创面收集液对表皮干细胞增殖分化的影响及其分子机制初探

发布时间：2018-06-07 15:47

本文选题：创面 + 表皮干细胞　；参考：《第三军医大学》2007年硕士论文

【摘要】： 有关创面愈合的研究是一个既古老又新颖的课题。皮肤创伤愈合又是一个异常复杂的生物学过程,有多种修复细胞、炎症细胞及细胞因子参与。这些细胞和细胞因子之间有着复杂的调控关系。它涉及细胞运动、粘附、通讯、增殖和分化等细胞生物学的多个方面。既往对创面的研究往往集中在真皮层的修复与创面床的改建上,而较少涉及表皮再生。随着对各种干细胞研究的不断深入,研究表明,多种干细胞对创伤的修复起到关键的作用。表皮干细胞(epidermal stem cells , ESC)作为皮肤组织的特异性干细胞,不仅维持表皮组织日常的新陈代谢,而且与创面的愈合紧密相关[1,2],被认为是皮肤及其附属器修复的源泉细胞。然而,ESC与创面愈合间的确切关系尚知而不详。尽管我们在以往的相关研究中发现了ESC的异位现象与创面愈合密切相关,但ESC为何发生异位、以及细胞间的网络信号系统是如何调控这一过程的,均有待于作出回答,这也是实现创面完美愈合最终目标的重要环节。本研究以体外培养的ESC为实验对象,通过全层皮肤创面收集液(wound harvested effusion ,WHE)的干预,观察了其对ESC增殖、分化的影响;并在ESC表型改变的过程中,动态地观察了表皮干细胞内Ca2 +浓度的变化,以及MAPKs信号通路系统对细胞内Ca2 +的影响,初步探讨了两者之间可能存在的调控作用。本研究的主要研究内容与结论如下: 为避免在体复杂因素的干扰,以全层创面收集液作为单一影响因子探讨其对表皮干细胞生物学行为的影响,用聚氨酯海绵收集成年Wistar大鼠背部全层创面渗出液;快速粘附分离法体外分离、培养新生Wistar大鼠的表皮干细胞,待表皮干细胞呈克隆生长时,加入创面收集液干预,分别于干预后的0、6、12、18、24、30、36、42、48、54、60、72 h采用流式细胞仪及β1整合素、角蛋白19、14、10免疫组化染色进行细胞增殖、分化的鉴定。结果显示,表皮干细胞在创面收集液干预后仍可持续保持片状聚集生长的态势,但出现数量较多的K14阳性染色细胞群落,且随时间延长该阳性细胞群落呈上升趋势;另出现了散在的的K10染色阳性细胞。以上结果提示,创面收集液可以快速诱导体外培养表皮干细胞进入分化状态,且多表现为短暂扩充细胞表型,在该过程中表皮干细胞数量仍可维持一定的水平。为探讨创面收集液对表皮干细胞(ESC)内钙离子的作用以及丝裂原活化蛋白激酶(MAPK)通路对胞内游离钙的影响,将培养的表皮干细胞分为5组:①单纯对照组;②单纯创面收集液处理组;③PD98059阻断剂+创面收集液处理组;④SB203580阻断剂+创面收集液处理组;⑤PD98059与SB203580阻断剂+创面收集液处理组。应用特异性Ca2 +荧光指示剂Fluo-3/AM负载细胞,激光共聚焦显微镜检测细胞内Ca2 +荧光强度,以判断游离钙的浓度。结果显示,单纯创面收集液处理组ESC的Ca2 +荧光强度明显较单纯对照组增加;③、④两组均出现了不同的钙振荡现象;而⑤组则表现为Ca2 +荧光强度的迅速降低。实验结果说明,创面收集液可致ESC游离Ca2 +浓度的增加,MAPK信号通路对ESC胞内钙离子有反馈调节的作用。
[Abstract]:The study of wound healing is an old and novel topic. Skin wound healing is an extremely complex biological process, involving a variety of repair cells, inflammatory cells and cytokines. These cells and cytokines have complex regulatory relationships. It involves cell movement, adhesion, communication, proliferation, and differentiation. There are many aspects of cell biology. The previous study of the wound is often focused on the repair of the dermis and the remodeling of the wound bed, but less on the epidermal regeneration. With the continuous development of various stem cell studies, the research shows that multiple stem cells play a key role in the repair of the wound. Epidermal stem cells (ESC) is used as the key to the repair of the wound. Specific stem cells for skin tissue not only maintain the daily metabolism of the epidermal tissue, but also closely related to the healing of the wound. [1,2] is considered as the source of the skin and its appendage repair. However, the exact relationship between ESC and wound healing is unknown. Although we have found the heterotopia of ESC in previous related studies The phenomenon is closely related to the wound healing, but why ESC is ectopic and how the intercellular network signal system regulates this process needs to be answered, which is also an important link to achieve the ultimate goal of perfect healing of the wound.
In this study, the effects of wound harvested effusion (WHE) on the proliferation and differentiation of ESC were observed by the intervention of the full layer skin ESC effusion (WHE), and the changes of Ca2 + concentration in the epidermal stem cells were dynamically observed during the ESC phenotypic change, and the MAPKs signal pathway system was used for the intracellular Ca2 +. The possible regulatory effects between the two are discussed.
The main contents and conclusions of this study are as follows:
In order to avoid the interference of complex factors, the whole layer wound collecting fluid was used as a single influence factor to investigate the biological behavior of the epidermal stem cells. The full layer of the wound exudate of the back of the adult Wistar rat was collected by the polyurethane sponge, and the rapid adhesion and separation method was used to isolate the epidermal stem cells of the newborn Wistar rats in vitro. When the cell was cloned and growing, the wound collection solution was added to the cell, and the 0,6,12,18,24,30,36,42,48,54,60,72 h with the flow cytometry and the beta 1 integrin, and the cytochemical staining of keratin 19,14,10 were used to identify the cell proliferation and differentiation, respectively. The results showed that the epidermal stem cells still maintained the flaky aggregation after the wound collecting fluid. The growth trend, but there were a large number of K14 positive staining cell communities, and the positive cell community showed an upward trend with time, and the scattered K10 staining positive cells appeared. The above results suggested that the wound collection fluid could quickly induce the cultured epidermal stem cells to enter the differentiation state in vitro. Cell phenotype, in this process, the number of epidermal stem cells can still maintain a certain level.
To investigate the effect of wound collecting fluid on calcium ion in epidermal stem cell (ESC) and the effect of mitogen activated protein kinase (MAPK) pathway on intracellular free calcium, the cultured epidermal stem cells were divided into 5 groups: (1) simple control group; (2) simple wound collection solution group; (3) PD98059 blocker + wound collection solution group; (4) SB203580 blocker + The treatment group of wound collection solution, 5 PD98059 and SB203580 blocking agent + wound collection solution group. The specific Ca2 + fluorescent indicator Fluo-3/AM was used to load cells, and the intracellular Ca2 + fluorescence intensity was detected by laser confocal microscopy in order to determine the concentration of free calcium. The results showed that the Ca2 + fluorescence intensity of ESC in the Dan Chunchuang surface collection solution group was obviously better than that of the ESC. The pure control group increased; (3) the two groups all showed different calcium oscillations; while the group 5 showed a rapid decrease in the intensity of Ca2 + fluorescence. The experimental results showed that the wound collecting fluid could increase the concentration of ESC free Ca2 +, and the MAPK signaling pathway had feedback on the intracellular calcium ion of ESC.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R641;R329.2

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