本文選題：創(chuàng)面 + 表皮干細胞��； 參考：《第三軍醫(yī)大學》2007年碩士論文

【摘要】： 有關創(chuàng)面愈合的研究是一個既古老又新穎的課題。皮膚創(chuàng)傷愈合又是一個異常復雜的生物學過程,有多種修復細胞、炎癥細胞及細胞因子參與。這些細胞和細胞因子之間有著復雜的調控關系。它涉及細胞運動、粘附、通訊、增殖和分化等細胞生物學的多個方面。既往對創(chuàng)面的研究往往集中在真皮層的修復與創(chuàng)面床的改建上,而較少涉及表皮再生。隨著對各種干細胞研究的不斷深入,研究表明,多種干細胞對創(chuàng)傷的修復起到關鍵的作用。表皮干細胞(epidermal stem cells , ESC)作為皮膚組織的特異性干細胞,不僅維持表皮組織日常的新陳代謝,而且與創(chuàng)面的愈合緊密相關[1,2],被認為是皮膚及其附屬器修復的源泉細胞。然而,ESC與創(chuàng)面愈合間的確切關系尚知而不詳。盡管我們在以往的相關研究中發(fā)現了ESC的異位現象與創(chuàng)面愈合密切相關,但ESC為何發(fā)生異位、以及細胞間的網絡信號系統(tǒng)是如何調控這一過程的,均有待于作出回答,這也是實現創(chuàng)面完美愈合最終目標的重要環(huán)節(jié)。 本研究以體外培養(yǎng)的ESC為實驗對象,通過全層皮膚創(chuàng)面收集液(wound harvested effusion ,WHE)的干預,觀察了其對ESC增殖、分化的影響;并在ESC表型改變的過程中,動態(tài)地觀察了表皮干細胞內Ca2 +濃度的變化,以及MAPKs信號通路系統(tǒng)對細胞內Ca2 +的影響,初步探討了兩者之間可能存在的調控作用。 本研究的主要研究內容與結論如下: 為避免在體復雜因素的干擾,以全層創(chuàng)面收集液作為單一影響因子探討其對表皮干細胞生物學行為的影響,用聚氨酯海綿收集成年Wistar大鼠背部全層創(chuàng)面滲出液;快速粘附分離法體外分離、培養(yǎng)新生Wistar大鼠的表皮干細胞,待表皮干細胞呈克隆生長時,加入創(chuàng)面收集液干預,分別于干預后的0、6、12、18、24、30、36、42、48、54、60、72 h采用流式細胞儀及β1整合素、角蛋白19、14、10免疫組化染色進行細胞增殖、分化的鑒定。結果顯示,表皮干細胞在創(chuàng)面收集液干預后仍可持續(xù)保持片狀聚集生長的態(tài)勢,但出現數量較多的K14陽性染色細胞群落,且隨時間延長該陽性細胞群落呈上升趨勢;另出現了散在的的K10染色陽性細胞。以上結果提示,創(chuàng)面收集液可以快速誘導體外培養(yǎng)表皮干細胞進入分化狀態(tài),且多表現為短暫擴充細胞表型,在該過程中表皮干細胞數量仍可維持一定的水平。 為探討創(chuàng)面收集液對表皮干細胞(ESC)內鈣離子的作用以及絲裂原活化蛋白激酶(MAPK)通路對胞內游離鈣的影響,將培養(yǎng)的表皮干細胞分為5組:①單純對照組;②單純創(chuàng)面收集液處理組;③PD98059阻斷劑+創(chuàng)面收集液處理組;④SB203580阻斷劑+創(chuàng)面收集液處理組;⑤PD98059與SB203580阻斷劑+創(chuàng)面收集液處理組。應用特異性Ca2 +熒光指示劑Fluo-3/AM負載細胞,激光共聚焦顯微鏡檢測細胞內Ca2 +熒光強度,以判斷游離鈣的濃度。結果顯示,單純創(chuàng)面收集液處理組ESC的Ca2 +熒光強度明顯較單純對照組增加;③、④兩組均出現了不同的鈣振蕩現象;而⑤組則表現為Ca2 +熒光強度的迅速降低。實驗結果說明,創(chuàng)面收集液可致ESC游離Ca2 +濃度的增加,MAPK信號通路對ESC胞內鈣離子有反饋調節(jié)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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R641;R329.2

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本文編號：1991719