【作者】 薛茹月；

【导师】 杨力； Shu Chien；

【作者基本信息】 重庆大学 ， 生物医学工程， 2013， 博士

【摘要】 骨髓间充质干细胞（Mesenchymal stem cells, MSCs）作为成体干细胞中最重要的一种，是一类具有多向分化潜能（Multiple differentiation potentiality）的细胞，能够向神经细胞，肌细胞，成骨细胞，软骨细胞以及脂肪细胞等方向分化。因其多向分化的能力MSCs已成为组织再生、修复的重要种子细胞来源。目前的再生医学研究认为MSCs在心肌细胞受损后、脊髓损伤、骨损伤等组织修复和再生工程中发挥重要作用。然而，组织修复与再生研究中所用的生物材料的物理特性及细胞周围微环境，尤其是基底硬度对MSCs定向分化方面的作用机理，至今仍不明了。在组织修复与再生研究中，MSCs的定向诱导分化尤为重要，研究证明在复杂的组织微环境中，影响MSCs分化的因素不仅包括被广泛研究的生物化学因素，还包括传导力学信号的生物物理因素。不同的基底材料硬度（Matrix stiffness）和接种的细胞密度（Cell density）是否会调节MSCs分化以及如何影响其分化尚不清楚。本文着重研究基质硬度对MSCs向成骨和软骨分化的影响，在此基础上探讨不同细胞密度对其分化的影响，继而研究哪条信号通路在基底硬度诱导MSCs向成骨分化过程中起主要调控作用。本文的主要研究工作和结果如下：1. MSCs细胞形态和增殖对基底硬度及细胞密度的响应通过改变丙烯酰胺（Acrylamide）和甲叉双丙烯酰胺（Bis-acrylamide）的比例，使材料物理硬度控制在约0.5至100千帕之间。软基底上MSCs贴壁面积仅为硬基底上的40%且应力纤维少而弱。细胞在软硬凝胶上以低密度（1,000个/cm2）和高密度（20,000个/cm2）培养，结果显示，在低密度培养时，软基底显著抑制MSCs的增殖速度，且在软基底上P-Rb, P-P70显著降低，P27显著升高；高密度培养时，软硬基底对细胞增殖造成的差异消失。以上实验说明细胞增殖与基底硬度有关，但在高密度培养时，基底硬度对细胞增殖的影响消失，软硬基底上的细胞增殖没有显著差异。2.基底硬度和细胞密度对MSCs成骨和软骨分化的影响成骨分化的标记基因Alkaline Phosphatase (ALP), Type I collagen, alpha1(Col1α1)，Runt-related transcription factor2(Runx2)在硬基底（Hard,40±3.6kPa）上mRNA水平明显升高，表明单纯的物理刺激即基底硬度就能促进MSCs的成骨分化,在骨诱导培养液中也同样得到以上实验结果；另外软骨分化的标记基因SOX-9(SRY-related high mobility group-box gene9)，Aggrecan，Collagen II和Collagen X的mRNA水平在软基底（1.6±0.3kPa）上显著升高，SOX9的蛋白表达在软基底上明显升高，Alcian blue染色和Collagen II免疫荧光染色结果均显示软基底更有利于软骨分化。对成骨分化而言，高密度（20,000个/cm2）培养时成骨分化的标记基因ALP、Col1α1和Runx2的mRNA水平在软硬基底上无明显差异；而软骨分化则不受细胞密度的影响，无论高（20,000个/cm2）、低密度（1,000个/cm2）培养MSCs，SOX-9，Aggrecan和Collagen X的mRNA水平均在软基底上明显升高。本实验结果表明在组织工程应用中，低密度和硬基底培养有利于促进MSCs向成骨分化；软基底培养则有利于促进软骨分化，且此过程与细胞密度无关。3.不同硬度基底上RhoA和Rho-kinase (ROCK)及微管抑制剂对MSCs的影响通过对MSCs转染RhoAV14(constitutively active form of RhoA with GST tag)、加入Rho激酶特异性抑制剂Y-27632及微管抑制剂Paclitaxel，观察MSCs细胞形态变化、细胞增殖及细胞活性和成骨标记基因的表达。结果显示，转染RhoAV14刺激ERK磷酸化增强，促进MSCs增殖且上调软基底上成骨标记基因的表达。Y-27632影响细胞粘附，F-actin由中间均匀分布变为中间荧光微弱，仅有边缘荧光，同时Y-27632降低成骨分化标记基因的表达，上调软基底上神经元分化标记β3tubulin(TUBβ3)基因的表达。Paclitaxel作用后软硬基底上细胞增殖差异消失；细胞活性检测发现软基底上的细胞凋亡明显多于硬基底；检测Paclitaxel作用后成骨标记基因Runx2和神经分化标记基因TUBβ3，发现软基底上的Runx2明显受抑制，而硬基底上的Runx2抑制作用没有显著差异，TUBβ3加药组与对照组没有明显差异。综上，我们认为软硬基底调节的细胞分化过程中存在细胞骨架如微丝、微管以及RhoA和ROCK的参与。4. Smad1/5/8，ERK和AKT的活化通路参与硬基底促进的MSCs成骨分化过程为研究基底硬基底诱导MSCs向成骨分化的调节机制，对MSCs成骨分化调节相关信号因子进行了检测，发现硬基底在促进MSCs成骨分化的过程中激活Smad1/5/8，ERK，AKT蛋白的磷酸化，抑制P-38蛋白的磷酸化。而软基底促进软骨分化的过程中未检测到Smad1/5/8，AKT/ERK的磷酸化差异。分析以上结果推测Smad1/5/8、ERK和AKT的磷酸化在基底硬度调节的MSCs成骨分化中起关键作用，而软骨分化则是利用其他不同于成骨分化的通路。进一步利用重组腺病毒RasV12和RasN17分别外源性的激活和抑制ERK/AKT的磷酸化。结果RasN17能明显抑制ERK/AKT和Smad1/5/8的磷酸化；而RasV12只能刺激ERK的磷酸化，对AKT和Smad1/5/8的磷酸化作用不明显。腺病毒感染后的结果显示RasN17能显著抑制成骨分化的标志基因ALP，Col1α1和Runx2的mRNA水平。相反RasV12虽然能激活ERK磷酸化却并没有促进成骨分化的作用。提示基底硬度决定的细胞成骨分化机理除Ras-Smad1/5/8和ERK/AKT这条通路外应该还存在其他通路参与。综上所述，本文利用可调节力学特性的聚丙烯酰胺水凝胶模型培养MSCs，通过接种在不同的硬度基底上以及接种密度差异对比研究MSCs的成骨和软骨分化，发现细胞骨架、RhoA-ROCK参与其中，以及硬基底决定的成骨分化中关键信号通路Ras-Smad1/5/8/ERK/AKT。实验表明MSCs成骨及软骨分化方向的不同依赖于基底硬度和细胞密度对其的影响。进一步说明跟分化方向特异性相关的细胞外微环境因素有利于控制干细胞分化方向，从而可以调控细胞向需要的方向分化，为MSCs在组织工程和再生医学中的应用提供理论依据。更多还原

【Abstract】 Bone marrow-derived mesenchymal stem cells (BMSCs) are an importantmultipotent adult stem cells with the potential to differentiate into a variety of cell types,including neural cells, vascular smooth muscle cells (SMCs), osteoblasts, chondrocytesand adipocytes. Therefore, MSCs are an important cell source for tissue repair andregeneration. Currently, rehabilitation study shows that MSCs are valuable inmyocardial damage repair and regeneration; they also can directionally differentiate intonerve cells in spinal injury regeneration and new bone production after bone injury.However, it is not yet well understood