本文選題：皮膚軟組織擴張術(shù) + 表皮干細胞��； 參考：《第三軍醫(yī)大學》2006年碩士論文

【摘要】： 人體細胞生長在機體提供的微動力學環(huán)境中,機械應力不僅能引起細胞形態(tài)、結(jié)構(gòu)變化,還能調(diào)控細胞的功能狀態(tài),影響細胞的增殖、分化及凋亡,在某些生理和病理過程中起著重要作用。業(yè)已證明,適當?shù)臋C械應力可促進細胞增殖與分化,通過外力刺激因素獲得“額外”組織的臨床技術(shù)均是基于這一理論,并已在臨床上收到了令人滿意的療效。如骨科的肢體牽引延長術(shù)、骨折的加壓固定術(shù)、顱頜面外科的顱頜骨牽引術(shù)以及整形外科的皮膚軟組織擴張術(shù)(skin soft-tissue expansion,SSTE)等。同時研究表明,不同種類的細胞有明顯的異質(zhì)性,對相同的應力刺激所表現(xiàn)出的效應不同,而同一細胞對不同的應力響應也不同,因此,其他研究領(lǐng)域的結(jié)果難于指導某一領(lǐng)域的臨床實踐。目前開展的相關(guān)基礎(chǔ)研究比較活躍的領(lǐng)域主要集中在骨細胞(研究應力成骨)、血管平滑肌細胞(研究高血壓)、血管內(nèi)皮細胞、腎小球系膜細胞(研究腎小球性高血壓)、以及肺上皮細胞等,而與SSTE相關(guān)的研究文獻報道甚少。更未見應力與表皮干細胞(epidermal stem cells,ESCs)相關(guān)性的文獻。 自1976年Radovan發(fā)明可控式擴張器以來,SSTE已廣泛應用于臨床,數(shù)以萬計的患者從中受益,現(xiàn)已成為整形外科的常規(guī)治療手段之一。雖然SSTE經(jīng)過了30年的發(fā)展,在實驗研究、臨床應用、擴張方法、并發(fā)癥防治等諸多方面取得了長足的進展,然而,其擴張周期較長、并發(fā)癥較多的缺憾仍尚未從更本上獲得解決。因此,如何縮短擴張時間、減少并發(fā)癥即成為SSTE研究領(lǐng)域的焦點問題。業(yè)已證明,擴張產(chǎn)生的“額外”皮膚來源于皮膚的機械蠕變、彈性擴張和生物性增殖,其中盡快獲得生物性增殖、充分有效的刺激細胞的有絲分裂活動而獲得所需要的“額外”皮膚量,可能是克服上述難題的根本所在,但迄今為止,皮膚細胞在擴張過程中的增殖分化行為尚不甚清楚。ESC作為皮膚組織的特異性干細胞,不僅是維持皮膚日常新陳代謝的主要功能細胞,而且與創(chuàng)面的修復亦緊密相關(guān)。但目前就有關(guān)ESC在SSTE過程中所起到的具體作用,或與皮膚擴張間的關(guān)系尚鮮為人知。為此,深入認識SSTE過程中ESC的分布、增殖與分化特征以及相關(guān)的調(diào)控機制,無疑對充實、完善SSTE基礎(chǔ)理論以及對指導臨床更加完美地應用SSTE有著極其重要的意義。 為此,本研究擬以離體與在體兩種手段開展以下實驗觀察: 利用表皮干細胞及其增殖、分化過程中不同的細胞表面標志物以及增殖細胞核抗原(PCNA)標記增殖期細胞的特點,采用免疫組織化學法觀察擴張過程中具有增殖能力的表皮干細胞、短暫擴充細胞的分布與數(shù)量差異,初步觀察皮膚擴張術(shù)對表皮干細胞增殖分化的影響,可進一步研究表皮干細胞在皮膚擴張中的作用。 此外,用機械應力作用于體外培養(yǎng)的細胞,研究其受應力作用后的各種變化,是目前細胞生物學領(lǐng)域發(fā)展十分迅速的一種方法。通過分離培養(yǎng)人表皮干細胞,建立可行的壓應力細胞培養(yǎng)體系裝置,觀察和研究壓應力對表皮干細胞增殖分化的影響。 【目的】觀察大鼠活體皮膚軟組織擴張術(shù)對其擴張表皮細胞增殖分化特征的影響以及間歇壓應力對離體表皮干細胞增殖分化特征的影響并探討其可能的作用機制。 【方法】 1) 36只Wistar大鼠制作皮膚擴張動物模型,并隨機分為擴張組和非擴張對照組。利用表皮干細胞表達角蛋白(keratin 19,K19)、p63,短暫擴充細胞表達K14及增殖細胞核抗原(PCNA)的特點,分別于擴張過程中與擴張后的不同時相點,采用鏈霉卵白素-生物素(SP)免疫組織化學染色法檢測擴張皮膚表皮干細胞和短暫擴充細胞的分布特征,及常規(guī)HE染色觀察擴張皮膚表皮的厚度改變。 2)通過我們已建立的鼠表皮干細胞體外分離、培養(yǎng)的方法,進行人表皮干細胞的體外分離、培養(yǎng),并利用鏈霉卵白素-生物素(SP)免疫組織化學染色法和細胞周期分析進行鑒定;采用培養(yǎng)細胞加壓裝置施以不同壓應力(4kPa,6kPa,8kPa,10kPa,12kPa)對表皮干細胞加壓,利用表皮干細胞特異性表達角蛋白19及終末分化細胞表達角蛋白10的特點,以鏈霉卵白素-生物素(SP)免疫組織化學染色法檢測加壓前后表皮干細胞和終末分化細胞的變化情況。 【結(jié)果】 1)擴張皮膚的表皮層明顯增厚,不僅其基底層K19、p63、K14和PCNA陽性細胞的數(shù)量明顯增多,而且在棘層和顆粒層可見K19、p63、K14及PCNA陽性細胞的表達; 2)分離、培養(yǎng)的表皮干細胞K19免疫組化染色陽性,細胞周期分析有84.80%的細胞處于G_1期;8kPa以上的間歇壓應力作用粘附于硅膠膜上的表皮干細胞1周后,其數(shù)量明顯增多,免疫組化染色發(fā)現(xiàn)其中有角蛋白10陽性細胞。 【結(jié)論】本研究發(fā)現(xiàn)皮膚軟組織擴張術(shù)的應力能誘導大鼠表皮干細胞增殖與分化,皮膚擴張的過程既有應力誘導引起的增殖效應,又有應力造成的微損傷啟動的創(chuàng)傷修復效應,是非典型的創(chuàng)面修復的病理生理過程,而在此過程中具有自我更新、無限增殖潛能和再生修復能力的表皮干細胞發(fā)揮著關(guān)鍵性的作用;同時也證實8kPa以上的間歇壓應力能誘導表皮干細胞增殖分化,進一步提示表皮干細胞對機械應力的響應機制可能是皮膚軟組織擴張術(shù)獲得“額外皮膚”的主要的生物學基礎(chǔ)。
[Abstract]:Human cells grow in the microdynamic environment provided by the body. Mechanical stress can not only cause cell morphology and structural changes, but also regulate cell function state, affect cell proliferation, differentiation and apoptosis. It plays an important role in some physiological and pathological processes. It has been proved that proper mechanical stress can promote cell proliferation and differentiation. The clinical techniques of obtaining "extra" tissue through external force factors are based on this theory and have received satisfactory clinical effects. Such as limb traction in the Department of orthopedics, compression fixation of fractures, craniofacial traction in craniofacial surgery, and plastic surgery for skin soft tissue expansion (skin soft-tissue exp) Ansion, SSTE) and so on. At the same time, the