【作者】 王晓；

【导师】 姜笃银；

【作者基本信息】 山东大学 ， 外科学， 2019， 博士

【摘要】 间充质干细胞(Mesenchymal stem cells,MSCs)具有强大的自我更新能力和多向分化潜能,在再生医学领域具有广阔的发展前景。目前,骨髓MSCs、脂肪MSCs和脐带MSCs的功能及作用机制得到了较为广泛的研究,并且已逐步应用到了临床。同成人MSCs相比,胎儿的MSCs具有以下特点:不仅增殖能力更高、多向分化潜能更强,而且免疫原性更低。这些优势使得胎儿MSCs引起广泛的关注。胎儿真皮MSCs(Fetal dermal MSCs,FDMSCs)提取自孕中期胎儿真皮组织,是介导胎儿皮肤无瘢痕愈合最主要的细胞,在皮肤组织的修复再生领域具有潜在的优势。近年来,MSCs的旁分泌作用引起了学者们的广泛关注和深入研究,许多研究成果和临床数据表明MSCs主要通过旁分泌机制发挥作用。通过旁分泌作用,MSCs向细胞外释放生长因子、细胞因子、炎性介质、外泌体等一系列生物活性物质。其中,外泌体介导的信号传递是MSCs发挥作用的主要机制之一。外泌体是直径30-150nm的圆形小囊泡,携带多种生物活性物质,一般包括蛋白质、核酸、脂质,由脂质双分子层包裹,被细胞分泌至细胞外基质中,通过向靶细胞传递这些物质介导细胞间的通讯。外泌体通过改变靶细胞的蛋白质和基因表达,调控细胞增殖、细胞侵袭、血管生成、免疫反应等过程,参与机体的各种生理病理过程。外泌体还可作为疾病的特异性生物标志物,用于多种疾病的诊断、作为运输载体靶向传递药物。在组织修复过程中,MSCs来源的外泌体发挥着同MSCs相似的作用:促进修复、促进内源性再生、促进血管生成、调节免疫反应等。然而MSCs的外泌体促进皮肤创面愈合的具体分子机制还不清楚,亟待进一步探究。我们推测,外泌体作为FDMSCs发挥其修复作用的重要媒介,能够促进皮肤创面愈合。为了验证这一假说,我们提取了 FDMSCs的外泌体(FDMSC-derived exosome,FDMSC-Ex)并将其应用于小鼠皮肤损伤模型,发现FDMSC-Ex能够显著加快小鼠创面的愈合速度;为了进一步探究FDMSC-Ex促进创面愈合的分子机制,我们将FDMSC-Ex应用于成人真皮成纤维细胞(Adult dermal fibroblasts,ADFs),检测ADFs的增殖、迁移、合成细胞外基质(Extracellular matrix,ECM)的能力及相应信号通路的改变。FDMSC-Ex孵育后,ADFs的增殖、迁移和ECM蛋白合成能力增强,同时发现Notch通路被激活;应用抑制剂DAPT阻断Notch通路后,ADFs的增殖和迁移能力显著下降。为了进一步探索Notch通路激活的原因,我们检测了 FDMSC-Ex中Notch通路5种配体蛋白(Jagged 1、Jagged 2、Dll-1、Dll-3、D11-4)的表达情况,发现FDMSC-Ex中只表达Jagged 1而不表达其他四种配体蛋白。为了验证Jagged 1在FDMSC-Ex介导的促进愈合功能中的作用,使用Jagged 1多肽模拟Jagged 1对Notch通路的激活作用,使用siRNA敲除Jagged 1在FDMSC-Ex中的表达,结果证实,Jagged 1是FDMSC-Ex激活ADFs Notch通路的关键因子,介导FDMSC-Ex发挥促进愈合的作用,由此阐明FDMSC-Ex促进皮肤组织创面愈合的分子机制。本研究探索了 FDMSC-Ex促进创面愈合的分子机制,分析MSCs外泌体的临床应用前景,为今后FDMSC-Ex用于临床难愈性创面、大面积烧伤、皮肤缺损等创面的治疗提供了理论基础。研究目的提取FDMSC-Ex,研究FDMSC-Ex对皮肤创面愈合和ADFs功能的影响,阐明FDMSC-Ex促进皮肤创面愈合作用的分子机制。研究方法1.提取FDMSC-Ex。从人胎儿皮肤组织提取FDMSCs并进行多向分化潜能、细胞表面标记物的检测;从FDMSCs的条件培养基中提取FDMSC-Ex,通过透射电镜观察形态并测量直径、通过Western blot检测其标记物(Tsgl01、Alix和CD63)、并进行了外泌体粒径分析。2.动物实验。构建小鼠背部10mm×10mm皮肤全层创面模型,术后即刻在创面周围分4个点共注射200μl 1mg/ml的FDMSC-Ex。分别于术后第7、14天观察皮肤创面愈合情况,拍照,并取创周皮肤标本进行组织学检测。3.FDMSC-Ex摄取实验。使用PKH26标记FDMSC-Ex,然后将标记好的FDMSC-Ex加入ADFs中孵育24小时,DAPI染核后使用荧光显微镜观察。4.ADFs 增殖实验。将 FDMSC-Ex 按照 1 μg/ml、10μg/ml、1OOμg/ml 的浓度梯度加入ADFs中进行孵育,24小时后使用CCK-8的方法检测细胞的增殖能力。5.ADFs迁移实验。使用划痕实验和transwell实验方法,向ADFs中加入1μg/ml、10μg/ml、100μg/ml的FDMSC-Ex干预细胞。孵育24小时后,显微镜下测量单层细胞机械划痕两侧的宽度(划痕实验);transwell实验则在细胞固定、结晶紫染色后去除小室内层细胞后,计数穿过小室膜到达小室外侧的细胞数目,检测ADFs的迁移能力。6.ADFs合成ECM蛋白能力的检测。使用RT-PCR检测FDMSC-Ex孵育后ADFs合成ECM蛋白(Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、弹性蛋白、纤维粘连蛋白、α-平滑肌肌动蛋白)的能力。7.Notch信号通路的检测。使用Western blot检测Notch通路中活性Notchl、Jagged 1和下游蛋白Hes 1的表达量改变;为了进一步验证Notch通路的作用,使用抑制剂DAPT阻断Notch通路后,再次检测ADFs的增殖和迁移。检测FDMSC-Ex中Notch通路配体蛋白表达情况,分析引起Notch通路活性改变的原因。8.Jagged 1作用的检测。使用Jagged 1多肽模拟Jagged 1对Notch通路的激活作用,使用siRNA敲除Jagged 1在FDMSC-Ex中的表达,检测Jagged 1在Notch通路激活和FDMSC-Ex促进创面愈合中的作用。研究结果1.成功提取并鉴定FDMSC-Ex。2.FDMSC-Ex促进小鼠背部皮肤创面愈合。3.FDMSC-Ex 能被 ADFs 摄取。4.FDMSC-Ex增强ADFs的增殖、迁移、合成ECM的能力。5.FDMSC-Ex通过激活Notch信号通路提高ADFs促进创面愈合的能力。阻断Notch通路后,ADFs的增殖与迁移能力显著降低。6.FDMSC-Ex中的Jagged 1蛋白激活Notch通路促进创面愈合。结论1.FDMSC-Ex促进小鼠背部皮肤创面愈合。2.FDMSC-Ex增强ADFs的增殖、迁移、合成ECM的能力。3.FDMSC-Ex通过激活Notch信号通路提高ADFs创面愈合的能力。阻断Notch通路后,FDMSC-Ex促进ADFs增殖与迁移的能力显著降低。4.FDMSC-Ex中的Jagged 1蛋白能够激活Notch通路,提高ADFs的增殖、迁移能力,在促进创面愈合中起到重要作用。更多还原

