动态压力作用下体外海藻酸盐微囊化培养兔关节软骨细胞差异蛋白质组学研究
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摘要
应力作用下软骨细胞代谢和功能的改变是导致软骨组织不断生长改建的主要原因。在正常人体关节软骨所受到的各种力中,压力是最主要的,研究表明,软骨受到动态和静态压缩载荷影响了软骨移植物的生物合成。但到目前为止,有关动态压力作用下调控软骨细胞生物学行为变化的分子机制尚不清楚,通过差异蛋白质组学研究可以识别参与细胞生命活动过程中的重要蛋白质,为深入探索其机制提供信息。本研究力图构建类似于体内软骨组织的体外软骨细胞立体培养动态压力加载模型,并借助于蛋白质组学技术,寻找和发现动态压力作用下调控软骨细胞代谢和功能改变的特定的蛋白质分子,为动态压力作用下软骨改建机理的更深入研究提供依据。
研究方法:1)取4W龄乳兔膝关节软骨组织,体外分离、培养软骨细胞、对培养的软骨细胞进行蕃红“O”、甲苯胺蓝及II型胶原免疫组化染色等表型鉴定;2)将鉴定具有正常表型的P1代软骨细胞和海藻酸盐复合,形成凝胶小球培养,观察藻酸钙凝胶内软骨细胞生长情况。通过MTT检测确定细胞活性,使用二甲基亚甲兰法测定样品硫酸化糖胺多糖(GAG)的含量,同时对在藻酸钙凝胶内的软骨细胞进行组织学及组织化学染色以鉴定其表型;3)选取浓度分别为1%、2%、3%海藻酸盐,并通过INSTRON3365测定三种浓度海藻酸盐形成凝胶的机械刚度,将P1代软骨细胞分别与这三种浓度的海藻酸钙复合,培养28天,通过MTT法,二甲基亚甲兰法及RT-PCR测定并比较三种条件下软骨细胞增殖状况,GAG含量变化及II型胶原,聚集蛋白聚糖,I型胶原的基因表达状况从而筛选出最有利于软骨细胞生长的3D海藻酸盐凝胶培养环境;4)仿照Shram系统构建了体外立体培养的软骨细胞动态压力加载模型;并参照Shram等压力加载条件给予0.2MPa,0.66Hz静水压持续作用4h,提取培养相同时间的加压组和未加压组藻酸钙凝胶中的软骨细胞总蛋白,进行双向电泳分离,电泳后的凝胶银染,用分析软件对图谱进行分析,从而建立动态压力作用下藻酸盐立体培养条件下兔关节软骨细胞差异表达蛋白质图谱;5)使用基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)对得到的肽段进行分析,并结合蛋白质序列数据库,鉴定出差异表达的蛋白点。
结果:采用消化法进行幼兔膝关节软骨细胞原代培养,经甲苯胺兰,II型胶原染色鉴定阳性,所培养细胞生长状态良好其软骨细胞表形能够保持3代稳定,且反映其生长增殖能力的生长曲线正常,在体外培养时可经历与在体状况相似的成熟过程,并具有与在体的关节软骨相似的生物学活性。2)在海藻酸钙小球内培养的软骨细胞能够保持球形状态,且随时间的延长,细胞逐渐增殖,GAG含量增多。经蕃红“O”、甲苯胺蓝及II型胶原免疫组化染色均为阳性。鉴定证实为具有正常表型的软骨细胞。3)浓度对海藻酸钙凝胶的机械刚度具有显著的影响。1%的海藻酸钙凝胶的压缩模量为19.44±2.72显著低于2%(167.09±12.43)和3%(226.24±19.82)。在培养的21天,软骨细胞在较软凝胶内增殖较快,但在21天后,在最软的凝胶内细胞增殖显著减少;基质积累随海藻酸盐刚度改变而不同。在培养第四周,软骨细胞在中间刚度组表现出最高的GAG合成,而软骨特征性基因CoLII、Agg的表达在具有中等刚度的凝胶内最稳定;4)通过双向电泳技术我们建立了动态压力作用下立体培养的软骨细胞差异表达蛋白质图谱。未加压组检测到1632±54个蛋白点,加压组检测到1698±13个蛋白点。通过软件分析,找到了10个差异蛋白点,其中2个在加压培养后表达消失;2个在加压培养后出现表达;6个表达增强。5)将这10个蛋白点通过质谱(MALDI-TOF-MS)分析,获得相应的肽质量指纹图谱,进一步通过蛋白质序列数据库鉴定了8个蛋白点,其中有意义的蛋白点6个。它们分别是:prolyl 4-hydroxylase alpha I,pyruvate kinase,L-lactate dehydrogenase A,Prolyl 4-hydroxylase subunit beta,destrin isoform,alpha enolase。
结论:1)采用酶消化法获得幼兔关节软骨细胞的方法简便可行。体外培养的原代或P1,P2代软骨细胞具有良好的软骨细胞表型特征,适用于实验研究。2)软骨细胞在藻酸钙凝胶小球中生长维持了球状形态,细胞少量增殖并合成GAG,II型胶原,说明海藻酸钙凝胶适合于软骨细胞的生长。3)具有中等刚度的海藻酸钙凝胶更有利于软骨细胞的生长和软骨表型的维持。4)利用差异蛋白质组学方法研究动态压力作用对立体培养的软骨细胞的影响是可行的;5)通过差异蛋白质组学研究我们发现了动态压力作用下调控软骨细胞生物学行为的6个有意义的关键蛋白质,并推测了其在软骨改建中的作用。为今后研究这些差异蛋白在软骨细胞受压力刺激后代谢和功能变化的生物学作用奠定了基础。
