MSC与成纤维细胞的增殖及分化在创面愈合中的作用及其机制研究

发布时间：2018-05-22 17:18

本文选题：MSC + 三维悬浮培养　；参考：《第三军医大学》2015年博士论文

【摘要】：背景:皮肤是人体的防御器官,皮肤的完整性对于机体抵抗病原微生物具有重要意义(1-3)。烧伤、创伤病人往往伴有皮肤组织的损伤,皮肤完整性的破坏导致皮肤屏障的功能破坏,是增加机体受病原微生物感染的重要原因之一(4-6),促进皮肤创面的愈合对于防止病情恶化、预防感染的发生及改善预后都有积极的意义(7、8)。有研究发现,烧伤、创伤等外科意外发生后,创伤皮肤的表面会聚集大量MSC,在创面微环境各种炎性因子的诱导下向成纤维细胞和表皮细胞进行分化,通过一系列细胞进程来修复创面(9-13)。在烧伤患者特别是大面积烧伤患者,由于大面积皮肤缺损的出现,后期感染甚至脓毒血症发生的几率大大增加,早期促进创面的愈合对于预防感染的发生和改善预后十分重要(13-17)。近年来皮肤干细胞移植治疗的发展为加速创面的愈合开辟了一条前景光明的道路,但自身干细胞的来源有限,一定程度上又限制了皮肤干细胞移植治疗的发展(16-19)。如何能够在体外快速大量的扩增MSC,为皮肤干细胞移植的发展提供帮助,也成为了科研工作者的一个研究方向(20-22)。此外皮肤真皮纤维细胞以及表皮角质细胞在皮肤创面的愈合过程中作用也不容忽视,因此本文分为两部分分别针对如何为干细胞移植治疗提供“原料”和具备“原料”后如何“加工“完成创面的修复进行研究,为促进创面的愈合给予理论和实验依据。方法:第一部分我们以MSC为研究对象,采用三维悬浮(RCCS)模式对细胞进行诱导分化,然后通过CCK8实验、流式细胞仪观测细胞周期,观察MSC增殖能力的变化,同时WB法对其“干性”和“成骨分化”分化相关基因和细胞表面标志进行检测判断MSC分化情况,最后我们采用生化检测和稳定同位素氨基酸标记技术研究细胞的代谢和蛋白质组学的变化,并探讨RCCS环境对MSC增殖与分化调控的分子机制。第二部分我们将成纤维细胞以及角质细胞选作研究对象,采用机械拉伸模拟皮肤表面张力的变化,观察张力变化对成纤维细胞增殖及迁移的影响及其与表皮细胞间的相互作用关系,探讨其分子机制及其在皮肤创面愈合中发挥的作用。结果:第一部分研究发现,与对照组相比三维悬浮培养模式下CCK8和细胞周期检测结果均显示MSC增殖增强,流式细胞仪检测细胞表面标志及WB检测发现其“干性”相关基因表达增强,“成骨分化”相关基因表达减弱,MSC分化受到抑制。进一步生化检测细胞代谢情况发现,三维悬浮培养的MSC细胞内、外乳酸含量增加,LDH活性增加,葡萄糖消耗增加。SILAC检测结果显示细胞线粒体表达的基因减弱,糖酵解基因表达增强,细胞能量代谢处于有氧氧化减弱,糖酵解增强的能量代谢失衡状态。第二部分研究发现与无牵拉和静态牵拉作用力组相比,在周期性机械牵拉力下,细胞增殖曲线及细胞周期结果均显示HF的增殖受到抑制。WB检测HF-ECM发现,周期性机械牵拉力导致HF的ECM中I型胶原显著降低、而纤维连接蛋白明显增加。将表皮细胞(Ha Ca T)接种于周期性牵拉力作用后的HF-ECM上,表皮细胞的增殖仍然受到抑制。将表皮细胞接种于经过I型胶原或纤维连接蛋白包被的基质上,分别检测细胞的增殖曲线、细胞周期和细胞迁移能力,结果发现I型胶原比纤连蛋白更能促进细胞的增殖,而在影响细胞迁移的能力上却不如纤连蛋白。用免疫共沉淀、哺乳动物双杂交和基因干扰的方法研究发现,周期性机械牵拉力诱导的抑制细胞增殖作用与intergrinβ-CASK信号通路有关。结论:第一部分研究发现三维悬浮培养模式可以在体外快速大量扩增MSC,这种MSC具有高“干性”低“分化”特性,可能与三维悬浮模式能够诱导细胞处于有氧呼吸减弱,糖酵解增强的能量代谢状态有关。该法是简单有效的体外扩增MSC的手段,可以用于皮肤创面干细胞移植的研究。第二部分研究表明皮肤表面张力的改变可以调节HF-ECM成分,通过“人纤维细胞-ECM-角质细胞”之间的“细胞对话”影响表皮细胞的增殖以及迁移,从而加速创面持续愈合。本研究在一定程度上阐明了成纤维细胞和表皮细胞在创面修复中的作用及其机制,为皮肤创面愈合的的进一步发展提供了理论基础和实验依据。
[Abstract]:Background: skin is the defense organ of the human body. Skin integrity is of great significance to the body's resistance to pathogenic microbes (1-3). Burns, trauma patients often accompanied by skin tissue damage, skin integrity damage leads to the damage of the skin barrier function, is one of the important reasons for increasing the organism infection of pathogenic microorganism (4-6), promoting skin The healing of skin wound is of positive significance in preventing the deterioration of the disease, preventing the occurrence of infection and improving the prognosis (7,8). It is found that after the occurrence of surgical accidents such as burns and trauma, the surface of the wound skin will gather a large number of MSC, and the differentiation of fibroblasts and epidermal cells under the induction of various inflammatory factors in the microenvironment is differentiated. A series of cell processes are used to repair the wound (9-13). In burn patients, especially in large area burns, the incidence of late infection and even sepsis is greatly increased due to the appearance of large area skin defects. Early healing of the wound is important to prevent infection and improve the prognosis (13-17). The development of cell transplantation has opened up a promising road for accelerating wound healing, but the source of its own stem cells is limited, and to some extent it restricts the development of skin stem cell transplantation (16-19). How to expand MSC rapidly and rapidly in vitro can help the development of skin stem cell transplantation. A research direction for researchers (20-22). In addition, skin dermal fibroblasts and epidermal keratinocytes can not be ignored during the healing process of skin wounds. Therefore, the two parts are divided into how to provide "raw materials" for stem cell transplantation treatment and how to "process" the completion of the wound after having the "raw material". In order to promote the healing of the wound, the theoretical and experimental basis was given. Method: in the first part, we used MSC as the research object to induce the differentiation of the cells by the three-dimensional suspension (RCCS) model. Then the cell cycle