【作者】 刘立跃；

【导师】 王金福；

【作者基本信息】 浙江大学 ， 细胞生物学， 2011， 博士

【摘要】 人骨髓间充质干细胞(human mesenchymal stem cells, hMSCs)是存在于骨髓基质系统中的一种组织干细胞。由于其具有多向分化潜能和低免疫原性而被作为种子细胞广泛应用于组织工程,特别是骨组织工程。多种诱因可以诱导hMSCs向成骨细胞分化,如激素、机械力学刺激、细胞因子等。体内骨组织中的组织间隙液能对骨生成细胞产生流体剪切力(fluid shear stress, FS S),这种流体剪切力能促进骨生成细胞的增殖和分化,对于骨骼的生长发育和正常形态功能的维持非常重要。因此,人们在进行组织工程骨的研究过程中,利用生物反应器模拟体内环境产生FSS作用于体外培养的hMSCs,以此促进组织工程骨的生成。尽管已有研究证明FSS能促进hMSCs向成骨细胞分化。但是关于FSS促进hMSCc成骨分化的机理目前还尚不完全清楚。在本项研究中我们利用灌流培养体系产生FSS作用于接种在多孔隙PLGA-3D支架上的hMSCs,研究FSS对hMSCs分化的影响及相关信号转导机制。主要包括以下三个部分：第一部分：检测了4.2dynes/cm2间歇性FSS、4.2dynes/cm2持续性FSS和0.34dynes/cm2的FSS三种不同的FSS对hMSCs成骨分化的影响,并且检验了ERK1/2在FSS促进hMSCs成骨分化的过程中的作用以及三种不同FSS对ERK1/2活性的影响。利用RT-PCR检测不同培养条件下hMSCs内ALP、COLIα、OCN和Runx2等与成骨分化相关的特异基因的表达情况,采用改良钙钴染色和ALP活性检测试剂盒分别进行定性和定量分析不同FSS对hMSCs的ALP活性的影响。采用Western-blot检测ERK1/2的磷酸化水平。并且检测在用PD98059抑制ERK1/2活性的基础上,FSS对各成骨分化特异性基因的表达和ALP活性的影响。结果表明4.2dynes/cm2间歇性FSS能有效地促进hMSCs向成骨细胞分化,而4.2dynes/cm持续性FSS和0.34dynes/cm2的FSS对hMSCs没有明显的诱导分化作用,并且4.2dynes/cm2间歇性FSS较其他两种FSS能更有效地提高ERK1/2的活性。同时我们的实验还证明了ERK1/2在FSS促进hMSCs成骨分化的过程中起着重要的调节作用。第二部分：主要验证了β1整合素是否在FSS促进hMSCS成骨分化的过程中充当力学受体感受力学刺激以及FSS所引起的ERK1/2活化是否通过提高Runx2转录活性而调节与成骨分化相关基因的表达。采用western-blot检测在FSS作用下,hMSCs内ERK1/2、pi整合素和FAK等的活化情况或蛋白水平,利用免疫共沉淀检测了FSS对Runx2的酪氨酸磷酸化水平的影响,并且检测了在用小分子多肽RGDS特异性阻断β1整合素和胞外基质之间的连接后,FSS对FAK和ERK1/2的活性的影响。最后检测在用PD98059抑制ERK1/2活性后,FSS对Runx2的磷酸化水平和β1整合素的表达的影响。结果我们发现在FSS的作用下细胞内的ERK1/2、FAK和Runx2磷酸化水平显著升高,β1整合素的表达水平也显著升高。而用小分子多肽RGDS特异性阻断β1整合素后,FSS未能引起的ERK1/2和FAK的磷酸化水平升高。并且经PD98059处理后,FSS亦未能提高Runx2的活性和上调β1整合素的表达。以上结果表明在FSS促进hMSCs成骨分化的过程中,FSS所引起的ERK1/2和FAK活化依赖β1整合素感受力学刺激,活化的ERK1/2不仅能通过提高Runx2的转录活性而启动与成骨分化相关的基因表达,还能反馈调节β1整合素的表达。第三部分：在第一、二部分研究中,我们发现ERK1/2对因FSS引起的Runx2和p1整合素表达升高具有重要的调节作用,但是ERK1/2是通过那些信号途径来调节其表达的呢?目前还尚无相关报道。由于BMPs/Smadl/5/8信号通路是调节Runx2表达的重要信号通路,而NFkB已被证明是调节BMP2、BMP4和β1整合素表达的重要转录因子。我们猜测FSS引起的ERK1/2活化可能通过提高NFkB转录活性而上调节β1整合素和BMP2、BMP4表达,再通过活化BMPs/Smadl/5/8信号通路而调节Runx2的表达。为了验证以上猜测,我们分别采用RT-PCR和Western-blot检测了FSS对hMSCs内的BMP2和BMP4表达以及对Smadl/5/8活性的影响,并且在用人重组noggin蛋白阻断BMPs/Smad1/5/8信号通路后检测FSS对Runx2表达和Smad1/5/8活性的影响。其次用Western-blot和免疫荧光染色分别检测了FSS对NFkB活性和核转移的影响,并且检测在用BAY11-7082抑制NFkB核转移后,FSS对BMP2、BMP4、Runx2和β1整合素表达的影响以及对Smad1/5/8活性的影响。最后采用PD98059抑制ERK1/2活性后检测FSS对NFkB和Smad1/5/8活性的影响以及对BMP2和BMP4表达的影响。结果发现FSS能使hMSCs内的BMP2和BMP4的mRNA水平和Smad1/5/8活性显著升高,经noggin处理后,FSS未能使hMSCs内的Runx2表达水平和Smadl/5/8的升高。此外,FSS能使p65NFkB的活性和核转移率显著升高,而抑制NFkB核转移后,FSS所引起的β1整合素、Runx2、BMP2和BMP4表达以及Smadl/5/8的磷酸化也受到抑制。PD98059不仅能抑制FSS所导致的p65NFkB活化和核转移还能抑制FSS所引起的BMP2和BMP4的表达以及Smad1/5/8活化。以上结果表明在FSS影响hMSCs成骨分化的信号转导机制中,ERK1/2能通过调节NFkB的转录活性而影响β1整合素和BMPs的表达,BMPs进而影响Smadl/5/8的活化,调节Runx2的表达,ERK1/2在该信号转导机制中起着非常关键的调节作用。本项研究不仅验证了间歇性FSS较持续性FSS能有效地促进hMSCs向成骨细胞分化,而且证明了在FSS促进hMSCs成骨分化的信号转导机制中,D1整合素感受FSS所产生的力学刺激进而引起ERK1/2活化,活化的ERK1/2既能通过提高Runx2的转录活性而启动与成骨分化相关基因的表达,又能通过调节NFkB活性调节β1整合素和BMPs的表达,再通过BMPs/Smad信号通路调节Runx2的表达。本研究为阐明FSS促进hMSCs成骨分化的信号转导机制提供了重要信息,对于骨组织工程的发展具有重要的指导意义。更多还原