Mechanical stresses are known to play important role on articular cartilage functions in vivo and also on cartilage explants and chondrocytes monolayer culture. However,the molecular events of chondrocytes in respose to dynamic stress are still not well understood so far.Differential proteomics can be applied to evaluate the significant proteins in cellular metabolism, which would aid in better understanding of the molecular mechanism of cartilage remolding under dynamic loading.Therefore, in the present study, we tried to set up an in vitro 3D loading model system similar to native joint cartilage and found out the major proteins involved in the chondrocytes cultured in alginate beads under dynamic stress loading by using comparative proteomics technology .
Methods:1) rabbit chondrocytes were isolated from the articular cartilage of a 4-week-old rabbit by enzyme digestion method , cultured in DMEM supplemented with 10% FBS.the chondrocytes were identified with Toluidine blue and immunohistochemistry of collagen type II staining;2)P1 chondrocytes suspended in calcicum alginate beads were observed dynamically under phrase contrast microscope,the vability and proliferation of cells were evaluated by MTT,The quantity of GAG was determined by a modification of the dimethyl-methylene blue method,histological and immunohistochemistry staining were employed to evaluate cell morphology, matrix deposition and the presence of cartilage specific type II collagen. 3) alginate hydrogels ranging in stiffness were formed via varying the alginate solids concentration from 1% to 3%, The compressive modulus of each condition were characterized using INSTRON 3365,The articular chondrocyte were encapsulated in each hydrogel condition, and growth, morphology,and gene expression were assessed; 4) A dynamic compression unit was set up followed the method of Sharam,chondrocytes cultured in 2% alginate beads were exposed to 0.2 MPa,0.66Hz cyclic loadings for 4h,the total proteins extracted from either loading or unloadind cells were separated by two-dimensional gel eletrophoresis(2-DE).5)PMF was analyzed using Matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),the differentially expressed proteins involved in chondrocytes cultured in alginate beads under dynamic stress loading were identified with protein and pertide databases.
Results:
1) The chondrocytes obtained here showed favorable growth and function character. The phenotype expression of chondrocytes could be kept steadily for three passages. The growth curve which could reflect the proliferation ability of the cells was obtained.in vitro cultured chondrocytes could also experienced the same mature process as they growed in vivo and had the similiar biological activity as those cells in vivo.
