Wnt5a抑制毛囊生长的体外及体内实验研究

发布时间：2018-07-16 15:12

【摘要】：毛囊是一个复杂的皮肤附属器官,它由上皮(毛母质与多层同心圆状结构)和真皮(毛乳头和结缔组织鞘)两大部分组成,其中包含有20余种细胞,包括角质形成细胞、黑色素细胞、Langerhans细胞、Merkel细胞、真皮鞘细胞和毛乳头细胞,这些细胞在体内经过复杂的相互作用,共同参与了毛囊的发育、生长、分化与周期性的调节。毛囊依靠周期性循环来实现毛发的生长与更新,根据毛囊形态上的变化可将其分为三个阶段:生长期(anagen)毛囊持续延长;退化期(catagen)毛乳头开始上移,毛母质细胞消失,毛囊逐渐缩短;静止期(telogen)时毛乳头到达真皮与皮下组织交界处,仅靠极小的上皮细胞索与毛囊相连。在毛囊生长的不同周期,一些活性蛋白、细胞因子及激素均可对毛囊周期或生长产生促进或抑制作用。寻找能够影响毛发生长的成分对阐明毛囊生长周期调控机制、发展治疗脱发的有效手段具有重要意义。目前研究表明,毛囊的发生发育及周期维持受到多种信号途径的调节,如Wnt、Bmp、Shh等,其中经典Wnt/β-catenin途径日益引起人们的关注,该途径被认为是毛囊周期维持不可或缺的通路。目前在哺乳动物中已发现19个Wnt蛋白家族成员,而且小鼠皮肤毛囊发育过程中同时存在多种Wnt的表达,其中包括了经典Wnts,也包括非经典Wnts。但是,迄今为止对该家族两大类Wnts在毛囊周期调控中的作用是否存在差异尚不明确。 Wnt5a (wingless-type MMTV integration site family, member 5A)作为Wnt家族非经典Wnts的代表,在细胞的增殖、分化、迁移、运动、极性调节中有重要作用,其参与的信号网络复杂交错,除经典Wnt途径外还可激活多条非经典Wnt信号途径,如Wnt/Ca2+途径、PCP途径、Wnt/Ror2途径等。最近研究发现非经典Wnt5a途径能通过促进β-catenin降解,从而抑制经典Wnt/β-catenin途径的激活。为了更加清楚地了解Wnt5a在毛囊周期调控中的作用,本研究首先利用原位杂交、免疫组织化学、半定量RT-PCR和Western blot技术,观察Wnt5a在小鼠毛囊周期中的时空表达规律;其次在体外毛囊器官培养水平,采用腺病毒介导方式,检测Wnt5a对毛囊生长的调控效应;然后采用拔毛同步化动物模型,经皮内注射腺病毒,进一步从在体水平验证Wnt5a对毛囊生长的影响,并运用基因芯片检测、定量RT-PCR、IHC等技术,分析该效应可能涉及的作用机制。本研究主要结果如下: 一、Wnt5a在小鼠毛囊周期中的表达规律 1.以地高辛标记的Wnt5a RNA为探针,采用背皮whole-mount原位杂交技术观察到:①生长期毛球部有较强的Wnt5a mRNA信号,分布在matrix、IRS和DP,尤其是强增殖能力的matrix内可观察到大量的阳性细胞。大量Wnt5a由生长期毛囊细胞分泌,强烈提示它可能参与了对毛囊生长过程的调控。②进入退化期后,发生凋亡的毛球部阳性信号逐渐减少。③静止期毛囊中仅在DP见到少量Wnt5a mRNA表达。Wnt5a mRNA表达表现出显著的周期性变化。 2.采用背皮组织的免疫组织化学方法观察到:①Wnt5a蛋白在生长期毛囊中分布广泛,除matrix、IRS、DP外,precortex细胞、ORS中也有分布,且表达量随毛囊生长逐渐增多。Wnt5a蛋白分布范围较mRNA更弥散,说明分泌Wnt5a的细胞和能接受Wnt5a信号的细胞可能不完全一致,提示Wnt5a在毛囊中的作用途径可能同时存在自分泌和旁分泌两种方式。②到退化期,上皮索的表达逐渐减少。③进入静止期,少量Wnt5a蛋白的表达仅局限于bulge区、secondary hair germ和DP。 3.通过背皮组织半定量RT-PCR和Western blot技术观察到,Wnt5a mRNA和蛋白的表达趋势与原位杂交和IHC一致,均表现为生长期最丰富,退化期减弱,静止期最低。二、体外器官水平检测Wnt5a对毛囊生长的作用 1.采用触须组织的免疫组织化学方法,观察到Wnt5a蛋白表达情况与背皮相似,说明这两种毛囊类型中Wnt5a可能发挥相似调控作用。 2.利用腺病毒介导的方式,进行触须毛囊的体外器官培养,观察到:Wnt5a处理组毛囊的毛干伸出长度变短,提示Wnt5a对毛囊生长有抑制作用。三、动物在体水平进一步验证Wnt5a对毛囊生长的抑制作用 1.建立小鼠拔毛诱导同步化模型,经皮内注射腺病毒,观察到Wnt5a组注射部位出现明显抑毛区,HE染色显示该区毛囊为静止期形态,而周边毛囊已经进入生长中期,提示Wnt5a抑制了由于拔毛所诱导的毛囊生长,干扰了毛囊anagen的进程。一段时间后,抑毛区会逐渐缩小直至消失,长出正常毛发,说明单一的Wnt因素只能短暂抑制毛囊生长。 2.采用小鼠全基因组芯片检测及定量RT-PCR,发现注射Wnt5a后β-catenin的水平显著下调,提示Wnt5a的抑毛效应可能是通过抑制经典Wnt/β-catenin信号途径而达到的。同时检测到Rac2的表达显著上调,提示Wnt5a可能激活了沿Rac2的PCP途径信号链。 3.经皮内注射干扰β-catenin的腺病毒Ad-SimBC后,小鼠毛囊表型及HE染色改变类似于Wnt5a处理组,提示它们可能涉及到相似的分子机制,即抑制了经典Wnt/β-catenin信号途径。 4.对采用免疫组织化学染色技术检测Wnt5a处理组和SimBC处理组,观察到:经典Wnt/β-catenin信号途径的几个分子β-catenin、Lef1、Dvl,增殖指标Ki67和分化指标AE15表达均下降,进一步说明Wnt5a的抑毛效应可能是通过抑制经典Wnt/β-catenin途径导致的。总的来说,本课题通过一系列实验首先观察了Wnt5a在毛囊周期中的表达规律,然后分别在体外器官培养水平和动物在体水平,着重研究Wnt5a对毛囊生长的调控作用及可能的机制,旨在为深入研究不同Wnt信号在毛囊周期调控中的作用机理提供实验依据;为毛发的生长调节、皮肤组织工程提供一定的理论基础。
