Wnt3a对小鼠黑素细胞谱系黑素生成的作用研究

发布时间：2018-04-26 21:36

  本文选题：Wnt3a + Wnt/β-catenin途径　； 参考：《第三军医大学》2012年博士论文

【摘要】：研究背景： 黑素细胞在黑素小体内合成黑素，并将黑素颗粒传递给临近的角质形成细胞，产生有颜色的皮肤或毛发。皮肤中黑素细胞的主要作用是防护人的皮肤免受紫外线的损伤。最新研究表明，白癜风白斑表皮无黑素细胞，但毛囊外根鞘隆突区(Bulge)内黑素干细胞仍然存在，在治疗恢复区皮损中，Bulge中黑素干细胞数量明显增多，并出现有功能的黑素细胞；而白癜风恢复时首先表现为在毛囊口产生色素点，然后逐渐向外扩大形成色素岛，最后色素岛相互融合，白斑色素恢复正常；而掌跖及黏膜等无毛囊的部位，白癜风很难恢复。由此可见，毛囊中静止的黑素干细胞可在治疗作用下重新活化、分裂增殖，由无功能状态转变为功能状态，成为白癜风治疗恢复时黑素细胞的来源。并且，研究发现，Bulge中黑素干细胞的提前分化是衰老性白发产生的基础。因此，黑素细胞的作用不仅仅是产生黑素颗粒，它对色素减退性皮肤病(如白癜风)的复色过程，毛干的正常维持，白发形成或逆转都有重要意义。 黑素细胞来源于神经嵴起源的黑素母细胞。在胚胎晚期，神经嵴细胞分化为黑素母细胞，后者通过迁移进入表皮基底层。在哺乳动物有毛的皮肤部位，黑素母细胞进一步由表皮迁移进入发育中的毛囊，并最终定居在此。在小鼠，黑素母细胞在E10.5～12.5进入真皮，E13.5进入表皮，E14.5开始从表皮基底部向发育中的毛囊迁移，E15.5进入毛芽，此后，黑素母细胞分为两个不同的走向：一部分继续迁移，进入毛囊底部毛球部，分化为成熟黑素细胞(melanocytes，MCs)，表达MITF、TRP1、TRP2和Tyr，负责毛发的色素沉着；另一部分迁移到毛囊上部隆突区(Bulge)并定居下来，分化为黑素干细胞(melanocyte stem cells，McSCs)，只表达DCT，不产生黑素，是毛发黑素细胞和黑素的来源。 目前已知，正常毛发是Bulge内两类干细胞共同工作的结果：毛囊干细胞是新毛发生和生长的基础，黑素干细胞则提供毛发黑素的来源。成熟毛囊中的黑素细胞谱系可分为三类不同的细胞类型：Bulge中主要处于静息状态的黑素干细胞、分化中的TA细胞(transient amplifying cells)以及位于毛球部的终末分化的黑素细胞，前两者为未成熟黑素细胞，最后一类则是成熟黑素细胞。研究表明，毛囊黑素细胞谱系的生物学活性与毛囊周期密切相关。在毛囊静止期末，巢内两类干细胞被分别激活，并进入初始分化进程，毛囊从静止期转入生长早期，此时毛囊细胞干和黑素干细胞均发生快速不对称分裂，其子代细胞中，部分返回Bulge，恢复静息干细胞状态，而TA细胞则向毛囊远端迁移，最后进入毛球，在此，两类细胞分别进一步增殖分化为上皮类细胞及黑素细胞。随着毛囊和毛发生长，，黑素细胞中黑素小体出现并逐渐增加，随后，黑素小体成熟并被转运到毛囊干细胞分化生成的接受细胞中，毛发呈现颜色。在生长晚期，毛球下端的上皮细胞凋亡，毛囊逐渐缩短；同时黑素细胞退化，主要黑素合成酶(TYR/TRP1)活性明显下降，黑素生成减少，部分分化的黑素细胞凋亡。在退化期和静止期，毛囊干细胞和黑素干细胞主要处于静息状态，但在静止期末，它们又被重新激活并触发下一个毛囊周期，开始新一轮增殖、分化和迁移的单向路径，并再次完成毛发生长、黑素生成和传递。 Wnt3a是Wnt家族的成员之一，通过经典Wnt/β-catenin途径起作用。已有的研究表明，Wnt3a对黑素细胞发育有重要作用。靶向敲除Wnt1和Wnt3a基因的小鼠出生后没有黑素细胞。体外研究发现，神经嵴细胞在无Wnt1和Wnt3a条件情况下，分化为神经元细胞而非黑素细胞；当用Wnt3a处理后，可诱导其分化为黑素细胞而非神经细胞。Wnt3a也可促进神经嵴来源细胞或胚胎干细胞向黑素母细胞分化，并进一步分化为黑素细胞。 虽然目前对Wnt3a在黑素细胞发育过程的重要作用已有一定了解，但Wnt3a对生后的黑素细胞谱系是否也有重要作用仍不清楚。 研究目的： 本课题拟研究Wnt3a在生后小鼠毛囊周期中的时空表达情况，以及Wnt3a在黑素细胞谱系中的表达情况，并进一步探讨体内、体外情况下过表达Wnt3a对黑素细胞谱系(成熟黑素细胞、未成熟黑素细胞——黑素干细胞和TA细胞)黑素合成的影响，并探索可能的机制。 