. In the pure ligustrazine group, the rabbit knee joint cavity was injected with 0.3 mL of ligustrazine once a week for 4 weeks. In the cell transplantation group, the rabbit knee joint cavity was injected with one-off bone marrow mesenchymal stem cells(0.3 mL, 3×10~8 cells/L), and the knee joint was injected with 0.3 mL of normal saline once a week for 4 weeks. In the combination group, one-off bone marrow mesenchymal stem cells(0.3 mL, 3×10~8 cells/L) were injected into the rabbit knee joint, while intra-articular injection of ligustrazine, 0.3 mL, once a week, for 4 weeks, were simultaneously performed. The knee joint fluid of each group was collected at 6 and 12 weeks after the intervention.RESULTS AND CONCLUSION:(1) The knee joint cartilage of the blank control group had no obvious wear as well as no obvious hyperplasia and inflammatory changes in the surrounding synovial membrane. The knee joint of the ligustrazine group was seriously worn, and the cartilage damage was defined as grade 3, but the subchondral bone was not exposed, accompanied with obvious surrounding synovial hyperplasia. In the cell transplantation and combination groups, the knee joint was slightly worn, the cartilage damage was confirmed as grade 1 or 2, and the surrounding synovial hyperplasia and inflammatory changes were not obvious. We could not distinguish the severity of wear between the cell transplantation and combination groups under the general view.(2) Compared with the cell transplantation group, the levels of interleukin-1β and interleukin-6 in the knee joint fluid of rabbits were significantly lower in the ligustrazine group and combination group(P <0.05). At the 6~(th) week, when the cartilage destruction was not obvious in the early stage of rabbit knee osteoarthritis, the levels of inflammatory factors in the knee joint fluid were the lowest in the ligustrazine group. At the 12~(th) week, the levels of inflammatory factors in the joint fluid were the lowest in the combination group. To conclude, ligustrazine cannot obviously promote the chondrogenic differentiation and regeneration of bone marrow mesenchymal stem cells, but it can significantly reduce the level of inflammatory factors and relieve clinical symptoms in patients with knee osteoarthritis.
BACKGROUND:Mesenchymal stem cells or ligustrazine alone has limited efficacy in the treatment of osteoarthritis. Whether bone marrow mesenchymal stem cell transplantation combined with ligustrazine can achieve better results has yet to be reported.OBJECTIVE:To define whether ligustrazine has the function to promote the chondrogenic differentiation and regeneration of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were isolated from New Zealand white rabbits(1 week old, provided by the Animal Experimental Center of Guangxi Medical University in China) and cultured to the third generation. Forty healthy New Zealand white rabbits(5-6 months old, provided by the Animal Experimental Center of Guangxi Medical University) were randomly divided into four groups(n=10 per group). Except for the blank control group, models of knee osteoarthritis were made in the right hind limbs of rabbits in the extension position using the plaster fixation method in the other three groups. On the 2~(nd) day after the successful modeling, the rabbits in the blank control group were injected with 0.3 mL of normal saline once a week for 4 weeks. In the pure ligustrazine group, the rabbit knee joint cavity was injected with 0.3 mL of ligustrazine once a week for 4 weeks. In the cell transplantation group, the rabbit knee joint cavity was injected with one-off bone marrow mesenchymal stem cells(0.3 mL, 3×10~8 cells/L), and the knee joint was injected with 0.3 mL of normal saline once a week for 4 weeks. In the combination group, one-off bone marrow mesenchymal stem cells(0.3 mL, 3×10~8 cells/L) were injected into the rabbit knee joint, while intra-articular injection of ligustrazine, 0.3 mL, once a week, for 4 weeks, were simultaneously performed. The knee joint fluid of each group was collected at 6 and 12 weeks after the intervention.RESULTS AND CONCLUSION:(1) The knee joint cartilage of the blank control group had no obvious wear as well as no obvious hyperplasia and inflammatory changes in the surrounding synovial membrane. The knee joint of the ligustrazine group was seriously worn, and the cartilage damage was defined as grade 3, but the subchondral bone was not exposed, accompanied with obvious surrounding synovial hyperplasia. In the cell transplantation and combination groups, the knee joint was slightly worn, the cartilage damage was confirmed as grade 1 or 2, and the surrounding synovial hyperplasia and inflammatory changes were not obvious. We could not distinguish the severity of wear between the cell transplantation and combination groups under the general view.(2) Compared with the cell transplantation group, the levels of interleukin-1β and interleukin-6 in the knee joint fluid of rabbits were significantly lower in the ligustrazine group and combination group(P <0.05). At the 6~(th) week, when the cartilage destruction was not obvious in the early stage of rabbit knee osteoarthritis, the levels of inflammatory factors in the knee joint fluid were the lowest in the ligustrazine group. At the 12~(th) week, the levels of inflammatory factors in the joint fluid were the lowest in the combination group. To conclude, ligustrazine cannot obviously promote the chondrogenic differentiation and regeneration of bone marrow mesenchymal stem cells, but it can significantly reduce the level of inflammatory factors and relieve clinical symptoms in patients with knee osteoarthritis.