[Abstract]:The hair follicle is a complex skin accessory. It consists of two parts of the epithelium (the hair mother and the concentric circular structure) and the dermis (papillae and connective tissue sheath). It contains more than 20 kinds of cells, including keratinocytes, melanocytes, Langerhans cells, Merkel cells, dermal sheath cells and dermal papilla cells, and these cells. Through complex interaction in the body, it participates in the development, growth, differentiation and periodic regulation of hair follicles. Hair follicles rely on periodic circulation to realize hair growth and renewal. According to the morphological changes of the hair follicles, the hair follicles can be divided into three stages: the growth period (anagen) hair follicles continue to extend, and the degenerative period (catagen) of the hair nipple begins to move up. The hair mother cells disappear and the hair follicle gradually shortens; at telogen, the dermal papilla reaches the boundary between the dermis and the subcutaneous tissue and is connected to the hair follicle only by the tiny epithelial cells. In the different cycles of the follicle growth, some active proteins, cytokines and hormones can promote or inhibit the cycle or growth of the hair follicle. The composition of the hair growth is of great significance for clarifying the regulation mechanism of hair follicle growth cycle and developing the effective means of treating hair loss. At present, the research shows that the development and maintenance of hair follicles are regulated by various signal pathways, such as Wnt, Bmp, Shh and so on. Among them, the classical Wnt/ beta -catenin pathway is becoming more and more concerned. This approach is recognized. 19 Wnt protein family members have been found in mammals, and there are many Wnt expressions in the development of mouse hair follicles, including the classic Wnts, including the non classical Wnts., but so far the family of the family of the two major types of Wnts in the hair follicle cycle regulation It is not clear whether there is a difference in whether it exists.
Wnt5a (Wingless-type MMTV integration site family, member 5A), as the representative of the non classical Wnts of the Wnt family, plays an important role in cell proliferation, differentiation, migration, movement, and polarity regulation. The signal network involved is complex and interlaced. In addition to the classic Wnt pathway, many non classical Wnt signal pathways can be activated. Wnt/Ror2 pathway and so on. Recent studies have found that the non classical Wnt5a pathway can inhibit the activation of the classical Wnt/ beta -catenin pathway by promoting the degradation of beta -catenin. In order to understand the role of Wnt5a in the regulation of hair follicle cycle more clearly, this study first uses in situ hybridization, immunohistochemistry, semi quantitative RT-PCR and Western blot technology, to observe W. The temporal and spatial expression of nt5a in the cycle of mouse hair follicle; secondly in the culture level of hair follicle in vitro, using adenovirus mediated method to detect the regulation effect of Wnt5a on hair follicle growth, and then use the hair synchronization animal model and intradermal injection of adenovirus to further verify the effect of Wnt5a on the growth of hair follicles from the body level, and use the base. The mechanism of the effect may be analyzed by means of chip detection, quantitative RT-PCR, IHC and so on.