【Abstract】 Mesenchymal stem cells(MSCs)have a significant promise for regenerative medicine for the self-renewal and multipotent capacity.MSCs can be isolated from bone marrow,adipose tissue,umbilical cord tissue.Fetal MSCs are a new potential source of MSCs.Compared with adult MSCs,fetal MSCs exhibit lower immunogenicity,higher proliferation and differentiation potential.FDMSCs are derived from accidental aborted fetus,and they are thought as the main functional cells involved in scarless wound healing.Furthermore,owing to the histological origin of FDMSCs,they may deserve unique properties on skin regeneration.In summary,FDMSCs are better candidates than adult MSCs in wound healing.Recent literature suggests that the regenerative effect of MSCs is mainly mediated through paracrine signaling to regulate recipient cells.MSCs are high secretive capacity cells which can secrete lots of biologically active paracrine molecules including cytokines,chemokines,growth factors and exosomes.Accumulating evidences showed that the tissue repairing effect of MSCs is mediated by exosomes.In the last decades,researchers have shown increased interests in exosomes.Exosomes are 30-150nm small membranous vesicles secreted by most cell types.There are nuclear acids,lipids,and proteins in exosomes,and their main function is to transfer bioactive molecular in cell-cell communication.Exosomes participate in multiple physiological and pathological processes by regulating cell migration,invasion,gene expression,angiogenesis and immune response.Moreover,recent studies have shown the role of exosomes in disease diagnosis and drug delivery.Exosomes derived from MSCs(FDMSC-Ex)are involved in the acceleration of wound healing by activating endogenous repairing process,regulating inflammation,promoting angiogenesis and altering the target cell status.However,the molecular mechanism of promoting wound healing by MSC exosomes is still not clear.Therefore,we used a promising MSCs type,FDMSCs,to investigate the effect of their exosomes on cutaneous wound healing in vivo.Then we applied the FDMSC-Ex on adult dermal fibroblasts in vitro,and analyzed the proliferation,migration and secreting capacity of adult dermal fibroblasts.Then the Notch signal pathway associated to wound healing process was detected to illustrate the underlying mechanism of the FDMSC-Ex in promoting wound healing.We found that Notch signaling is activated after incubating with FDMCS-Ex;and in FDMSC-Ex,the ligands of the Notch pathway were undetectable except for Jagged 1,and the results of Jagged 1 mimic