引文
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[10]Kuang B,Dai J,Wang QY,et al.Combined degenerative and regenerative remodeling responses of the mandibular condyle to experimentally induced disordered occlusion.Am J Orthod Dentofacial Orthop.2013;143(1):69-76.
[11]谢平金,史桐雨,梁贵洪,等.川芎嗪对膝骨性关节炎大鼠软骨BMP-2、Smad1及BMP-2m RNA、Smad1mRNA表达的影响[J].中国骨质疏松杂志,2018,24(6):727-731.
[12]方斌,刘文刚,赵自明,等.参麦注射液关节内注射治疗家兔膝骨关节炎的实验研究[J].风湿病与关节炎,2013,2(8):27-30.
[13]聂林.骨关节炎的动物模型[J].中华实验外科杂志,1990,7(2):96-97.
[14]Okazaki R,Sakai A,Ootsuyama A,et al.Apoptosis and p53expression in chondrocytes relate to degeneration in articular cartilage of immobilized knee joints.J Rheumatol.2003;30(3):559-566.
[15]江捍平,王大平.骨关节炎动物模型[J].中国现代医学杂志,2004,14(6):153-154.
[16]Perilli E,Le V,Ma B,et al.Detecting early bone changes using in vivo micro-CT in ovariectomized,zoledronic acid-treated,and sham-operated rats.Osteoporos Int.2010;21(8):1371-1382.
[17]Zhu S,Chen K,Lan Y,et al.Alendronate protects against articular cartilage erosion by inhibiting subchondral bone loss in ovariectomized rats.Bone.2013;53(2):340-349.
[18]Bellido M,Lugo L,Roman-Blas JA,et al.Subchondral bone microstructural damage by increased remodelling aggravates experimental osteoarthritis preceded by osteoporosis.Arthritis Res Ther.2010;12(4):R152.
[19]彭冰,胡正国,宋才渊,等.小鼠骨质疏松合并骨性关节炎模型的建立及评价[J].广西中医药大学学报,2015,18(2):8-10.
[20]Bahat-Stroomza M,Barhum Y,Levy YS,et al.Induction of adult human bone marrow mesenchymal stromal cells into functional astrocyte-like cells:potential for restorative treatment in Parkinson's disease.J Mol Neurosci.2009;39(1-2):199-210.
[21]Kaka GR,Tiraihi T,Delshad A,et al.In vitro differentiation of bone marrow stromal cells into oligodendrocyte-like cells using triiodothyronine as inducer.Int J Neurosci.2012;122(5):237-247.
[22]周斌,樊粤光.骨髓间充质干细胞与软骨修复研究进展[J].中国中医骨伤科杂志,2007,15(10):67-69.
[23]Ruiz-Ibán Má,Díaz-Heredia J,García-Gómez I,et al.The effect of the addition of adipose-derived mesenchymal stem cells to a meniscal repair in the avascular zone:an experimental study in rabbits.Arthroscopy.2011;27(12):1688-1696.
