大鼠骨内膜骨髓细胞增殖和成软骨分化能力的体外研究
|
摘要
目的对比骨内膜骨髓细胞(endosteal bone marrow cells,EBMCs)与中央骨髓细胞(center bone marrow cells,CBMCs)增殖及成软骨分化,为组织工程技术寻找较CBMCs更优良的种子细胞。方法取健康雄性SD大鼠的双侧股骨及胫骨,采用酶消化法分离EBMCs,采取密度梯度离心法分离CBMCs予体外增殖克隆,通过流式细胞术(flow cytometry,FCM)检测细胞表面分子CD29、CD34、CD44、CD45的表达。集落形成单位(colony forming unit,CFU)检测细胞克隆形成,5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,Brd U)检测细胞增殖能力,定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,qRT-PCR)检测细胞周期抑制因子相关基因p15、p16、p21、p27的表达。成软骨诱导培养21d后行阿尔新蓝染色并提取mRNA行qRT-PCR检测对比成软骨分化相关基因SOX9、aggrecan、Col2α1的表达。结果 EBMCs与CBMCs表面分子CD29、CD44均呈阳性表达,CD34、CD45均呈阴性表达,但EBMCs表达较CBMCs表达低,二者比较差异有统计学意义(P<0.05)。CFU检测中碱性磷酸酶(alkaline phosphatase,ALP)染色及结晶紫染色可见EBMCs比CBMCs颗粒沉淀数量更多,颗粒直径更大。Brd U检测细胞增殖能力,可见CBMCs的荧光信号倍增量(1.47±0.16),明显低于EBMCs的荧光信号倍增量(2.56±0.36),二者比较差异具有统计学意义(P<0.001)。细胞周期抑制因子p15、p16、p21、p27的表达,EBMCs中分别为0.315±0.052、0.158±0.023、0.416±0.051、0.100±0.022,明显低于CBMCs的1.000±0.000、0.481±0.064、1.342±0.135、0.461±0.156,二者比较差异有统计学意义(P<0.001)。阿尔新蓝染色可见EBMCs成软骨分化后染色蓝染较CBMCs更明显;EBMCs中成软骨骨分化相关基因SOX9,aggrecan,Col2α1的表达分别为1.783±0.305、0.918±0.201、0.384±0.108明显高于CBMCs的1.000±0.000、0.297±0.082、0.124±0.007,二者比较差异均具有统计学意义(P<0.001)。结论 EBMCs较CBMCs有更强的增殖分化能力,更适合做软骨组织工程的种子细胞。
Objective To compare the difference of proliferation and chondrogenic differentiation between endosteal bone marrow cells(EBMCs)and center bone marrow cells(CBMCs),and find better seed cells for tissue engineering than CBMCs.Methods EBMCs and CBMCs were isolated from bilateral femur and tibia of healthy male SD rats by enzyme digestionand density gradient centrifugation,respectively. And CBMCs were given proliferation cloning in vitro.The expressions of cell surface molecules CD29,CD34,CD44 and CD45 were detected by flow cytometry(FCM).Colony forming unit(CFU)was taken to detect the cell clone formation,5-Bromodeoxyuridine(BrdU)was used to detect the ability of cell proliferation,and quantitative reverse transcription-polymerase chain reaction(qRTPCR)was used to detect the expressions of gene p15,p16,p21,p27 associated with cell cycle inhibitors.After cultured with chondrocytes for 21 days,alcain blue wasused to stain,mRNA was extracted and compared among the expressions of chondrogenic differentiation related gene SOX9,aggrecan and Col2α1.Results The surface molecules CD29 and CD44 of EBMCs and CBMCs were both positive expressions,CD34 and CD45 were negative expressions,the expressions of EBMCs were lower than these of CBMCs(P<0.05).Alkaline phosphatase(ALP)staining and crystal violet staining in the CFU detection showed EBMCs had more precipitates and larger particles than CBMCs.BrdU was used to detect the cell proliferation ability.The proliferation of fluorescence signal in CBMCs,was significantly lower than that in EBMCs(1.47±0.16,vs.2.56±0.36,P<0.001).The expressions of cell cycle inhibitor p15,p16,p21 and p27 in EBMCs were significantly lower than those in CBMCs[(0.315±0.052,0.158±0.023,0.416±0.051,0.100±0.022),vs.(1.000±0.000,0.481±0.064,1.342±0.135,0.461±0.156),P<0.001].Alcian blue staining showed the blue-staining in EBMCs after chondrogenic differentiation was much more obvious than that in CBMCs.The expressions of chondrogenic differentiation related gene SOX9,aggrecan,Col2α1 in EBMCs were significantly higher than those in CBMCs [(1.783±0.305,0.918±0.201,0.384±0.108)vs.(1.000±0.000,0.297±0.082,0.124±0.007),P<0.001].Conclusion EBMCs have stronger proliferation and differentiation ability than CBMCs,which are more suitable for the seed cells of cartilage tissue engineering.