study shows that different types of cells have distinct heterogeneity, and the effects on the same stress stimulation are different, and the same cell response to different stress is different. Therefore, the results of other research fields are difficult to guide the clinical practice in a certain field. The relevant basic research is active at present. The field is mainly focused on bone cells (Study of stress osteogenesis), vascular smooth muscle cells (hypertension), vascular endothelial cells, glomerular mesangial cells (glomerular hypertension), and lung epithelial cells, while SSTE related literature is rarely reported. No stress is associated with epidermal stem cells (ESCs). The literature of sex.
Since the invention of controllable expander in Radovan in 1976, SSTE has been widely used in clinical practice. Tens of thousands of patients have benefited from it and have become one of the conventional treatments for plastic surgery. Although SSTE has developed for 30 years, considerable progress has been made in many aspects, such as experimental research, clinical application, expansion methods, prevention and treatment of complications. Therefore, how to shorten the time of expansion and reduce the complications has become the focus of SSTE research. It has been proved that the "extra" skin caused by expansion is derived from the mechanical creep, elastic expansion and biological proliferation of the skin, which can be obtained as soon as possible. Biological proliferation, full and effective stimulation of cell mitosis to obtain the required "extra" skin volume may be the fundamental problem to overcome the above problems, but so far, the proliferation and differentiation of skin cells during the expansion process is not very clear to the specific stem cells of.ESC skin tissue, not only to maintain the skin. The main functional cells of daily metabolism are also closely related to the repair of the wound. However, the specific role of ESC in the process of SSTE, or the relationship with the skin extension, is little known at present. Therefore, the understanding of the distribution, proliferation and differentiation characteristics and related regulatory mechanisms of ESC in the process of SSTE is undoubtedly enriched, It is of great significance to improve the basic theory of SSTE and guide the clinical application of SSTE more perfectly.
For this purpose, the following experiments were carried out in two ways: in vitro and in vivo.
Using epidermal stem cells and their proliferation, differentiation of cell surface markers and proliferating cell nuclear antigen (PCNA) to mark the characteristics of proliferating cells, the proliferation ability of epidermal stem cells in the process of dilation was observed by immunohistochemistry. The distribution and quantity difference of the cells were extended briefly, and the skin dilatation was observed preliminarily. The effect of epidermal stem cells on skin expansion can be further studied.
In addition, it is a very rapid development method in the field of cell biology to study the changes of the cells cultured in vitro by mechanical stress and to study the various changes of the cells in the field of cell biology. By separating and cultivating human epidermal stem cells, a viable pressure cell culture system is established to observe and study the proliferation of epidermal stem cells by pressure stress. The influence of chemistry.