was observed by the CCK8 experiment and the flow cytometry was used to observe the changes of the proliferation ability of the MSC. At the same time, the WB method was used to "dry" and "Cheng". Bone differentiation "differentiation related genes and cell surface markers are used to detect the differentiation of MSC. Finally, biochemical detection and stable isotope amino acid labeling technique are used to study the changes in cell metabolism and proteomics, and the molecular mechanism of the regulation of MSC proliferation and differentiation in the RCCS environment is discussed. The second part will be fibrous thin. Cell and keratinocytes were selected as the research object. Mechanical tensile was used to simulate the changes of skin surface tension, and the effects of tension on the proliferation and migration of fibroblasts and the interaction with the epidermal cells were observed. The molecular mechanism and its role in the healing of skin wound were discussed. Compared with the control group, the results of CCK8 and cell cycle detection in three dimensional suspension culture showed that the proliferation of MSC was enhanced. The expression of "dry" related genes was enhanced by flow cytometry, and the expression of "dry" related genes was enhanced. The expression of "osteogenic differentiation" related genes was weakened, and the differentiation of MSC was inhibited. Further biochemical detection of cell metabolism was carried out. It was found that the content of outer lactic acid in MSC cells increased, the activity of LDH increased, and the.SILAC detection results showed that the gene of mitochondria expressed in the cell was weakened, the expression of glycolytic gene was enhanced, the energy metabolism in the cell was weakened and the energy metabolism of glycolysis Jie Zengqiang was unbalance. The second part of the study was the study of hair metabolism. Compared with the non traction and static pulling force group, the proliferation curve and the cell cycle results of the periodic mechanical pull force showed that the proliferation of HF was inhibited by.WB detection HF-ECM, and the periodic mechanical traction led to the decrease of I collagen in ECM of HF and the increase of fibrin connexin. The epidermal cells (Ha Ca T) were connected. The proliferation of epidermal cells was still inhibited on HF-ECM after the periodic traction force. The epidermal cells were inoculated on the matrix of I collagen or fibronectin coated substrate, and the cell proliferation curve, cell cycle and cell migration ability were detected respectively. The results showed that type I collagen was more capable of promoting cell proliferation than fibronectin. The ability to affect cell migration is not as good as fibronectin. Using immunoprecipitation, mammalian two hybrids and gene interference, it is found that the inhibition of cell proliferation induced by periodic mechanical traction is related to the intergrin beta -CASK signaling pathway. Conclusion: the first part of the study found that the three-dimensional suspension culture mode could be in body MSC, which has a high "dry" low "differentiation" characteristic, may be associated with a three dimensional suspension model that can induce cells to be in the state of reduced aerobic respiration and glycolysis enhanced energy metabolism. This method is a simple and effective means to expand MSC in vitro and can be used for the study of skin wound stem cell transplantation. Second The study shows that the changes in skin surface tension can regulate the HF-ECM components and influence the proliferation and migration of epidermal cells through the "cell dialogue" between the -ECM- keratinocytes of human fibroblasts, thus accelerating the continuous healing of the wound surface. It provides a theoretical basis and experimental basis for further development of skin wound healing.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R641

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