[24]Friedenstein AJ,Chailakhyan RK,Gerasimov UV.Bone marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers.Cell Tissue Kinet.1987;20(3):263-272.
[25]Fortier LA1,Nixon AJ,Williams J,et al.Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells.Am J Vet Res.1998;59(9):1182-1187.
[26]Im GI,Kim DY,Shin JH,et al.Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow.J Bone Joint Surg Br.2001;83(2):289-294.
[27]田园,李运鹏,韩若东.川芎嗪对大鼠脑缺血再灌注后细胞凋亡及caspase12表达的影响[J].临床和实验医学杂志,2014,13(1):16-19.
[28]吕红斌,徐大启,胡建中,等.川芎嗪关节腔内注射治疗兔骨关节炎模型的研究[J].中国医师杂志,2007,9(9):1163-1165.
[29]付长龙,梅阳阳,林洁,等.川芎嗪干预类风湿关节炎的机制探讨[J].风湿病与关节炎,2016,5(9):45-47.
[30]李应池,王晓霞,邱桐,等.川芎嗪对鼠骨关节炎关节软骨MMP-1、TIMP-1表达的影响[J].中华中医药学刊,2011,29(9):2120-2124.
[31]胡建中,罗承耀,康明,等.川芎嗪关节腔内注射对膝骨关节炎的治疗作用[J].中南大学学报(医学版),2006,31(4):591-594.
[32]罗森.川芎嗪注射液治疗膝骨性关节炎的临床观察[J].内蒙古中医药,2018,15(8):96-97.
[33]张利省.盐酸川芎嗪注射液联合美洛昔康对类风湿性关节炎患者症状改善及日常生活能力的影响[J].中国药物经济学,2017,12(9):93-95.
[2]Bonaiuti D,Arioli G,Diana G,et al.SIMFER Rehabilitation treatment guidelines in postmenopausal and senile osteoporosis.Eura Medicophys.2005;41(4):315-337.
[3]中华医学会骨科学分会.骨关节炎诊治指南(2007年版)[J].中国临床医生,2008,36(1):28-30.
[4]王万铁,徐正衸,林丽娜,等.川芎嗪对肝缺血-再灌注损伤家兔血浆TXA2/PGI2水平的影响[J].中国临床药学杂志,1999,8(3):167-170.
[5]胡建中,罗承耀,康明,等.川芎嗪关节腔内注射对膝骨关节炎的治疗作用[J].中南大学学报(医学版),2006,31(4):591-594.
[6]罗丹.川芎嗪联合来氟米特对Ⅱ型胶原诱导性关节炎大鼠的治疗作用及其机理研究[D].北京:北京中医药大学,2014.
[7]Loeser RF,Goldring SR,Scanzello CR,et al.Osteoarthritis:a disease of the joint as an organ.Arthritis Rheum.2012;64(6):1697-1707.
[8]Park DY,Jin LH,Min BH,et al.Subchondral bone scan uptake correlates with articular cartilage degeneration in osteoarthritic knees.Int J Rheum Dis.2017;20(10):1393-1402.
[9]Thomas NP,Li P,Fleming BC,et al.Attenuation of cartilage pathogenesis in post-traumatic osteoarthritis(PTOA)in mice by blocking the stromal derived factor 1 receptor(CXCR4)with the specific inhibitor,AMD3100.J Orthop Res.2015;33(7):1071-1078.
[10]Kuang B,Dai J,Wang QY,et al.Combined degenerative and regenerative remodeling responses of the mandibular condyle to experimentally induced disordered occlusion.Am J Orthod Dentofacial Orthop.2013;143(1):69-76.
[11]谢平金,史桐雨,梁贵洪,等.川芎嗪对膝骨性关节炎大鼠软骨BMP-2、Smad1及BMP-2m RNA、Smad1mRNA表达的影响[J].中国骨质疏松杂志,2018,24(6):727-731.
[12]方斌,刘文刚,赵自明,等.参麦注射液关节内注射治疗家兔膝骨关节炎的实验研究[J].风湿病与关节炎,2013,2(8):27-30.