[Objective] to observe the effect of soft tissue dilatation on the proliferation and differentiation of expanded epidermal cells in rats and the effect of intermittent pressure stress on the proliferation and differentiation of epidermal stem cells and explore the possible mechanism of action.
[method]
1) 36 Wistar rats were made of skin dilatation animal model and were randomly divided into dilated group and non dilated control group. Using epidermal stem cells to express keratin (keratin 19, K19), p63, briefly expand the expression of K14 and proliferating cell nuclear antigen (PCNA), respectively, respectively, in Yu Kuo Zhang Guocheng and the different phase after dilation, using streptomycin oooxin - Biotin (SP) immunohistochemical staining was used to detect the distribution of epidermal stem cells and transient expanded cells in dilated skin, and the thickness of the epidermis of dilated skin was observed by routine HE staining.
2) through the isolation and culture of rat epidermal stem cells in vitro, the human epidermal stem cells were isolated and cultured in vitro, and identified by streptomycin SP immuno histochemical staining and cell cycle analysis. Different compressive stresses (4kPa, 6kPa, 8kPa, 10kPa, 12kPa) were applied to the culture cell compression device. The changes of epidermal stem cells and terminal differentiation cells before and after pressurization were detected by pressure of epidermal stem cells, using epidermal stem cells specifically expressing keratin 19 and the expression of keratin 10 in terminal differentiated cells, and using streptomycin and biotin (SP) immunohistochemical staining.
[results]
1) the epidermis of dilated skin was thickened obviously, not only the number of K19, p63, K14 and PCNA positive cells in the basal layer increased significantly, but also the expression of K19, p63, K14 and PCNA positive cells in the spinous and granular layers.
2) isolated, cultured epidermal stem cells K19 immunohistochemical staining positive, cell cycle analysis of 84.80% cells in the G_1 stage, 8kPa above the intermittent pressure stress on the epidermal stem cells adhered to the silica gel membrane for 1 weeks, the number of them increased significantly, immunohistochemical staining found that there were keratin 10 positive cells.
[Conclusion] this study found that the stress of skin soft tissue expansion can induce the proliferation and differentiation of rat epidermal stem cells. The process of skin dilatation not only has the effect of stress induced proliferation, but also the healing effect of the micro injury caused by stress. It is the pathophysiological process of the atypical wound repair. In this process, it has a pathophysiological process. Epidermal stem cells play a key role in self renewal, unlimited proliferation potential and regeneration and repair. It is also proved that the intermittent pressure stress above 8kPa can induce the proliferation and differentiation of epidermal stem cells, and further hints that the response mechanism of epidermal stem cells to mechanical stress may be the "extra skin" of skin soft tissue expansion. The main biological basis.
【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】：2006
【分類號】：R329

【引證文獻】

相關(guān)博士學位論文 前1條

1 葛丹;NSCs三維培養(yǎng)及其微流控動態(tài)模型的構(gòu)建[D];大連理工大學;2013年

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本文編號：2020307