how the directional differentiation of MSCsrespond to the physical characteristics of biomaterial and microenvironment aroundcells, especially matrix elasticity.The differentiation of MSCs is especially important in tissue repair andregeneration. There is evidence that the tissue microenvironment includes not only thewidely studied biochemical cues, but also the biophysical factors that deliver signals forMSC differentiation. It is not clear whether and how matrix elasticity and cell seedingdensity collectively regulate MSCs. In this study, we investigated the collective effectsof substrate elasticity and cell seeding densities on ostogenesis and chondrogenesis. Inaddition, we studied the underlying mechanism during osteogenesis induced by matrixelasticity. The main experiments and results are as follows:1. Morphology and proliferation of MSCs on different matrix elasticity with differentcell densitiesTo investigate the role of matrix elasticity in the proliferation and differentiation ofMSCs, we utilize tunable elasticity polyacrylamide hydrogels which can be manipulatedby adjusting the relative concentrations of acrylamide and bis-acrylamide. The physicalelasticities can be controlled between0.5kPa and100kPa. We cultured MSCs on softmatrix(1.6±0.3kPa for5%/0.05%acryl/bis) and hard matrix(40±3.6kPa for10%/0.5%acryl/bis), following by the observation of cell morphology and spreading area. Theresults showed that cells cultured on soft matrix have restricted spreading area, which isonly approximately40%of that on hard matrix24hours after culture. To determine theeffects of cell density and matrix elasticity on hMSC proliferation, the cell cycledistribution was determined. We also detected the cell cycle positive and negativeregulatory factor Phospho-Rb, P-P70S6kinase and P-27. The result showed that soft matrix with low cell density inhibited MSCs’ proliferation, and obviously decreased theexpression of P-Rb, P-P70and up-regulated p-27. However, both soft and hard matricescaused significant decreases cell percentage in the synthetic phase in high-density.These results suggest that the effect of matrix elasticity on hMSCs proliferation dependson cell density and that high cell density was able to override theproliferation-promotion effect of hard matrix.2. Effects of matrix elasticity and cell density on osteogenesis and chondrogenesisTo determine the role of matrix elasticity on MSCs differentiation, we culturedMSCs on soft and hard hydrogel in growth medium. The result showed that hard matrixincreased the expression of osteogenic marker genes including Alkaline Phosphatase(ALP)，Type I collagen, alpha1(Col1α1) and runt-related transcription factor2(Runx2).This demonstrated that hard matrix promote osteogenesis of MSCs, in both inductionand growth medium. In addition, we found that chondrogenesis was promoted on softmatrix(1.6±0.3kPa). Western blotting confirmed that SOX-9was significantly higher onsoft matrix. Alcian blue staining and collagen II immunostaining also confirmed theresult, the mRNA expressions of SOX-9, ACAN, Collagen II and Collagen X weresignificantly higher on soft matrix. There are studies indicating that cell densityinfluence MSC differentiation, so we detected the MSCs differentiation with diversecell density on soft and hard hydrogels. At high cell density (20,000/cm2), the cellsshowed no significant difference in osteogenic marker expressions between hard andsoft matrices; matrix elasticity affects chondrogenic process at both high and low cellseeding