2) chondrocytes cultured in alginate mantained the spherical morphology. The number of viability cells and glycosaminoglycan(GAG) production increased with time .Histological examination reveal chondrocytes retained their differentiation state in alginate beads.
3) The contents of the alginate had a significant influence on compressive mechanical properties. 1% alginate conditions had significantly lower moduli(19.44±2.72) than the 2%(167.09±12.43) and 3%(226.24±19.82) alginate conditions. chondrocytes were proliferating more rapidly on lower stiffness gels(1%,2%) than on the higher stiffness gels(3%). After day 21, no further cell proliferation, but a slight reduction in cell number, especially, on the softest gel,the reduction is most obvious.Matrix accumulation also varied significantly with type of alginate used, At week 4, the 2% group resulted in the highest GAGs production , the expression of cartilage-specific genes, such as type II collagen and aggrecan ,in the middle stinffness alginate constructs showed the highest level expression;
4)two-dimensional gel eletrophoresis showed significant differences between the dynamic stress group and unloading group. There are 1698±13 protein spots in the loading group and 1632±54 in the unloading group. 10 proteins were diferentially expressed by analyzing with soft ware compared with unloading group,among them, 2 proteins were new appearance, 2 proteins disappeared,6 proteins were up-regulated.
5)Among the 10 diferentially expressed proteins,the PMFs of 8 proteins were obtained through MALDI-TOF-MS analysis.Fouthermore ,by searching in protein and pertide databases,six meaningfull proteins were identified,they were:prolyl 4-hydroxylase alpha I, pyruvate kinase,L-lactate dehydrogenase A,Prolyl 4-hydroxylase subunit beta,destrin isoform,alpha enolase.
Conclusions :
1) The method used in the present work for isolation and culture of chondocytes is simple and feasible.The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the P0, P1, P2 passages,which is suitable for most experiments.
2) Alginate induced chondrocytes cluster-like growth and increased the deposition of GAG as well as phenotype stability,which indicated that alginate is favored for chondrocytes growth.
3) The physical properties of alginate gel influence the embedded cells bio-behave,alginate with the middle stiffness led to the highest GAG synthesize and the most stable phonotype maintaining.
4) it’s feasible to investigate the influence of dynamic compression on chondrocytes through the differential proteomics approach.