Objective To compare the difference of proliferation and chondrogenic differentiation between endosteal bone marrow cells(EBMCs)and center bone marrow cells(CBMCs),and find better seed cells for tissue engineering than CBMCs.Methods EBMCs and CBMCs were isolated from bilateral femur and tibia of healthy male SD rats by enzyme digestionand density gradient centrifugation,respectively. And CBMCs were given proliferation cloning in vitro.The expressions of cell surface molecules CD29,CD34,CD44 and CD45 were detected by flow cytometry(FCM).Colony forming unit(CFU)was taken to detect the cell clone formation,5-Bromodeoxyuridine(BrdU)was used to detect the ability of cell proliferation,and quantitative reverse transcription-polymerase chain reaction(qRTPCR)was used to detect the expressions of gene p15,p16,p21,p27 associated with cell cycle inhibitors.After cultured with chondrocytes for 21 days,alcain blue wasused to stain,mRNA was extracted and compared among the expressions of chondrogenic differentiation related gene SOX9,aggrecan and Col2α1.Results The surface molecules CD29 and CD44 of EBMCs and CBMCs were both positive expressions,CD34 and CD45 were negative expressions,the expressions of EBMCs were lower than these of CBMCs(P<0.05).Alkaline phosphatase(ALP)staining and crystal violet staining in the CFU detection showed EBMCs had more precipitates and larger particles than CBMCs.BrdU was used to detect the cell proliferation ability.The proliferation of fluorescence signal in CBMCs,was significantly lower than that in EBMCs(1.47±0.16,vs.2.56±0.36,P<0.001).The expressions of cell cycle inhibitor p15,p16,p21 and p27 in EBMCs were significantly lower than those in CBMCs[(0.315±0.052,0.158±0.023,0.416±0.051,0.100±0.022),vs.(1.000±0.000,0.481±0.064,1.342±0.135,0.461±0.156),P<0.001].Alcian blue staining showed the blue-staining in EBMCs after chondrogenic differentiation was much more obvious than that in CBMCs.The expressions of chondrogenic differentiation related gene SOX9,aggrecan,Col2α1 in EBMCs were significantly higher than those in CBMCs [(1.783±0.305,0.918±0.201,0.384±0.108)vs.(1.000±0.000,0.297±0.082,0.124±0.007),P<0.001].Conclusion EBMCs have stronger proliferation and differentiation ability than CBMCs,which are more suitable for the seed cells of cartilage tissue engineering.
