低强度脉冲超声波对牙周膜细胞成骨分化影响的初步研究
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摘要
hPDLCs是一组具有多向分化潜能的异质性多能干细胞,包含成纤维细胞、成骨细胞、成牙骨质细胞及未分化间充质细胞等多种细胞成分;它可分化为成骨细胞、成牙骨质细胞并形成牙槽骨、牙骨质及牙周膜,是牙周组织再生的细胞学基础,尤其对于牙周病治疗具有极其重要的研究价值。
牙周病治疗终极目的是牙周组织再生和形成新附着。促进牙周组织再生的关键之一是牙槽骨新生,即诱导hPDLCs成骨分化并行使功能。在牙周病损区域中,hPDLCs数量和功能活性都是有限的。如何促进该区域牙周膜细胞增殖及骨向分化对牙周病组织修复具有不可估量的研究价值。
近年来,LIPUS因其具有缩短骨折愈合时间、增加骨密度、改善力学特性,促使软骨骨化成熟等生物作用而被作为一种无创、安全、有效的治疗方法广泛用于骨折延迟愈合和骨不连等疾病治疗。
大量研究证实LIPUS作为一种机械信号,具备以下组织作用机制: 1.促进组织中具有增殖分化潜能的前体细胞增殖及成骨分化;2.增强功能细胞生物活性;3.改善血液循环。
因此,本研究首次将LIPUS引入诱导hPDLCs成骨分化以促进牙周组织再生治疗的研究具有重要意义。
目的:
1.建立hPDLCs的LIPUS体外辐照模型,为下一步研究LIPUS对hPDLCs成骨分化影响提供实验基础。
2.通过连续动态观察LIPUS对hPDLCs合成分泌碱性磷酸酶、骨钙素的影响,探讨LIPUS是否具有hPDLCs骨向诱导分化能力并遴选适宜参数。
3、以多个具有重要生理功能并可能在力学转导过程中处于调控枢纽地位的分子为切入点,研究LIPUS对hPDLCs integrinβ1、c-fos、c-jun、cbfa1mRNA表达的影响,为揭示LIPUS对hPDLCs成骨分化调控机制提供一些实验依据。
方法:
1、采用组织块法原代培养hPDLCs并传代建立有限细胞系,进行形态学观察、细胞组织学来源鉴定、绘制细胞生长曲线,为下一步LIPUS辐照提供实验细胞。
2、采用第4代hPDLCs消化后接种于6孔板中,LIPUS辐照参数为:工作频率1.5MHz;脉冲重复频率1.0KHz;脉冲占空比1:4,幅宽:200us;按输出强度90mW/cm2、60mW/cm2、30mW/cm2;同一声强辐照时间为10min,20min,30min分组,连续作用5天后检测各组ALP活性并遴选适宜的辐照参数用于后续试验。
3、采用第5代hPDLCs,按90mW/cm2-20min/d连续辐照15天,分别于5、7、9、11、13、15天时间点,采用生化法和放射免疫法检测碱性磷酸酶和骨钙素合成分泌情况。
4、取第5代hPDLCs,以90mW/cm2-20min/d LIPUS辐照处理5d,镜下见细胞汇合约90%,收集需要检测的细胞,采用RT-PCR检测integrinβ1、c-fos、c-jun、cbfa1的mRNA表达情况。
结果:
1、本实验培养的hPDLCs细胞形态、生长曲线均与文献报道相近,免疫组化证实所培养细胞来源于中胚层,结合取材部位说明本实验培养细胞为牙周膜细胞,可用于本实验研究
2、经LIPUS辐照后,各组ALP活性均有不同程度提高,其中90mW/cm2-20min/d组改变最为明显。
3、LIPUS连续辐照15天研究发现,细胞裂解液中的ALP于第5天开始增加,第11天达峰值,第13天下降较明显;分泌至上清液中的ALP于第9天开始增加,第11天达峰值,第13天时开始下降。同时,上清液中骨钙素于第9天显著增加,第15天达峰值。
4、以90mW/cm2-20min/d LIPUS辐照处理5d,引起integrinβ1、c-fos、c-jun、cbfa1mRNA表达不同程度上调。
结论:
1、LIPUS对hPDLCs骨向分化具有促进作用,本实验条件下较适宜作用参数为90mW/cm2-20min/d
2、LIPUS(90mW/cm2-20min/d)连续作用15d,牙周膜细胞ALP合成于第5天开始增加,第9天开始ALP向胞外分泌增加,第11天时合成及分泌同时达峰值,于第13天时均有下降。骨钙素作为成骨分化晚期标志蛋白,分泌于第9天显著增加,第15天达峰值,LIPUS诱导合成高峰稍晚于碱性磷酸酶。
3、LIPUS辐照下integrinβ1、c-fos、c-jun、cbfa1表达均有上调,提示上述基因可能参与了LIPUS诱导hPDLCs骨向分化的调控过程。推测其可能机制为:LIPUS以机械应力信号为主要形式作用于hPDLCs上,首先影响“细胞外基质-整合素-细胞骨架”力学信号转导系统,通过MAPK信号通路将物理信号传入核内,诱导即早基因c-fos、c-jun和成骨控制基因cbfa1转录翻译增加,可能进一步形成AP-1或与cbfa1相互调节影响成骨相关基因的表达,如碱性磷酸酶、骨钙素表达增加并发挥其生物学效应,从而实现LIPUS对hPDLCs的成骨分化诱导。
Purpose:The focus of this current study is to explore such effects on osteogenic differentiation of hPDLCs by LIPUS and potential regulatory mechanisms,to provide the experimental evidences for the promising prospects of LIPUS in periodontal tissue regeneration.
