机械应力对表皮干细胞增殖、分化影响的研究

发布时间：2018-06-15 03:05

本文选题：皮肤软组织扩张术 + 表皮干细胞　；参考：《第三军医大学》2006年硕士论文

【摘要】： 人体细胞生长在机体提供的微动力学环境中,机械应力不仅能引起细胞形态、结构变化,还能调控细胞的功能状态,影响细胞的增殖、分化及凋亡,在某些生理和病理过程中起着重要作用。业已证明,适当的机械应力可促进细胞增殖与分化,通过外力刺激因素获得“额外”组织的临床技术均是基于这一理论,并已在临床上收到了令人满意的疗效。如骨科的肢体牵引延长术、骨折的加压固定术、颅颌面外科的颅颌骨牵引术以及整形外科的皮肤软组织扩张术(skin soft-tissue expansion,SSTE)等。同时研究表明,不同种类的细胞有明显的异质性,对相同的应力刺激所表现出的效应不同,而同一细胞对不同的应力响应也不同,因此,其他研究领域的结果难于指导某一领域的临床实践。目前开展的相关基础研究比较活跃的领域主要集中在骨细胞(研究应力成骨)、血管平滑肌细胞(研究高血压)、血管内皮细胞、肾小球系膜细胞(研究肾小球性高血压)、以及肺上皮细胞等,而与SSTE相关的研究文献报道甚少。更未见应力与表皮干细胞(epidermal stem cells,ESCs)相关性的文献。自1976年Radovan发明可控式扩张器以来,SSTE已广泛应用于临床,数以万计的患者从中受益,现已成为整形外科的常规治疗手段之一。虽然SSTE经过了30年的发展,在实验研究、临床应用、扩张方法、并发症防治等诸多方面取得了长足的进展,然而,其扩张周期较长、并发症较多的缺憾仍尚未从更本上获得解决。因此,如何缩短扩张时间、减少并发症即成为SSTE研究领域的焦点问题。业已证明,扩张产生的“额外”皮肤来源于皮肤的机械蠕变、弹性扩张和生物性增殖,其中尽快获得生物性增殖、充分有效的刺激细胞的有丝分裂活动而获得所需要的“额外”皮肤量,可能是克服上述难题的根本所在,但迄今为止,皮肤细胞在扩张过程中的增殖分化行为尚不甚清楚。ESC作为皮肤组织的特异性干细胞,不仅是维持皮肤日常新陈代谢的主要功能细胞,而且与创面的修复亦紧密相关。但目前就有关ESC在SSTE过程中所起到的具体作用,或与皮肤扩张间的关系尚鲜为人知。为此,深入认识SSTE过程中ESC的分布、增殖与分化特征以及相关的调控机制,无疑对充实、完善SSTE基础理论以及对指导临床更加完美地应用SSTE有着极其重要的意义。为此,本研究拟以离体与在体两种手段开展以下实验观察: 利用表皮干细胞及其增殖、分化过程中不同的细胞表面标志物以及增殖细胞核抗原(PCNA)标记增殖期细胞的特点,采用免疫组织化学法观察扩张过程中具有增殖能力的表皮干细胞、短暂扩充细胞的分布与数量差异,初步观察皮肤扩张术对表皮干细胞增殖分化的影响,可进一步研究表皮干细胞在皮肤扩张中的作用。此外,用机械应力作用于体外培养的细胞,研究其受应力作用后的各种变化,是目前细胞生物学领域发展十分迅速的一种方法。通过分离培养人表皮干细胞,建立可行的压应力细胞培养体系装置,观察和研究压应力对表皮干细胞增殖分化的影响。【目的】观察大鼠活体皮肤软组织扩张术对其扩张表皮细胞增殖分化特征的影响以及间歇压应力对离体表皮干细胞增殖分化特征的影响并探讨其可能的作用机制。【方法】 1) 36只Wistar大鼠制作皮肤扩张动物模型,并随机分为扩张组和非扩张对照组。利用表皮干细胞表达角蛋白(keratin 19,K19)、p63,短暂扩充细胞表达K14及增殖细胞核抗原(PCNA)的特点,分别于扩张过程中与扩张后的不同时相点,采用链霉卵白素-生物素(SP)免疫组织化学染色法检测扩张皮肤表皮干细胞和短暂扩充细胞的分布特征,及常规HE染色观察扩张皮肤表皮的厚度改变。 2)通过我们已建立的鼠表皮干细胞体外分离、培养的方法,进行人表皮干细胞的体外分离、培养,并利用链霉卵白素-生物素(SP)免疫组织化学染色法和细胞周期分析进行鉴定;采用培养细胞加压装置施以不同压应力(4kPa,6kPa,8kPa,10kPa,12kPa)对表皮干细胞加压,利用表皮干细胞特异性表达角蛋白19及终末分化细胞表达角蛋白10的特点,以链霉卵白素-生物素(SP)免疫组织化学染色法检测加压前后表皮干细胞和终末分化细胞的变化情况。【结果】 1)扩张皮肤的表皮层明显增厚,不仅其基底层K19、p63、K14和PCNA阳性细胞的数量明显增多,而且在棘层和颗粒层可见K19、p63、K14及PCNA阳性细胞的表达; 2)分离、培养的表皮干细胞K19免疫组化染色阳性,细胞周期分析有84.80%的细胞处于G_1期;8kPa以上的间歇压应力作用粘附于硅胶膜上的表皮干细胞1周后,其数量明显增多,免疫组化染色发现其中有角蛋白10阳性细胞。【结论】本研究发现皮肤软组织扩张术的应力能诱导大鼠表皮干细胞增殖与分化,皮肤扩张的过程既有应力诱导引起的增殖效应,又有应力造成的微损伤启动的创伤修复效应,是非典型的创面修复的病理生理过程,而在此过程中具有自我更新、无限增殖潜能和再生修复能力的表皮干细胞发挥着关键性的作用;同时也证实8kPa以上的间歇压应力能诱导表皮干细胞增殖分化,进一步提示表皮干细胞对机械应力的响应机制可能是皮肤软组织扩张术获得“额外皮肤”的主要的生物学基础。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

【引证文献】