引文
[1]Tang QO,Carasco CF,Gamie Z,et al.Preclinical and clinical data for the use of mesenchymal stem cells in articular cartilage tissue engineering[J].Expert Opin Biol Ther,2012,12(10):1361-1382
[2]Fernandes AM,Herlofsen SR,Karlsen TA,et al.Similar properties of chondrocytes from osteoarthritis joints and mesenchymal stem cells from healthy donors for tissue engineering of articular cartilage[J].PLoS One,2013,8(5):e62994
[3]Kon E,Filardo G,Roffi A,et al.New trends for knee cartilage regeneration:from cell-free scaffolds to mesenchymal stem cells[J].Curr Rev Musculoskelet Med,2012,5(3):236-243
[4]Madry H,Grün UW,Knutsen G.Cartilage repair and joint preservation:medical and surgical treatment options[J].Dtsch Arztebl Int,2011,108(40):669-677
[5]Siclari VA,Zhu J,Akiyama K,et al.Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow[J].Bone,2013,53(2):575-586
[6]胡鑫,尹小朋,龚忠诚,等.滑膜间充质干细胞与软骨细胞移植修复膝关节软骨缺损[J].中国组织工程研究,2017,21(9):1408-1413
[7]杨萌,邵博,龚忠诚,等.三维环境下软骨细胞诱导滑膜间充质干细胞向软骨样细胞的分化[J].中国组织工程研究,2016,20(11):1544-1550
[8]Afizah H,Hui JH.Mesenchymal stem cell therapy for osteoarthritis[J].J Clin Orthop Trauma,2016,7(3):177-182
[9]Wang Y,Yuan M,Guo QY,et al.Mesenchymal stem cells for treating articular cartilage defects and osteoarthritis[J].Cell Transplant,2015,24(9):1661-1678
[10]Mardones R,JofréCM,Minguell JJ.Cell therapy and tissue engineering approaches for cartilage repair and/or regeneration[J].Int J Stem Cells,2015,8(1):48-53
[11]邵博,龚忠诚,刘慧,等.大鼠来源滑膜间充质干细胞的成软骨分化[J].中国组织工程研究,2014,18(15):2338-2344
[12]Yang Z,Zhu L,Li F,et al.Bone marrow stromal cells as a therapeutic treatment for ischemic stroke[J].Neurosci Bull,2014,30(3):524-534
[13]Ye ZL,Hou XX,Chen RL,et al.Effects of methylthiouracil on the proliferation and apoptosis of rat bone marrow stromal cells[J].Exp Ther Med,2014,7(6):1738-1744
[14]Zheng L,Yang J,Fan H,et al.Material-induced chondrogenic differentiation of mesenchymal stem cells is material-dependent[J].Exp Ther Med,2014,7(5):1147-1150
[15]Strong AL,Jiang Q,Zhang Q,et al.Design,synthesis,and osteogenic activity of daidzein analogs on human mesenchymal stem cells[J].ACS Med Chem Lett,2013,5(2):143-148
[16]Caplan AI.Review:mesenchymal stem cells:cell-based reconstructive therapy in orthopedics[J].Tissue Eng,2005,11(7-8):1198-1211
[17]Chen FH,Tuan RS.Mesenchymal stem cells in arthritic diseases[J].Arthritis Res Ther,2008,10(5):223
[18]Al-Nbaheen M,Vishnubalaji R,Ali D,et al.Human stromal(mesenchymal)stem cells from bone marrow,adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential[J].Stem Cell Rev,2013,9(1):32-43
[19]Jaramillo-Ferrada PA,Wolvetang EJ,Cooper-White JJ.Differential mesengenic potential and expression of stem cell fate modulators in mesenchymal stromal cells from human-term placenta and bone marrow[J].J Cell Physiol,2012,227(9):3234-3242
[20]宋珂,石琦,青莹,等.大鼠骨髓间充质干细胞分离培养及鉴定的相关研究[J].临床口腔医学杂志,2015,31(9):531-536
[21]赵亚光,李毅,宋卓悦,等.人骨骼肌来源不同表面标志物间充质干细胞特定亚群的多向分化潜能[J].中国组织工程研究,2017,21(13):2108-2113