【Abstract】 Human mesenchymal stem cells (hMSCs) are tissue stem cells that reside in bone marrow microenvironment and have been widely used in studies of tissue engineering because of their multiple differentiation potent and low immunogenicity, especially bone tissue engineering. Various factors can induce the osteogenic differentiation of hMSCs, such as hormones, cytokines and mechanical stimulation. Since bone is a porous tissue, mechanical loading can lead to the deformation of bone, induce strain gradients as well as local pressure gradients, and accordingly lead to the flow of marrow which causes fluid shear stress (FSS) loading on cells residing in bone marrow including MSCs. FSS is very important to the growth and homeostasis of bone and can promote the proliferation and osteogenic differentiation of preosteoblasts. Therefore, biomaterials and bioreactors are used to imitate the micro-structure of bone and produce FSS exerting on hMSCs to promote the formation of engineering bone tissue. Although it has been proved that FSS can induce hMSCs to differentiate into osteoblasts, the mechano-transduction of FSS in hMSCs remains largely unknown.In this study we investigated the effects of different FSS on the osteogenic differentiation of hMSCs and the mechanism of FSS promoting the ostoegenic differentiation using perfusion culture system and PLGA-3D scaffolds. Firstly, we examined the effects of intermittent FSS at4.2dynes/cm2, continuous FSS at4.2 dynes/cm2and low FSS at0.34dynes/cm2on the osteogenic differentiation of hMSCs. The phosphorylaton levell of ERK1/2was also measured to investigate the effects of different FSS application manners on the activation of signaling molecules using Western-blot. RT-PCR was used to measure the mRNA level of ALP, Runx2, COLIa and OCN. The ALP activity was examined with ALP measurement kit and Modified Gomori Staining. The results showed that intermittent FSS was favored to the osteogenesis of hMSCs compared with continuous FSS and low FSS. The expression level of osteogenic genes and the ALP activity in hMSCs cultured under intermittent FSS application were significantly higher than those in hMSCs cultured under continuous FSS and low FSS application. Moreover, intermittent FSS could more markedly up-regulated the activation of ERK1/2in hMSCs than continuous FSS and low FSS.Secondly, it was investigated whether β1integrins acted as mechano-receptors in FSS-induced osteogenic differentiation of hMSCs to sense the stimulation of FSS and activate ERK1/2by activating FAK and the effect of ERK1/2on the activation of Runx2was also measured. Western-blot was used to evaluate the phosphorylation level or protein level of ERK1/2, FAK and β1integrins. And immunoprecipitation was performed to detect the phospho-Runx2level. To access the roles of ERK1/2and β1integrins in the FSS-induced osteogenic differentiation of hMSCs, PD98059and RGDS peptide were used to individually block ERK1/2pathway and β1integrin pathway. The results showed that FSS enhanced significantly the phosphorylation of ERK1/2, Runx2and FAK. And FSS-induced activation of ERK1/2and FAK was inhibited by the blockade of the connection between β1integrins and ECM with RGDS peptide. Additionally, our study also found that FSS could up-regulate the expression level of β1integrins, and this up-regulation could be abolished by PD98059. Therefore, it could be inferred that β1integrins should detect the stimulation of FSS and thus activate ERK1/2through activating of FAK, and the FSS-activated ERK1/2feed back to up-regulated the expression of β1integrins.Finally, it was estimated how FSS-activated ERK1/2modulated the expression of β1integrins and Runx2as it was proved that FSS-induced up-regulation of β1integrins and Runx2was dependent on the activation of