方法： 1. Wnt3a在小鼠黑素细胞谱系中的表达研究 (1)本课题以Dct-LacZ转基因小鼠为动物模型，通过X-gal染色技术观察小鼠黑素细胞谱系在生后小鼠皮肤中的分布情况，并采用RT-PCR和Western blot方法对小鼠各周期中皮肤全层Wnt3a的mRNA和蛋白表达进行半定量分析； (2)采用X-gal染色结合免疫荧光技术，免疫荧光双标技术，观察Wnt3a在小鼠黑素细胞谱系中的表达情况。 (3)运用X-gal染色结合免疫组织化学技术、免疫荧光技术，检测Wnt/β-catenin信号途径在生长期毛囊黑素细胞谱系中是否存在。 2. Wnt3a对小鼠黑素细胞谱系黑素生成的体外研究 (1) HEK293细胞扩增AdWnt3a，AdGFP和AdSimMITF，并通过氯化铯梯度离心纯化病毒。 (2)选取melan-a黑素细胞为成熟黑素细胞模型，通过免疫细胞化学、Western blot方法检测腺病毒介导下Wnt3a在细胞中的表达情况； TOP/FOP-flash双荧光素酶报告基因方法分析AdWnt3a感染后细胞中Wnt/β-catenin信号的激活情况；MTT、细胞计数、BrdU以及流式检测细胞周期方法检测Wnt3a对黑素细胞增殖的影响；黑素含量测定、酪氨酸酶活性分析、RT-PCR以及Western blot方法检测Wnt3a对黑色合成的影响；并通过AdSimMITF阻断内源性MITF，分析Wnt3a对黑素细胞作用的可能机制。 (3)选取iMC23黑素细胞前体细胞为未成熟黑素细胞模型，通过形态学观察、酪氨酸酶活性分析、Western blot方法检测Wnt3a对未成熟黑素细胞分化以及黑素合成的影响。 (4)培养小鼠皮肤原代黑素细胞，通过形态学观察分析Wnt3a对原代黑素细胞的影响。 3. Wnt3a对小鼠黑素细胞谱系黑素生成的体内研究 (1)通过小鼠静止期皮肤在体注射腺病毒模型，皮内注射AdWnt3a，观察皮肤表型及组织学观察。 (2)通过小鼠拔毛诱导同步化模型，皮内注射AdSimMITF、联合注射AdSimMITF+AdWnt3，观察表型改变及组织学观察。 结果与讨论： 1. Wnt3a在小鼠黑素细胞谱系中的表达研究 (1)在生长期，Wnt3a蛋白的分布较为广泛，在表皮、外根鞘、内根鞘以及毛球部均有表达；在退化期，Wnt3a蛋白仅在外根鞘有表达；在静止期，几乎检测不到Wnt3a蛋白的表达。Wnt3a mRNA相对表达情况与免疫组化结果一致，在生长期达到峰值，退化期减弱，静止期最弱。实验结果证明了Wnt3a在毛囊周期中的动态表达情况，提示Wnt3a可能对毛囊生长、毛囊中多种组织/细胞的生物学活性起到一定的作用。 (2)在生长期毛囊中，毛囊外根鞘的黑素干细胞/TA细胞、毛球部的黑素细胞均有Wnt3a蛋白表达。在退化期毛囊中，毛囊中黑素细胞凋亡，虽然Wnt3a在外根鞘有微弱表达，但在黑素干细胞中未观察到Wnt3a蛋白。在静止期，无黑素细胞存在，黑素干细胞不表达Wnt3a蛋白。当毛囊进入下一个生长期时，Wnt3a蛋白再次表达于黑素细胞谱系。Wnt3a蛋白在黑素细胞谱系中的周期性表达，提示Wnt3a可能对调节黑素细胞谱系的生物学活性相关。 (3)在生长期毛囊黑素细胞中，β-catenin和Lef-1均呈细胞核表达，黑素干细胞中未检测到β-catenin细胞核表达与Lef-1的表达。实验结果证明生长期黑素细胞中Wnt/β-catenin信号途径处于活化状态，提示Wnt/β-catenin信号途径可能对黑素细胞的增殖、分化或黑素合成起到一定作用。 2. Wnt3a对小鼠黑素细胞谱系黑素生成的体外研究 (1)通过HEK293成功扩增了高滴度的AdWnt3a、AdGFP和AdSimMITF。 (2) Wnt3a对小鼠成熟黑素细胞(melan-a细胞)的体外研究结果：AdWnt3a可有效感染黑素细胞，成功表达Wnt3a蛋白，并且能够激活细胞内Wnt/β-catenin信号途径；Wnt3a对黑素细胞的增殖具有抑制作用，同时伴随着S期细胞减少、G1期细胞增加现象；Wnt3a可通过上调MITF以及下游靶基因TYR和TRP1的表达，从而促进黑素合成。 (3) Wnt3a对小鼠未成熟黑素细胞(iMC23细胞)的体外研究结果：Wnt3a可促进未成熟黑素细胞前体细胞分化并产生黑素，在此过程中MITF可作为Wnt3a下游分子参与分化过程。 (4) Wnt3a对小鼠原代黑素细胞的体外研究结果：Wnt3a可促进原代黑素细胞分化并产生黑素。 