【作者】 刘晓玲；

【导师】 池岛乔；

【作者基本信息】 沈阳药科大学 ， 药理学， 2019， 博士

【摘要】 细胞外基质(Extracellular matrix,ECM)是细胞赖以生存的环境,不仅能够支持细胞结构,影响细胞的黏附,也可与细胞表面的受体结合并传导机械信号改变细胞的生物学行为。胶原是ECM的主要组分,广泛存在于多种组织细胞中,影响着细胞行为。在脂肪组织细胞外部基质中含量最多的组分是Ⅰ型胶原。但是Ⅰ型胶原是否影响脂肪组织细胞的生物学行为还未有考察。在本研究中,我们使用Ⅰ型胶原培养培养体系考察其对小鼠3T3-L1脂肪前体细胞增殖、迁移以及分化行为的影响,并在此基础上研究了药物水飞蓟宾的作用。细胞骨架染色的结果表明,与未包被组的大而扁平的形态相比,Ⅰ型胶原分子包被组的细胞表现出较长的成纤维状的形态。胶原促进细胞的增殖。细胞迁移实验的结果显示Ⅰ型胶原促进细胞迁移。进一步的机制研究表明,Ⅰ型胶原通过促进NF-κB p65的活化增强3T3-L1细胞的迁移。在体内,适度的细胞迁移有利于伤口的修复,但是过度的迁移易引起多种纤维化相关的疾病。水飞蓟宾是一种天然药物,对肝脏纤维化等疾病的治疗有一定作用。本实验室前期研究证明了水飞蓟宾能够抑制3T3-L1细胞的脂肪分化,但是其对细胞迁移的影响还未有研究。结果表明,Ⅰ型胶原降低了细胞对水飞蓟宾的敏感性,水飞蓟宾通过抑制NF-κB p65的活化逆转胶原对细胞迁移的促进作用。接下来,我们考察了水飞蓟宾对NF-κB p65活性调节的具体机制。在多种细胞中,NF-κB p65的活性受到NAD+依赖性的去乙酰化酶Sirt1的调节,Sirt1可使活化的NF-κB p65去乙酰化,降低其转录活性。在本研究中,水飞蓟宾通过促进Sirt1的表达,抑制NF-κB p65的核转录,以及其乙酰化和磷酸化修饰的水平。Sirt1可以直接NF-κB p65的乙酰化,但是它是如何影响其磷酸化修饰水平呢?实验室前期结果表明水飞蓟宾影响多种细胞中的自噬水平。细胞自噬在纤维化疾病中有着重要的调节作用。在本研究中,水飞蓟宾通过促进细胞内自噬的水平,逆转胶原对细胞迁移的增强作用。自噬降低了胶原对NF-κB p65磷酸化水平的调节,但是不影响其乙酰化水平。自噬对NF-κB p65的部分调节作用,使我们猜想Sirt1与自噬之间可能存在一定的联系。研究结果表明水飞蓟宾可以通过促进AMP依赖性激酶(AMP-activated protein kinase,AMPK)的磷酸化诱导自噬,并且这种作用受到Sirt1的调控。进一步研究发现,发现水飞蓟宾通过激活PPARα(过氧化物酶体增殖物激活受体α)来促进sirt1的表达,进而提高AMPK的磷酸化,增强细胞内的自噬水平。在脂肪组织形成时,脂肪前体细胞迁移到特定的组织部位,在此进行增殖并分化为成熟的脂肪细胞。在此过程中常常伴随着ECM组分的变化。但是目前还没有相关的研究考察ECM是否影响脂肪细胞的分化过程。因此,接下来我们考察了 Ⅰ型胶原对脂肪细胞分化过程是否存在着影响。YAP是Hippo信号通路的关键效应蛋白,能读取细胞外部的机械信号,并将这些信号转导至不同的生物学信号通路中,调节细胞的行为,尤其在细胞的分化与转分化方面发挥重要的作用。因此,接下来在研究细胞分化与转分化方面,我们考察了 YAP的作用。在体外通过激素刺激可诱导3T3-L1细胞分化成内含脂滴的脂肪细胞。Ⅰ型胶原分子包被能够在分化的不同时期,抑制甘油三酯脂滴的形成和脂肪生成因子的表达。并且,在细胞分化的过程中,细胞核内YAP的表达减少。同样,在高脂饲养小鼠的脂肪组织内,YAP的表达也是降低的。与之相反的是在Ⅰ型胶原包被的培养体系中,核内YAP的表达增加。这表明细胞的分化程度与YAP的表达密切相关。YAP的抑制剂维替泊芬以及YAP的小干扰RNA能够促进细胞分化。但是当细胞转染了 YAP质粒或YAP上游抑制性激酶LATS1的小干扰RNA时,其分化程度受到了抑制。Ⅰ型胶原通过促进YAP的入核,抑制细胞分化。在从小鼠皮下脂肪组织提取的原代脂肪前体细胞中,也发现了相同的结果。在伤口愈合的过程中,机械压力或转化生长因子-β1(Transforming growth factor-β1,TGF-β1)能诱导间质细胞转分化成a-SMA阳性的肌成纤维细胞,收缩伤口,引起ECM重构。过度的ECM重构与纤维化相关疾病密切相关。在体内,胶原多肽经螺旋、修饰形成胶原分子,胶原分子进一步组装成胶原纤维。明胶是胶原纤维的变性或者降解中间产物,在明胶酶的作用下,能被水解为胶原分子。ECM重构常伴随着胶原分子的组装,胶原纤维的降解和明胶的水解。3T3-L1细胞是一种间质来源的细胞,具有多能干细胞特性,可被诱导转分化成肌成纤维细胞。因此,为了研究ECM组分对于这种转分化影响我们分别采用了胶原分子包被、胶原凝胶(纤维)和明胶分子包被三种培养体系,考察其对3T3-L1细胞向肌成纤维细胞转分化的影响以及水飞蓟宾的作用。研究结果表明,三种形式的胶原培养均能够促进细胞转分化成a-SMA阳性的肌成纤维细胞,伴随着细胞内TGF-β1的合成与释放的增多。TGF-β1中和抗体的加入降低了 a-SMA的表达。这表明TGF-β1的激活促进了细胞的转分化。在这一过程中,胶原、明胶促进了 YAP的入核,YAP的活化促进了 TGF-β1的合成与释放。水飞蓟宾的加入减少了 YAP的入核,TGF-β1的释放,进而逆转了细胞的转分化。综上,Ⅰ型胶原通过促进NF-κB p65的活化增强细胞的迁移;Ⅰ型胶原通过促进YAP的入核,抑制细胞分化为脂肪细胞同时促进细胞向肌成纤维细胞的转分化。水飞蓟宾通过激活PPARα/Sirt1/自噬逆转细胞的迁移;通过抑制YAP的入核,减少细胞向肌成纤维细胞的转分化。这些结果提示我们,胶原在肥胖、纤维化等疾病的发生与发展中发挥重要作用,水飞蓟宾可能会对胶原的作用有反向的调节。更多还原

【Abstract】 Extracellular matrix(ECM)is very important for cell survival and function.ECM not only supports cell structure and influences the cell attachment but also changes cell behaviors by binding to receptors on cell surface for the transduction of mechanical signal.A the main component of ECM,Collagen,widely exists in many tissues,affecting cell behaviors.Collagen I(col Ⅰ)is one of the most abundant ECM components in adipose tissue.However,whether col Ⅰ exerts a direct influence on cell behaviors in adipose tissue remain to be elucidated.In this study,murine 3T3-L1 preadipocytes were incubated on col Ⅰ-cultured system to see whether col Ⅰ has influence on cell proliferation,migration and differentiation,and the effect of silibinin on that.3T3-L1 cells were cultured on collagen I(col