ERK1/2in the previous two sections of this study. It is s widely accepted that BMPs/Smad pathway modulates the expression of Runx2and NFkB also acts as important transcription factor of BMP2, BMP4and β1integrins. We suspect that FSS-activated ERK1/2should mediate the expression of BMP2, BMP4and β1integrins by modulating the activation of NFkB, and high expression of BMP2and BMP4would consequently lead to the activation of BMPs/Smad1/5/8pathway to initiate the expression of Runx2. To verify above suspicion, a series of works were done in this study:determining whether FSS could increase the expression of BMP2and BMP4and activate Smad1,5and8; examining the effect of FSS on the nuclear translocation of p65NF-kB and the activation of p65 and IKBa; using PD98059to inhibit ERK1/2activation to determine whether ERKl/2had effect on FSS-induced activation of NF-kB and Smadl/5/8; determining whether BMPs/Smad pathway was essential to FSS-induced upregulation of Runx2by the inhibition of human recombinant noggin to BMPs; testing the requirement of NF-kB for FSS-induced up-regulation of Runx2and integrin β1by blockade of NF-kB nuclear translocation with BAY11-7082. The results of our study showed that BMPs/Smad pathway was involved in the mechanotransduction of FSS in hMSCs and was critical to FSS-induced upregulation of Runx2. The activation of NF-kB p65increased significantly with FSS application and was mediated by ERKl/2. Furthermore, blockade of the activation of NF-kB inhibited FSS-induced up-regulation of β1integrins, Runx2and BMPs. Such, our data supports an opinion that ERKl/2mediates the expression of β1integrins by modulating the activation of NFkB and regulates the expression of Runx2via crosstalking with BMPs/Smad pathway by mediating the activation of NFkB.In summary, our study has not only confirmed that intermittent FSS could more effectively induce the osteogenic differentiation of hMSCs than continuous FSS but also demonstrated that two novel important signaling pathways involved in the mechanotransduction of FSS in hMSCs, combined with the classical signaling pathway, formed a moecular signaling network to enhance osteogenesis of hMSCs: First, β1integrins act as an important mechanoreceptor to sense the stimulation of extracelular FSS and in turn activate ERK1/2by activating FAK. The activated ERK1/2leads to the phosphorylation of Runx2, and the phosphorylated Runx2initiates the transcription of osteogenic genes to promote hMSCs to differentiate into osteoblasts. Second, the FSS-activated ERK1/2increases the expression of BMPs via activating NFkB, the increased BMPs results in the activation of BMPs/Smad pathway and finally leads to the expression of Runx2. Third, the FSS-activated ERK1/2influences the expression of integrin β1by mediating the activation of NFkB. Our study has provided better understanding how FSS promotes the osteogenic differentiation of hMSCs through a molecular signaling network and important information to elucidate the mechanism of FSS mechanotransduction in hMSCs. The understanding on the mechanism of FSS inducing the osteogenic differentiation of hMSCs will not only be helpful to develop the bone tissue engineering but also provide new targets for drug discovery for treatment of osteoporosis and related bone-wasting diseases.更多还原