Method:LIPUS of different paramaters were used to sonication the cultured hPDLCs.Using Alkali phosphatase activity assay and radioimmunoassay,we studied the influence of LIPUS on ALP activity and osteocalcin synthesis by hPDLCs.We observed integrinβ1、c-fos、c-jun、cbfα1 mRNA expression by RT-PCR.
Results:
1.The cultured cells were typical hPDLCs,in accordance with literature reported,and were suitable for further experiment.
2.LIPUS can promote the activity of ALP and OC with different paramaters.The most optimal parameter of LIPUS is 90mW/cm2-20min/d.
3.LIPUS can upregulate the expression of integrinβ1、c-fos、c-jun、cbfa1 mRNA of hPDLCs.
Conclusion:
1.By increasing the synthesis and activity of osteogenic marker proteins as ALP、OC,LIPUS could promote the osteogenic differentiation of hPDLCs in vitro.
2.Integrinβ1、c-fos、c-jun、cbfα1 mRNA expression were upregulated by LIPUS,which suggest those gene may participate in the regulation of LIPUS on hPDLCs osteogenic differentiation.
Thus,we speculate,LIPUS act on hPDLCs in the form of mechanical stress,which influence the“ECM-integrin-cytoskeleton”mechanical signaling system at first,transmit the signal into nucleus by ERK1/2 and JNK pathway,induce tigger gene expression such as integrinβ1、c-fos、c-jun、cbfa1 that may play as transcription factor in the upregulation of ALP、OC,which can represent the function status of hPDLCs and server in the osteogenic differentiation.
牙周病治疗终极目的是牙周组织再生和形成新附着。促进牙周组织再生的关键之一是牙槽骨新生,即诱导hPDLCs成骨分化并行使功能。在牙周病损区域中,hPDLCs数量和功能活性都是有限的。如何促进该区域牙周膜细胞增殖及骨向分化对牙周病组织修复具有不可估量的研究价值。
近年来,LIPUS因其具有缩短骨折愈合时间、增加骨密度、改善力学特性,促使软骨骨化成熟等生物作用而被作为一种无创、安全、有效的治疗方法广泛用于骨折延迟愈合和骨不连等疾病治疗。
大量研究证实LIPUS作为一种机械信号,具备以下组织作用机制: 1.促进组织中具有增殖分化潜能的前体细胞增殖及成骨分化;2.增强功能细胞生物活性;3.改善血液循环。
因此,本研究首次将LIPUS引入诱导hPDLCs成骨分化以促进牙周组织再生治疗的研究具有重要意义。
目的:
1.建立hPDLCs的LIPUS体外辐照模型,为下一步研究LIPUS对hPDLCs成骨分化影响提供实验基础。
2.通过连续动态观察LIPUS对hPDLCs合成分泌碱性磷酸酶、骨钙素的影响,探讨LIPUS是否具有hPDLCs骨向诱导分化能力并遴选适宜参数。
3、以多个具有重要生理功能并可能在力学转导过程中处于调控枢纽地位的分子为切入点,研究LIPUS对hPDLCs integrinβ1、c-fos、c-jun、cbfa1mRNA表达的影响,为揭示LIPUS对hPDLCs成骨分化调控机制提供一些实验依据。
方法:
1、采用组织块法原代培养hPDLCs并传代建立有限细胞系,进行形态学观察、细胞组织学来源鉴定、绘制细胞生长曲线,为下一步LIPUS辐照提供实验细胞。
2、采用第4代hPDLCs消化后接种于6孔板中,LIPUS辐照参数为:工作频率1.5MHz;脉冲重复频率1.0KHz;脉冲占空比1:4,幅宽:200us;按输出强度90mW/cm2、60mW/cm2、30mW/cm2;同一声强辐照时间为10min,20min,30min分组,连续作用5天后检测各组ALP活性并遴选适宜的辐照参数用于后续试验。
3、采用第5代hPDLCs,按90mW/cm2-20min/d连续辐照15天,分别于5、7、9、11、13、15天时间点,采用生化法和放射免疫法检测碱性磷酸酶和骨钙素合成分泌情况。
4、取第5代hPDLCs,以90mW/cm2-20min/d LIPUS辐照处理5d,镜下见细胞汇合约90%,收集需要检测的细胞,采用RT-PCR检测integrinβ1、c-fos、c-jun、cbfa1的mRNA表达情况。
结果:
1、本实验培养的hPDLCs细胞形态、生长曲线均与文献报道相近,免疫组化证实所培养细胞来源于中胚层,结合取材部位说明本实验培养细胞为牙周膜细胞,可用于本实验研究
2、经LIPUS辐照后,各组ALP活性均有不同程度提高,其中90mW/cm2-20min/d组改变最为明显。
3、LIPUS连续辐照15天研究发现,细胞裂解液中的ALP于第5天开始增加,第11天达峰值,第13天下降较明显;分泌至上清液中的ALP于第9天开始增加,第11天达峰值,第13天时开始下降。同时,上清液中骨钙素于第9天显著增加,第15天达峰值。
4、以90mW/cm2-20min/d LIPUS辐照处理5d,引起integrinβ1、c-fos、c-jun、cbfa1mRNA表达不同程度上调。
结论:
1、LIPUS对hPDLCs骨向分化具有促进作用,本实验条件下较适宜作用参数为90mW/cm2-20min/d
2、LIPUS(90mW/cm2-20min/d)连续作用15d,牙周膜细胞ALP合成于第5天开始增加,第9天开始ALP向胞外分泌增加,第11天时合成及分泌同时达峰值,于第13天时均有下降。骨钙素作为成骨分化晚期标志蛋白,分泌于第9天显著增加,第15天达峰值,LIPUS诱导合成高峰稍晚于碱性磷酸酶。
3、LIPUS辐照下integrinβ1、c-fos、c-jun、cbfa1表达均有上调,提示上述基因可能参与了LIPUS诱导hPDLCs骨向分化的调控过程。推测其可能机制为:LIPUS以机械应力信号为主要形式作用于hPDLCs上,首先影响“细胞外基质-整合素-细胞骨架”力学信号转导系统,通过MAPK信号通路将物理信号传入核内,诱导即早基因c-fos、c-jun和成骨控制基因cbfa1转录翻译增加,可能进一步形成AP-1或与cbfa1相互调节影响成骨相关基因的表达,如碱性磷酸酶、骨钙素表达增加并发挥其生物学效应,从而实现LIPUS对hPDLCs的成骨分化诱导。
Purpose:The focus of this current study is to explore such effects on osteogenic differentiation of hPDLCs by LIPUS and potential regulatory mechanisms,to provide the experimental evidences for the promising prospects of LIPUS in periodontal tissue regeneration.
Method:LIPUS of different paramaters were used to sonication the cultured hPDLCs.Using Alkali phosphatase activity assay and radioimmunoassay,we studied the influence of LIPUS on ALP activity and osteocalcin synthesis by hPDLCs.We observed integrinβ1、c-fos、c-jun、cbfα1 mRNA expression by RT-PCR.
Results:
1.The cultured cells were typical hPDLCs,in accordance with literature reported,and were suitable for further experiment.
2.LIPUS can promote the activity of ALP and OC with different paramaters.The most optimal parameter of LIPUS is 90mW/cm2-20min/d.
3.LIPUS can upregulate the expression of integrinβ1、c-fos、c-jun、cbfa1 mRNA of hPDLCs.
Conclusion:
1.By increasing the synthesis and activity of osteogenic marker proteins as ALP、OC,LIPUS could promote the osteogenic differentiation of hPDLCs in vitro.
2.Integrinβ1、c-fos、c-jun、cbfα1 mRNA expression were upregulated by LIPUS,which suggest those gene may participate in the regulation of LIPUS on hPDLCs osteogenic differentiation.
Thus,we speculate,LIPUS act on hPDLCs in the form of mechanical stress,which influence the“ECM-integrin-cytoskeleton”mechanical signaling system at first,transmit the signal into nucleus by ERK1/2 and JNK pathway,induce tigger gene expression such as integrinβ1、c-fos、c-jun、cbfa1 that may play as transcription factor in the upregulation of ALP、OC,which can represent the function status of hPDLCs and server in the osteogenic differentiation.
引文
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