Ⅰ,50 μg/ml)-coated dishes and the changes of the cell morphology,migration,proliferation and differentiation were examined.The staining of cell morphology study showed that compared with the large and flattened in cells on non-coated dishes,the cells cultured on col Ⅰ-coated dishes exhibited elongated and fibroblast-like ones.Col I promoted the proliferation of cells cultured on col Ⅰ-coated dishes.The results of cell migration assays showed that collagen I enhanced the cell migration.The detailed study on the mechanisms indicated that col Ⅰ enhanced the cell migration through the activation of NF-κB p65.In vivo,proper cell migration contributes to the wound healing;however,excessive cell migration often leads to fibrosis-related diseases.Silibinin,a natural flavonoid,has been used for the treatment of hepatic fibrosis.Previous studies show that silibinin inhibits the adipogenic differentiation,but little is known about its effect on cell migration.The results revealed that silibinin reversed the enhanced migration in cells cultured on col Ⅰ-coated dishes by the inhibition of NF-κB p65.Then we investigated the mechanisms underlying the regulation of silibinin on NF-kB p65 activity.In many kinds of cells,Sirt1,the NAD+-dependent deacetylase,inhibits the activation of NF-κB p65 by deacetylating and reduces its transcriptional activity.In this study,silibinin inhibited the nuclear translocation as well as the phosphorylation and acetylation of NF-κB p65 by up-regualting the expression of sirt1.Although Sirtl has a distinct inhibition on NF-κB p65 acetylation,the effect of Sirtl on NF-κB p65 phosphorylation remains to be elucidated.Previous studies show that silibinin influences autophagy levels in many kinds of cells.Autophagy plays an important role in fibrosis-related diseases.This study shows that silibinin reversed the enhanced migration of cells cultured on col Ⅰ-coated dishes through the increased level of autophagy.Autophagy decreased the level of NF-κB p65 phosphorylation,but had no effect on its acetylation.According to the partial effect of autophagy on NF-κB p65,we speculated that Sirtl might be related to autophagy induction.The results revealed that silibinin increased the level of AMPK phosphorylation to enhance autophagy,which was regulated by Sirtl.The further study indicated that silibinin promoted the autophagy level through the activation of PPARa that increased the expression of sirtl to enhance the phosphorylation of AMPK.Adipose tissue formation is initiated by migration of preadipocytes into different sites where they proliferate,before undergoing further differentiation into mature adipocytes.Adipogenesis is accompanied by the change of ECM component.Reports focused on whether ECM influences adipogenic differentiation is limited.Therefore,I further investigated the effect of col Ⅰ on the differentiation of preadipocytes.YAP,the key effector of Hippo pathway,plays an important role in cell behaviors,especially for cell differentiation and transdifferentiation through reading extracellular mechanical signals and transducing these signals to different biological pathways.Therefore,we investigated the role of YAP in the research of cell differentiation and transdifferentiation.In vitro,hormone