3.Wnt3a对小鼠黑素细胞谱系黑素生成的体内研究 (1)小鼠静止期皮肤在体注射模型结果：Wnt3a可促进毛囊干细胞激活，同时也促进黑素干细胞激活，分化生成黑素细胞并产生黑素。 (2)小鼠拔毛诱导同步化注射模型结果：阻断体内毛囊黑素细胞中MITF，可抑制黑素合成，但在Wnt3a存在的情况下，Wnt3a可挽回SimMITF对黑素合成的抑制作用，说明MITF是Wnt3a的直接靶基因。 结论： 本课题首次证明了Wnt3a在小鼠毛囊周期中的动态表达模式以及在黑素细胞谱系表达情况，并且从体外和体内两个方面证明了Wnt3a具有促进未成熟黑素细胞分化以及黑素合成、促进成熟黑素细胞黑素合成的作用。本课题着重于Wnt3a促进毛囊黑素干细胞分化以及促进黑素合成的作用以及相关的信号转导途径，研究结果能够进一步阐明黑素细胞谱系的功能并为各种色素疾患的临床治疗提供重要的理论依据。
[Abstract]:Research background:
Melanocytes synthesize melanin in the melanin body and transmit melanin particles to adjacent keratinocytes to produce coloured skin or hair. The main role of melanocytes in the skin is to protect human skin from UV damage. The latest research shows that vitiligo is not melanocyte, but the outer root sheath of the hair follicle (B Ulge) the endogenous melanin stem cells still exist. In the skin lesions of the recovery area, the number of melanin stem cells in the Bulge is significantly increased and the functional melanocytes appear. In the recovery of vitiligo, the pigment spots are produced at the mouth of the hair follicle, and then the pigment island is gradually expanded outward. Finally, the pigment island is fused and the pigment of leukoplakia is restored to normal. It is clear that the static melanin stem cells in the hair follicle can be reactivated, split and proliferate from non functional state to functional state, and become the source of melanocytes in the treatment and recovery of vitiligo. Furthermore, the study found that melanin stem cells in Bulge have been extracted. Pre differentiation is the basis of aging white hair. Therefore, the role of melanocytes is not only the production of melanin particles, but also is of great significance to the process of color reduction of pigmented dermatosis (such as vitiligo), the normal maintenance of hair dry, the formation and reversal of white hair.
