本文選題：MSC + 三維懸浮培養(yǎng)��； 參考：《第三軍醫(yī)大學》2015年博士論文

【摘要】：背景:皮膚是人體的防御器官,皮膚的完整性對于機體抵抗病原微生物具有重要意義(1-3)。燒傷、創(chuàng)傷病人往往伴有皮膚組織的損傷,皮膚完整性的破壞導致皮膚屏障的功能破壞,是增加機體受病原微生物感染的重要原因之一(4-6),促進皮膚創(chuàng)面的愈合對于防止病情惡化、預防感染的發(fā)生及改善預后都有積極的意義(7、8)。有研究發(fā)現(xiàn),燒傷、創(chuàng)傷等外科意外發(fā)生后,創(chuàng)傷皮膚的表面會聚集大量MSC,在創(chuàng)面微環(huán)境各種炎性因子的誘導下向成纖維細胞和表皮細胞進行分化,通過一系列細胞進程來修復創(chuàng)面(9-13)。在燒傷患者特別是大面積燒傷患者,由于大面積皮膚缺損的出現(xiàn),后期感染甚至膿毒血癥發(fā)生的幾率大大增加,早期促進創(chuàng)面的愈合對于預防感染的發(fā)生和改善預后十分重要(13-17)。近年來皮膚干細胞移植治療的發(fā)展為加速創(chuàng)面的愈合開辟了一條前景光明的道路,但自身干細胞的來源有限,一定程度上又限制了皮膚干細胞移植治療的發(fā)展(16-19)。如何能夠在體外快速大量的擴增MSC,為皮膚干細胞移植的發(fā)展提供幫助,也成為了科研工作者的一個研究方向(20-22)。此外皮膚真皮纖維細胞以及表皮角質(zhì)細胞在皮膚創(chuàng)面的愈合過程中作用也不容忽視,因此本文分為兩部分分別針對如何為干細胞移植治療提供“原料”和具備“原料”后如何“加工“完成創(chuàng)面的修復進行研究,為促進創(chuàng)面的愈合給予理論和實驗依據(jù)。方法:第一部分我們以MSC為研究對象,采用三維懸浮(RCCS)模式對細胞進行誘導分化,然后通過CCK8實驗、流式細胞儀觀測細胞周期,觀察MSC增殖能力的變化,同時WB法對其“干性”和“成骨分化”分化相關基因和細胞表面標志進行檢測判斷MSC分化情況,最后我們采用生化檢測和穩(wěn)定同位素氨基酸標記技術研究細胞的代謝和蛋白質(zhì)組學的變化,并探討RCCS環(huán)境對MSC增殖與分化調(diào)控的分子機制。第二部分我們將成纖維細胞以及角質(zhì)細胞選作研究對象,采用機械拉伸模擬皮膚表面張力的變化,觀察張力變化對成纖維細胞增殖及遷移的影響及其與表皮細胞間的相互作用關系,探討其分子機制及其在皮膚創(chuàng)面愈合中發(fā)揮的作用。結果:第一部分研究發(fā)現(xiàn),與對照組相比三維懸浮培養(yǎng)模式下CCK8和細胞周期檢測結果均顯示MSC增殖增強,流式細胞儀檢測細胞表面標志及WB檢測發(fā)現(xiàn)其“干性”相關基因表達增強,“成骨分化”相關基因表達減弱,MSC分化受到抑制。進一步生化檢測細胞代謝情況發(fā)現(xiàn),三維懸浮培養(yǎng)的MSC細胞內(nèi)、外乳酸含量增加,LDH活性增加,葡萄糖消耗增加。SILAC檢測結果顯示細胞線粒體表達的基因減弱,糖酵解基因表達增強,細胞能量代謝處于有氧氧化減弱,糖酵解增強的能量代謝失衡狀態(tài)。第二部分研究發(fā)現(xiàn)與無牽拉和靜態(tài)牽拉作用力組相比,在周期性機械牽拉力下,細胞增殖曲線及細胞周期結果均顯示HF的增殖受到抑制。WB檢測HF-ECM發(fā)現(xiàn),周期性機械牽拉力導致HF的ECM中I型膠原顯著降低、而纖維連接蛋白明顯增加。將表皮細胞(Ha Ca T)接種于周期性牽拉力作用后的HF-ECM上,表皮細胞的增殖仍然受到抑制。將表皮細胞接種于經(jīng)過I型膠原或纖維連接蛋白包被的基質(zhì)上,分別檢測細胞的增殖曲線、細胞周期和細胞遷移能力,結果發(fā)現(xiàn)I型膠原比纖連蛋白更能促進細胞的增殖,而在影響細胞遷移的能力上卻不如纖連蛋白。用免疫共沉淀、哺乳動物雙雜交和基因干擾的方法研究發(fā)現(xiàn),周期性機械牽拉力誘導的抑制細胞增殖作用與intergrinβ-CASK信號通路有關。結論:第一部分研究發(fā)現(xiàn)三維懸浮培養(yǎng)模式可以在體外快速大量擴增MSC,這種MSC具有高“干性”低“分化”特性,可能與三維懸浮模式能夠誘導細胞處于有氧呼吸減弱,糖酵解增強的能量代謝狀態(tài)有關。該法是簡單有效的體外擴增MSC的手段,可以用于皮膚創(chuàng)面干細胞移植的研究。第二部分研究表明皮膚表面張力的改變可以調(diào)節(jié)HF-ECM成分,通過“人纖維細胞-ECM-角質(zhì)細胞”之間的“細胞對話”影響表皮細胞的增殖以及遷移,從而加速創(chuàng)面持續(xù)愈合。本研究在一定程度上闡明了成纖維細胞和表皮細胞在創(chuàng)面修復中的作用及其機制,為皮膚創(chuàng)面愈合的的進一步發(fā)展提供了理論基礎和實驗依據(jù)。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R641

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