stimulation induces 3T3-L1 cells differentiation into adipocytes containing lipids.Col I inhibited the formation of lipids containing triglyceride and the expression of adipogenic factors in different adipogenic phase.Furthermore,the nuclear translocation of YAP was decreased gradually in the adipogenic processes.Similarly,the expression of YAP was also reduced in the adipose tissue isolated from high-fat diet fed mice.Conversely,the nuclear translocation of YAP was increased in the cells cultured on col Ⅰ-coated dishes,indicating that adipogenic differentiation might be inversely related to YAP activation.The treatment of verteporfin,an inhibitor of YAP,and YAP small-interfering RNA enhanced adipogenic differentiation.But the transfection of YAP plasmid or the silence of LATS1,an inhibitory kinase of YAP,reduced the adipogenic differentiation.Col I inhibited the cell differentiation by promoting nuclear translocation of YAP.Essentially the same results were also found in the adipocyte precursor cells derived from subcutaneous adipose tissue.In wound healing,mechanical pressure or transforming growth factor-β1(TGF-β1)promotes mesenchymal cells to transdifferentiate into α-SMA positive myofibroblasts,contracting the wound and leading to ECM remodeling.The excessive ECM remodeling has close relationship with fibrosis-related disorders.In vivo,molecular collagen,composed of collagen peptides in a helical structure assembles fibrillar collagen.Gelatin,denatured or degraded product of collagen,can be hydrolyzed into molecular collagen by gelatinase.ECM remodeling is accompanied by the assembly of collagen molecules,the degradation of collagen fibers and the hydrolysis of gelatin.The 3T3-L1 cell is the cell line of mesenchymal origin with characteristics of pluripotent stem cells and can be induced to transdifferentiate into myofibroblasts.Therefore,in order to investigate the effect of ECM components and silibinin on cell transdifferentiation,we adopted three collagenforms in culture systems;molecular collagen,fibrillar collagen and gelatin.The results showed that all the three forms of collagen were able to induce 3T3-L1 cells to transdifferentiate into α-SMA positive myofibroblasts with the increased synthesis and release of TGF-β1.The treatment of neutralizing antibody against TGF-β1 reduced the expression of α-SMA.This indicated that the activation of TGF-β1 promoted the cell transdifferentiation.During this process,collagen and gelatin increased the nuclear translocation of YAP that promoted the synthesis and release of TGF-β1.The treatment of silibinin reversed the cell transdifferentiation by decreasing the nuclear translocation of YAP and the release of TGF-β1.In conclusion,col Ⅰ enhanced the cell migration by activating NF-κB p65;col Ⅰ inhibited the adipogenic differentiation and promoted myofibroblast transdifferentiation through the activation of YAP.Silibinin reversed the enhanced cell migration through the activation of PPARa/sirtl/autophagy and reduced myofibroblast transdifferentiation by down-regulating the nuclear translocation of YAP.The results imply that col Ⅰ plays a vital role in obesity-or fibrosis-related diseases,and silibinin might exert a negative regulation on the effect of col Ⅰ.更多还原