[22]陈路,夏先学,蒋科.骨髓间充质干细胞与壳聚糖复合修复关节软骨损伤[J].中国组织工程研究,2015,45(19):7254-7258
[2]Fernandes AM,Herlofsen SR,Karlsen TA,et al.Similar properties of chondrocytes from osteoarthritis joints and mesenchymal stem cells from healthy donors for tissue engineering of articular cartilage[J].PLoS One,2013,8(5):e62994
[3]Kon E,Filardo G,Roffi A,et al.New trends for knee cartilage regeneration:from cell-free scaffolds to mesenchymal stem cells[J].Curr Rev Musculoskelet Med,2012,5(3):236-243
[4]Madry H,Grün UW,Knutsen G.Cartilage repair and joint preservation:medical and surgical treatment options[J].Dtsch Arztebl Int,2011,108(40):669-677
[5]Siclari VA,Zhu J,Akiyama K,et al.Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow[J].Bone,2013,53(2):575-586
[6]胡鑫,尹小朋,龚忠诚,等.滑膜间充质干细胞与软骨细胞移植修复膝关节软骨缺损[J].中国组织工程研究,2017,21(9):1408-1413
[7]杨萌,邵博,龚忠诚,等.三维环境下软骨细胞诱导滑膜间充质干细胞向软骨样细胞的分化[J].中国组织工程研究,2016,20(11):1544-1550
[8]Afizah H,Hui JH.Mesenchymal stem cell therapy for osteoarthritis[J].J Clin Orthop Trauma,2016,7(3):177-182
[9]Wang Y,Yuan M,Guo QY,et al.Mesenchymal stem cells for treating articular cartilage defects and osteoarthritis[J].Cell Transplant,2015,24(9):1661-1678
[10]Mardones R,JofréCM,Minguell JJ.Cell therapy and tissue engineering approaches for cartilage repair and/or regeneration[J].Int J Stem Cells,2015,8(1):48-53
[11]邵博,龚忠诚,刘慧,等.大鼠来源滑膜间充质干细胞的成软骨分化[J].中国组织工程研究,2014,18(15):2338-2344
[12]Yang Z,Zhu L,Li F,et al.Bone marrow stromal cells as a therapeutic treatment for ischemic stroke[J].Neurosci Bull,2014,30(3):524-534
[13]Ye ZL,Hou XX,Chen RL,et al.Effects of methylthiouracil on the proliferation and apoptosis of rat bone marrow stromal cells[J].Exp Ther Med,2014,7(6):1738-1744
[14]Zheng L,Yang J,Fan H,et al.Material-induced chondrogenic differentiation of mesenchymal stem cells is material-dependent[J].Exp Ther Med,2014,7(5):1147-1150
[15]Strong AL,Jiang Q,Zhang Q,et al.Design,synthesis,and osteogenic activity of daidzein analogs on human mesenchymal stem cells[J].ACS Med Chem Lett,2013,5(2):143-148
[16]Caplan AI.Review:mesenchymal stem cells:cell-based reconstructive therapy in orthopedics[J].Tissue Eng,2005,11(7-8):1198-1211
[17]Chen FH,Tuan RS.Mesenchymal stem cells in arthritic diseases[J].Arthritis Res Ther,2008,10(5):223
[18]Al-Nbaheen M,Vishnubalaji R,Ali D,et al.Human stromal(mesenchymal)stem cells from bone marrow,adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential[J].Stem Cell Rev,2013,9(1):32-43
[19]Jaramillo-Ferrada PA,Wolvetang EJ,Cooper-White JJ.Differential mesengenic potential and expression of stem cell fate modulators in mesenchymal stromal cells from human-term placenta and bone marrow[J].J Cell Physiol,2012,227(9):3234-3242
[20]宋珂,石琦,青莹,等.大鼠骨髓间充质干细胞分离培养及鉴定的相关研究[J].临床口腔医学杂志,2015,31(9):531-536
[21]赵亚光,李毅,宋卓悦,等.人骨骼肌来源不同表面标志物间充质干细胞特定亚群的多向分化潜能[J].中国组织工程研究,2017,21(13):2108-2113
[22]陈路,夏先学,蒋科.骨髓间充质干细胞与壳聚糖复合修复关节软骨损伤[J].中国组织工程研究,2015,45(19):7254-7258