5) Our research provided fundamental information on the differential expression of protein of chondrocytes cultured in alginate under dynamic stress. however, futher studies need to be done to better understand the molecular mechanisms of cartilage remodeling induced by dynamic stress loading.
研究方法:1)取4W龄乳兔膝关节软骨组织,体外分离、培养软骨细胞、对培养的软骨细胞进行蕃红“O”、甲苯胺蓝及II型胶原免疫组化染色等表型鉴定;2)将鉴定具有正常表型的P1代软骨细胞和海藻酸盐复合,形成凝胶小球培养,观察藻酸钙凝胶内软骨细胞生长情况。通过MTT检测确定细胞活性,使用二甲基亚甲兰法测定样品硫酸化糖胺多糖(GAG)的含量,同时对在藻酸钙凝胶内的软骨细胞进行组织学及组织化学染色以鉴定其表型;3)选取浓度分别为1%、2%、3%海藻酸盐,并通过INSTRON3365测定三种浓度海藻酸盐形成凝胶的机械刚度,将P1代软骨细胞分别与这三种浓度的海藻酸钙复合,培养28天,通过MTT法,二甲基亚甲兰法及RT-PCR测定并比较三种条件下软骨细胞增殖状况,GAG含量变化及II型胶原,聚集蛋白聚糖,I型胶原的基因表达状况从而筛选出最有利于软骨细胞生长的3D海藻酸盐凝胶培养环境;4)仿照Shram系统构建了体外立体培养的软骨细胞动态压力加载模型;并参照Shram等压力加载条件给予0.2MPa,0.66Hz静水压持续作用4h,提取培养相同时间的加压组和未加压组藻酸钙凝胶中的软骨细胞总蛋白,进行双向电泳分离,电泳后的凝胶银染,用分析软件对图谱进行分析,从而建立动态压力作用下藻酸盐立体培养条件下兔关节软骨细胞差异表达蛋白质图谱;5)使用基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)对得到的肽段进行分析,并结合蛋白质序列数据库,鉴定出差异表达的蛋白点。
结果:采用消化法进行幼兔膝关节软骨细胞原代培养,经甲苯胺兰,II型胶原染色鉴定阳性,所培养细胞生长状态良好其软骨细胞表形能够保持3代稳定,且反映其生长增殖能力的生长曲线正常,在体外培养时可经历与在体状况相似的成熟过程,并具有与在体的关节软骨相似的生物学活性。2)在海藻酸钙小球内培养的软骨细胞能够保持球形状态,且随时间的延长,细胞逐渐增殖,GAG含量增多。经蕃红“O”、甲苯胺蓝及II型胶原免疫组化染色均为阳性。鉴定证实为具有正常表型的软骨细胞。3)浓度对海藻酸钙凝胶的机械刚度具有显著的影响。1%的海藻酸钙凝胶的压缩模量为19.44±2.72显著低于2%(167.09±12.43)和3%(226.24±19.82)。在培养的21天,软骨细胞在较软凝胶内增殖较快,但在21天后,在最软的凝胶内细胞增殖显著减少;基质积累随海藻酸盐刚度改变而不同。在培养第四周,软骨细胞在中间刚度组表现出最高的GAG合成,而软骨特征性基因CoLII、Agg的表达在具有中等刚度的凝胶内最稳定;4)通过双向电泳技术我们建立了动态压力作用下立体培养的软骨细胞差异表达蛋白质图谱。未加压组检测到1632±54个蛋白点,加压组检测到1698±13个蛋白点。通过软件分析,找到了10个差异蛋白点,其中2个在加压培养后表达消失;2个在加压培养后出现表达;6个表达增强。5)将这10个蛋白点通过质谱(MALDI-TOF-MS)分析,获得相应的肽质量指纹图谱,进一步通过蛋白质序列数据库鉴定了8个蛋白点,其中有意义的蛋白点6个。它们分别是:prolyl 4-hydroxylase alpha I,pyruvate kinase,L-lactate dehydrogenase A,Prolyl 4-hydroxylase subunit beta,destrin isoform,alpha enolase。
结论:1)采用酶消化法获得幼兔关节软骨细胞的方法简便可行。体外培养的原代或P1,P2代软骨细胞具有良好的软骨细胞表型特征,适用于实验研究。2)软骨细胞在藻酸钙凝胶小球中生长维持了球状形态,细胞少量增殖并合成GAG,II型胶原,说明海藻酸钙凝胶适合于软骨细胞的生长。3)具有中等刚度的海藻酸钙凝胶更有利于软骨细胞的生长和软骨表型的维持。4)利用差异蛋白质组学方法研究动态压力作用对立体培养的软骨细胞的影响是可行的;5)通过差异蛋白质组学研究我们发现了动态压力作用下调控软骨细胞生物学行为的6个有意义的关键蛋白质,并推测了其在软骨改建中的作用。为今后研究这些差异蛋白在软骨细胞受压力刺激后代谢和功能变化的生物学作用奠定了基础。
Mechanical stresses are known to play important role on articular cartilage functions in vivo and also on cartilage explants and chondrocytes monolayer culture. However,the molecular events of chondrocytes in respose to dynamic stress are still not well understood so far.Differential proteomics can be applied to evaluate the significant proteins in cellular metabolism, which would aid in better understanding of the molecular mechanism of cartilage remolding under dynamic loading.Therefore, in the present study, we tried to set up an in vitro 3D loading model system similar to native joint cartilage and found out the major proteins involved in the chondrocytes cultured in alginate beads under dynamic stress loading by using comparative proteomics technology .