The expression of Wnt5a in the cycle of mouse hair follicle
1. with digoxin labeled Wnt5a RNA as a probe, the whole-mount in situ hybridization technique of dorsal skin was used to observe: (1) there was a strong Wnt5a mRNA signal in the growth period of the hair bulb, and a large number of positive cells were observed in matrix, IRS and DP, especially in the strong proliferative matrix. A large number of Wnt5a were secreted by the growth period hair follicle cells, strongly suggestive of it. It can participate in the regulation of the growth process of hair follicle. After entering the degenerative period, the positive signal of the apoptotic part of the hair bulb gradually decreases. (3) the expression of.Wnt5a mRNA expression of only a small amount of Wnt5a mRNA in the stillage hair follicle shows significant periodic changes.
2. the immunohistochemical method of the dorsal skin tissue was used to observe: (1) Wnt5a protein was widely distributed in the growth period hair follicles, except matrix, IRS, DP, precortex cells and ORS, and the expression of the protein was gradually increased with the growth of the follicle, and the distribution of.Wnt5a protein was more diffuse than mRNA, indicating the cells secreting Wnt5a and the cells that could receive Wnt5a signals. It may not be completely consistent, suggesting that the pathway of Wnt5a in hair follicles may exist in two ways of autocrine and paracrine. (2) the expression of the upper cord decreases gradually during the degenerative period. (3) the expression of the Wnt5a protein is limited to the bulge region, secondary hair germ and DP.
3. the expression trend of Wnt5a mRNA and protein was the same as in situ hybridization and IHC through the semi quantitative RT-PCR and Western blot techniques of the dorsal skin tissue. All of them showed that the growth period was the most abundant, the degenerate period weakened and the rest period was the lowest.
Two, in vitro organ level detection of Wnt5a on hair follicle growth.
1. the immunohistochemical staining of the tentacle tissue was used to observe the expression of Wnt5a protein as similar to the dorsal skin, indicating that Wnt5a may play a similar role in the two hair follicle types.
2. the organ culture of the tentacle hair follicle in vitro was carried out by adenovirus mediated method. It was observed that the hair length of hair follicles in the Wnt5a treatment group shortened, suggesting that Wnt5a could inhibit the growth of hair follicles.
Three, animal in vivo level further verified the inhibitory effect of Wnt5a on hair follicle growth.
1. the model of inducing hair induction synchronizing in mice was established. After intradermal injection of adenovirus, it was observed that the injection site of Wnt5a group had obvious hair suppression area. HE staining showed that the hair follicle in this area was stationary phase, and the peripheral hair follicle had entered the middle growth period, suggesting that Wnt5a inhibited the growth of hair follicle induced by plucking and interfered with the process of hair follicle anagen. After the time, the hair suppression area will gradually shrink to disappear and grow normal hair, which indicates that a single Wnt factor can only temporarily inhibit hair follicle growth.
2. the whole genome chip detection and quantitative RT-PCR were used to detect the level of beta -catenin after the injection of Wnt5a, suggesting that the inhibitory effect of Wnt5a might be achieved by inhibiting the classical Wnt/ beta -catenin signal pathway. Meanwhile, the expression of Rac2 was significantly up-regulated, suggesting that Wnt5a may activate the PCP pathway chain along Rac2.
3. after intradermal injection of adenovirus Ad-SimBC interfering with beta -catenin, the phenotype of hair follicles and HE staining changes in mice are similar to those in the Wnt5a treatment group, suggesting that they may involve a similar molecular mechanism, that is, the inhibition of the classical Wnt/ beta -catenin signal pathway.
4. the Wnt5a treatment group and SimBC treatment group were detected by immunohistochemical staining. The expression of several molecules beta -catenin, Lef1, Dvl, proliferation index Ki67 and differentiation index of the classical Wnt/ beta -catenin signal pathway were decreased, which further indicated that the inhibition effect of Wnt5a was probably caused by the inhibition of the classic Wnt/ beta -catenin pathway.
In general, this topic first observed the expression of Wnt5a in the hair follicle cycle through a series of experiments, then respectively in the level of organ culture in vitro and in the body level of animals, focusing on the regulatory role of Wnt5a on hair follicle growth and possible mechanisms, aiming at studying the mechanism of different Wnt signals in the regulation of hair follicle cycle. It provides experimental basis and provides a theoretical basis for skin growth engineering and skin tissue engineering.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R751

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