[13]聂林.骨关节炎的动物模型[J].中华实验外科杂志,1990,7(2):96-97.
[14]Okazaki R,Sakai A,Ootsuyama A,et al.Apoptosis and p53expression in chondrocytes relate to degeneration in articular cartilage of immobilized knee joints.J Rheumatol.2003;30(3):559-566.
[15]江捍平,王大平.骨关节炎动物模型[J].中国现代医学杂志,2004,14(6):153-154.
[16]Perilli E,Le V,Ma B,et al.Detecting early bone changes using in vivo micro-CT in ovariectomized,zoledronic acid-treated,and sham-operated rats.Osteoporos Int.2010;21(8):1371-1382.
[17]Zhu S,Chen K,Lan Y,et al.Alendronate protects against articular cartilage erosion by inhibiting subchondral bone loss in ovariectomized rats.Bone.2013;53(2):340-349.
[18]Bellido M,Lugo L,Roman-Blas JA,et al.Subchondral bone microstructural damage by increased remodelling aggravates experimental osteoarthritis preceded by osteoporosis.Arthritis Res Ther.2010;12(4):R152.
[19]彭冰,胡正国,宋才渊,等.小鼠骨质疏松合并骨性关节炎模型的建立及评价[J].广西中医药大学学报,2015,18(2):8-10.
[20]Bahat-Stroomza M,Barhum Y,Levy YS,et al.Induction of adult human bone marrow mesenchymal stromal cells into functional astrocyte-like cells:potential for restorative treatment in Parkinson's disease.J Mol Neurosci.2009;39(1-2):199-210.
[21]Kaka GR,Tiraihi T,Delshad A,et al.In vitro differentiation of bone marrow stromal cells into oligodendrocyte-like cells using triiodothyronine as inducer.Int J Neurosci.2012;122(5):237-247.
[22]周斌,樊粤光.骨髓间充质干细胞与软骨修复研究进展[J].中国中医骨伤科杂志,2007,15(10):67-69.
[23]Ruiz-Ibán Má,Díaz-Heredia J,García-Gómez I,et al.The effect of the addition of adipose-derived mesenchymal stem cells to a meniscal repair in the avascular zone:an experimental study in rabbits.Arthroscopy.2011;27(12):1688-1696.
[24]Friedenstein AJ,Chailakhyan RK,Gerasimov UV.Bone marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers.Cell Tissue Kinet.1987;20(3):263-272.
[25]Fortier LA1,Nixon AJ,Williams J,et al.Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells.Am J Vet Res.1998;59(9):1182-1187.
[26]Im GI,Kim DY,Shin JH,et al.Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow.J Bone Joint Surg Br.2001;83(2):289-294.
[27]田园,李运鹏,韩若东.川芎嗪对大鼠脑缺血再灌注后细胞凋亡及caspase12表达的影响[J].临床和实验医学杂志,2014,13(1):16-19.
[28]吕红斌,徐大启,胡建中,等.川芎嗪关节腔内注射治疗兔骨关节炎模型的研究[J].中国医师杂志,2007,9(9):1163-1165.
[29]付长龙,梅阳阳,林洁,等.川芎嗪干预类风湿关节炎的机制探讨[J].风湿病与关节炎,2016,5(9):45-47.
[30]李应池,王晓霞,邱桐,等.川芎嗪对鼠骨关节炎关节软骨MMP-1、TIMP-1表达的影响[J].中华中医药学刊,2011,29(9):2120-2124.
[31]胡建中,罗承耀,康明,等.川芎嗪关节腔内注射对膝骨关节炎的治疗作用[J].中南大学学报(医学版),2006,31(4):591-594.
[32]罗森.川芎嗪注射液治疗膝骨性关节炎的临床观察[J].内蒙古中医药,2018,15(8):96-97.
[33]张利省.盐酸川芎嗪注射液联合美洛昔康对类风湿性关节炎患者症状改善及日常生活能力的影响[J].中国药物经济学,2017,12(9):93-95.
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