density (1,000/cm2). This finding illustrated that low cell density and hardmatrix are benefit for osteogenesis, however, matrix rigidity affects chondrogenicprocess at both high and low cell seeding density.3. The role of RhoA/ROCK and Paclitaxel in MSCs proliferation and differentiationon matrixTo observe the MSCs morphology, proliferation and marker genes expressionvariation, we utilized RhoAV14(constitutively active form of RhoA with GST tag), Rhokinase inhibitor Y-27632and microtubule inhibitor Paclitaxel. RhoAV14transfectionup regulated ERK phosphorylation, MSCs proliferation and osteogenic marker genesexpression on soft matrix. Y-27632inhibited osteogenic marker genes expression andup regulated neurocyte marker gene β3tubulin expression, F-actin fluorescenceintensity from averaged at each fractional distance across the cell to a preferential distribution in the peripheral cortical layer. The MSCs’ proliferation difference wasdisappeared and the cell viability test showed that cell apoptosis on soft matrix wasmore than those on hard matrix with Paclitaxel treatment. Runx2mRNA expression wassignificantly down regulated on soft matrix, not on hard matrix. There were nosignificantly difference of TUBβ3mRNA expression between control and Paclitaxeltreatment. Based on above, the cell differentiation process which regulated by substrateelasticity exist cytoskeleton like microfilament, microtubule, RhoA and ROCKparticipation.4. Hard matrix induced Smad1/5/8, ERK, and AKT activation in osteogenesisTo further investigate the matrix elasticity influence on MSCs differentiation, wefound that the phosphorylation of Smad1/5/8, ERK and AKT were up regulated, whileP-38was down regulated on hard matrix, in contrast, our results showed that ERK andAKT activations were not affected by matrix elasticity during chondrogenesis,suggesting that the phosphorylation of Smad1/5/8、ERK and AKT may play importantrole in osteogenesis induced by matrix elasticity, other signal networks may be involvedin chondrogenesis. To further test the roles of ERK phosphorylation in matrix-regulatedosteogenesis, recombinant adenoviruses with constitutive active Ras (RasV12) anddominant negative Ras (RasN17) were used to modulate the signaling cascades.RasN17markedly inhibited the phosphorylation of Smad1/5/8, ERK, and AKT, RasV12up-regulated ERK activation, but had little effect on Smad1/5/8and AKTphosphorylations. The expressions of osteogenic markers, such as Runx2, Col1α1andALP mRNA, were significantly down-regulated after RasN17infection. However,RasV12did not cause any up-regulation of osteogenic markers. These results suggestthat there should be other signals besides Ras-Smad1/5/8/ERK/AKT involved inosteogenesis which induced by matrix elasticity.In summary, this study used hydrogel model which physical elasticity can becontrolled to culture the MSCs, to investigate the MSCs osteogenesis andchondrogenesis on soft and hard hydrogel, as well as the underlying signal transductionin osteogenesis. We found that cell cytoskeleton, RhoA-ROCK paticipate in MSCsdifferentiation and the phosphorylation of Smad1/5/8、ERK and AKT are involved inosteogenesis which determined by matrix elasticity. Our results demonstrated that thecollective effects of matrix elasticity and cell seeding density on MSCs differentiationare lineage dependent. Elucidation of the roles of lineage-specific extracellular cues will improve the control of stem cell fate to generate the desired cell types for potentialtherapeutic usage.更多还原