Melanocytes originate in melanocytes from the origin of the neural crest. In the late embryo, the neural crest cells differentiated into melanocytes, the latter migrated into the basal layer of the epidermis. In the hairy parts of the mammal, melanoma cells migrated further from the epidermis into the developing hair follicles and settled here. In mice, melanocytes. When E10.5 ~ 12.5 enters the dermis, E13.5 enters the epidermis, E14.5 begins to migrate from the basal part of the epidermis to the developing hair follicle, and E15.5 enters the hair bud. After that, the melanoma cells are divided into two different directions: a part continues to migrate, enter the hair bulb at the bottom of the hair follicle, and differentiate into the mature melanocytes (melanocytes, MCs), and express MITF, TRP1, TRP2 and Tyr, negative. The pigment was responsible for the hair; the other migrated to the upper part of the hair follicle (Bulge) and settled down, differentiated into melanocyte stem cells (McSCs), expressed only DCT, and did not produce melanin, which was a source of melanin and melanin.
It is known that normal hair is the result of the joint work of two types of stem cells in Bulge: hair follicle stem cells are the basis for the occurrence and growth of new hair, and melanin stem cells provide the source of melanin. The melanocyte lineage in the mature hair follicles can be divided into three different types of cell types: the main melanin stem cells in the resting state in Bulge are divided into two types. The TA cells (transient amplifying cells) and the final differentiation of melanocytes at the end of the hair bulb are immature melanocytes and the final class is mature melanocytes. The study shows that the biological activity of the follicle melanocyte lineage is closely related to the hair follicle cycle. At the end of the hair follicle, the two types of stem cells in the nest are in the nest. In the initial differentiation process, the follicle was transferred from the stationary phase to the early growth stage. At this time, the follicle cell stem and the melanin stem cells all had rapid asymmetric division. In their progeny cells, part of the cells returned to Bulge to restore the state of resting stem cells, while the TA cells migrated to the Mao Nangyuan end and finally entered the hair ball. Here, the two types of cells were entered respectively. With the growth of hair follicles and hair, melanosomes appear and gradually increase as the hair follicles and hair grow. Then, the melanosomes mature and are transported to the recipient cells of the hair follicle stem cells. The hair appears in color. In the late growth, the epithelial cells at the lower end of the hair bulb are apoptotic and the hair follicle gradually shortens. At the same time, melanocyte degradation, major melanin synthase (TYR/TRP1) activity decreased significantly, melanin production decreased, and partially differentiated melanocytes apoptosis. In degenerate and stationary phase, hair follicle stem cells and melanin stem cells were mainly in resting state, but at the end of the period, they were reactivated and triggered the next hair follicle cycle and began to be new. A one-way path of proliferation, differentiation and migration, and once again complete hair growth, melanogenesis and transmission.
Wnt3a, one of the members of the Wnt family, plays a role in the classic Wnt/ beta -catenin pathway. Previous studies have shown that Wnt3a plays an important role in the development of melanocytes. The mice targeting the knockout of Wnt1 and Wnt3a genes have no melanocytes after birth. In vitro studies have found that neural crest cells are differentiated into neuronal cells without Wnt1 and Wnt3a conditions. It is not a melanocyte; when treated with Wnt3a, it can be induced to differentiate into melanocytes instead of nerve cells.Wnt3a, and can also promote the differentiation of neural crest derived cells or embryonic stem cells to melanocytes, and further differentiate into melanocytes.
Although there is a certain understanding of the important role of Wnt3a in the development of melanocyte, it is not clear whether Wnt3a has an important role in the postnatal melanocyte lineage.
The purpose of the study is:
We intend to study the temporal and spatial expression of Wnt3a in the cycle of postnatal mouse hair follicles, and the expression of Wnt3a in the melanocyte lineage, and further explore the effect of overexpression of Wnt3a on melanogenesis of melanocyte lineage (mature melanocytes, melanin stem cells and TA cells) in vitro, in vitro and in vitro. And explore possible mechanisms.
Method锛

本文编号：1807813