Methods:1) rabbit chondrocytes were isolated from the articular cartilage of a 4-week-old rabbit by enzyme digestion method , cultured in DMEM supplemented with 10% FBS.the chondrocytes were identified with Toluidine blue and immunohistochemistry of collagen type II staining;2)P1 chondrocytes suspended in calcicum alginate beads were observed dynamically under phrase contrast microscope,the vability and proliferation of cells were evaluated by MTT,The quantity of GAG was determined by a modification of the dimethyl-methylene blue method,histological and immunohistochemistry staining were employed to evaluate cell morphology, matrix deposition and the presence of cartilage specific type II collagen. 3) alginate hydrogels ranging in stiffness were formed via varying the alginate solids concentration from 1% to 3%, The compressive modulus of each condition were characterized using INSTRON 3365,The articular chondrocyte were encapsulated in each hydrogel condition, and growth, morphology,and gene expression were assessed; 4) A dynamic compression unit was set up followed the method of Sharam,chondrocytes cultured in 2% alginate beads were exposed to 0.2 MPa,0.66Hz cyclic loadings for 4h,the total proteins extracted from either loading or unloadind cells were separated by two-dimensional gel eletrophoresis(2-DE).5)PMF was analyzed using Matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),the differentially expressed proteins involved in chondrocytes cultured in alginate beads under dynamic stress loading were identified with protein and pertide databases.
Results:
1) The chondrocytes obtained here showed favorable growth and function character. The phenotype expression of chondrocytes could be kept steadily for three passages. The growth curve which could reflect the proliferation ability of the cells was obtained.in vitro cultured chondrocytes could also experienced the same mature process as they growed in vivo and had the similiar biological activity as those cells in vivo.
2) chondrocytes cultured in alginate mantained the spherical morphology. The number of viability cells and glycosaminoglycan(GAG) production increased with time .Histological examination reveal chondrocytes retained their differentiation state in alginate beads.
3) The contents of the alginate had a significant influence on compressive mechanical properties. 1% alginate conditions had significantly lower moduli(19.44±2.72) than the 2%(167.09±12.43) and 3%(226.24±19.82) alginate conditions. chondrocytes were proliferating more rapidly on lower stiffness gels(1%,2%) than on the higher stiffness gels(3%). After day 21, no further cell proliferation, but a slight reduction in cell number, especially, on the softest gel,the reduction is most obvious.Matrix accumulation also varied significantly with type of alginate used, At week 4, the 2% group resulted in the highest GAGs production , the expression of cartilage-specific genes, such as type II collagen and aggrecan ,in the middle stinffness alginate constructs showed the highest level expression;
4)two-dimensional gel eletrophoresis showed significant differences between the dynamic stress group and unloading group. There are 1698±13 protein spots in the loading group and 1632±54 in the unloading group. 10 proteins were diferentially expressed by analyzing with soft ware compared with unloading group,among them, 2 proteins were new appearance, 2 proteins disappeared,6 proteins were up-regulated.
5)Among the 10 diferentially expressed proteins,the PMFs of 8 proteins were obtained through MALDI-TOF-MS analysis.Fouthermore ,by searching in protein and pertide databases,six meaningfull proteins were identified,they were:prolyl 4-hydroxylase alpha I, pyruvate kinase,L-lactate dehydrogenase A,Prolyl 4-hydroxylase subunit beta,destrin isoform,alpha enolase.
Conclusions :
1) The method used in the present work for isolation and culture of chondocytes is simple and feasible.The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the P0, P1, P2 passages,which is suitable for most experiments.
2) Alginate induced chondrocytes cluster-like growth and increased the deposition of GAG as well as phenotype stability,which indicated that alginate is favored for chondrocytes growth.
3) The physical properties of alginate gel influence the embedded cells bio-behave,alginate with the middle stiffness led to the highest GAG synthesize and the most stable phonotype maintaining.
4) it’s feasible to investigate the influence of dynamic compression on chondrocytes through the differential proteomics approach.
5) Our research provided fundamental information on the differential expression of protein of chondrocytes cultured in alginate under dynamic stress. however, futher studies need to be done to better understand the molecular mechanisms of cartilage remodeling induced by dynamic stress loading.
引文
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