by peptide and knockdown by siRNA suggested that Jagged 1 may lead the activation of the Notch signal in ADFs.Our results suggest that FDMSC-Ex can provide a useful tool to design treatment strategies for unhealed wound and large area burn wound.Research purposesTo isolate FDMSC-Ex and investigate the molecular mechanism of the wound-healing promoting effect of FDMSC-Ex.Research methods1)Isolation and identification of FDMSC-Ex.FDMSC-Ex were isolated using ExoQuick-TC kit(SBI,USA)following the instruction.The morphology of FDMSC-Ex was identified by transmission electron microscope and the marker protein(CD63,Alix,and Tsg101)and the diameter of FDMSC-Ex were detected.2)Animal assay.Full thickness 10mm×10mm dermal wounds were created in the skin on the back of the mouse.The wounds were treated with 200μl FDMSC-Ex(1mg/ml)at the edge of the wound.The wounds were evaluated at day 7/14 after surgery and the wound tissues were collected for histopathological study.3)Exosome labeling.Exosomes were labeled with PKH26 and incubated with ADFs for 24 hours.Then the cells were observed by fluorescence microscope after cell nucleus dyeing by DAPI.4)Cell proliferation.After incubating with FDMSC-Ex,CCK-8 assay was performed to evaluate the AFDs proliferation.5)Cell migration.After incubating with FDMSC-Ex,the migration of ADFs was observed by wound healing and transwell assay.6)ECM protein synthesis.The ECM protein synthesis ability was evaluated by RT-PCR after incubating with FDMSC-Ex.7)Notch signal pathway.The protein expression levels of Notch signal were evaluated by Western blot.To identify the function of Notch pathway in the wound healing process,Notch was blocked by the specific inhibitor,DAPT,and then the migration and proliferation of ADFs were detected again.8)The biological function analysis of Jagged 1 in FDMSC-Ex.Jagged 1 mimic by peptide and knockdown by siRNA were performed to detect the functional role of Jagged 1 in FDMSC-Ex.Research results1.FDMSC-Ex were isolated and identified.2.FDMSC-Ex can promote cutaneous wound healing in vivo.3.FDMSC-Ex internalization by ADFs.4.FDMSC-Ex enhanced proliferation,migration,and ECM synthesis capacity of ADFs.5.FDMSC-Ex activated Notch signaling pathway and DAPT can partly block the promoting effect of FDMSC-Ex on ADFs proliferation and migration.6.Jagged 1 in FDMSC-Ex can promote wound healing by activating Notch signaling.Conclusion1.FDMSC-Ex can promote cutaneous wound healing in vivo.2.FDMSC-Ex enhanced proliferation,migration,and ECM synthesis capacity of ADFs.3.FDMSC-Ex activated Notch signaling pathway and DAPT can partly block the promoting effect of FDMSC-Ex on ADFs proliferation and migration.4.Jagged 1 in FDMSC-Ex can activate Notch signaling,enhance proliferation and migration of ADFs to promote wound healing.更多还原