本文選題：Wnt3a + Wnt/β-catenin途徑��； 參考：《第三軍醫(yī)大學(xué)》2012年博士論文

【摘要】：研究背景： 黑素細(xì)胞在黑素小體內(nèi)合成黑素，并將黑素顆粒傳遞給臨近的角質(zhì)形成細(xì)胞，產(chǎn)生有顏色的皮膚或毛發(fā)。皮膚中黑素細(xì)胞的主要作用是防護(hù)人的皮膚免受紫外線的損傷。最新研究表明，白癜風(fēng)白斑表皮無黑素細(xì)胞，但毛囊外根鞘隆突區(qū)(Bulge)內(nèi)黑素干細(xì)胞仍然存在，在治療恢復(fù)區(qū)皮損中，Bulge中黑素干細(xì)胞數(shù)量明顯增多，并出現(xiàn)有功能的黑素細(xì)胞；而白癜風(fēng)恢復(fù)時(shí)首先表現(xiàn)為在毛囊口產(chǎn)生色素點(diǎn)，然后逐漸向外擴(kuò)大形成色素島，最后色素島相互融合，白斑色素恢復(fù)正常；而掌跖及黏膜等無毛囊的部位，白癜風(fēng)很難恢復(fù)。由此可見，毛囊中靜止的黑素干細(xì)胞可在治療作用下重新活化、分裂增殖，由無功能狀態(tài)轉(zhuǎn)變?yōu)楣δ軤顟B(tài)，成為白癜風(fēng)治療恢復(fù)時(shí)黑素細(xì)胞的來源。并且，研究發(fā)現(xiàn)，Bulge中黑素干細(xì)胞的提前分化是衰老性白發(fā)產(chǎn)生的基礎(chǔ)。因此，黑素細(xì)胞的作用不僅僅是產(chǎn)生黑素顆粒，它對色素減退性皮膚病(如白癜風(fēng))的復(fù)色過程，毛干的正常維持，白發(fā)形成或逆轉(zhuǎn)都有重要意義。 黑素細(xì)胞來源于神經(jīng)嵴起源的黑素母細(xì)胞。在胚胎晚期，神經(jīng)嵴細(xì)胞分化為黑素母細(xì)胞，后者通過遷移進(jìn)入表皮基底層。在哺乳動物有毛的皮膚部位，黑素母細(xì)胞進(jìn)一步由表皮遷移進(jìn)入發(fā)育中的毛囊，并最終定居在此。在小鼠，黑素母細(xì)胞在E10.5～12.5進(jìn)入真皮，E13.5進(jìn)入表皮，E14.5開始從表皮基底部向發(fā)育中的毛囊遷移，E15.5進(jìn)入毛芽，此后，黑素母細(xì)胞分為兩個(gè)不同的走向：一部分繼續(xù)遷移，進(jìn)入毛囊底部毛球部，分化為成熟黑素細(xì)胞(melanocytes，MCs)，表達(dá)MITF、TRP1、TRP2和Tyr，負(fù)責(zé)毛發(fā)的色素沉著；另一部分遷移到毛囊上部隆突區(qū)(Bulge)并定居下來，分化為黑素干細(xì)胞(melanocyte stem cells，McSCs)，只表達(dá)DCT，不產(chǎn)生黑素，是毛發(fā)黑素細(xì)胞和黑素的來源。 目前已知，正常毛發(fā)是Bulge內(nèi)兩類干細(xì)胞共同工作的結(jié)果：毛囊干細(xì)胞是新毛發(fā)生和生長的基礎(chǔ)，黑素干細(xì)胞則提供毛發(fā)黑素的來源。成熟毛囊中的黑素細(xì)胞譜系可分為三類不同的細(xì)胞類型：Bulge中主要處于靜息狀態(tài)的黑素干細(xì)胞、分化中的TA細(xì)胞(transient amplifying cells)以及位于毛球部的終末分化的黑素細(xì)胞，前兩者為未成熟黑素細(xì)胞，最后一類則是成熟黑素細(xì)胞。研究表明，毛囊黑素細(xì)胞譜系的生物學(xué)活性與毛囊周期密切相關(guān)。在毛囊靜止期末，巢內(nèi)兩類干細(xì)胞被分別激活，并進(jìn)入初始分化進(jìn)程，毛囊從靜止期轉(zhuǎn)入生長早期，此時(shí)毛囊細(xì)胞干和黑素干細(xì)胞均發(fā)生快速不對稱分裂，其子代細(xì)胞中，部分返回Bulge，恢復(fù)靜息干細(xì)胞狀態(tài)，而TA細(xì)胞則向毛囊遠(yuǎn)端遷移，最后進(jìn)入毛球，在此，兩類細(xì)胞分別進(jìn)一步增殖分化為上皮類細(xì)胞及黑素細(xì)胞。隨著毛囊和毛發(fā)生長，，黑素細(xì)胞中黑素小體出現(xiàn)并逐漸增加，隨后，黑素小體成熟并被轉(zhuǎn)運(yùn)到毛囊干細(xì)胞分化生成的接受細(xì)胞中，毛發(fā)呈現(xiàn)顏色。在生長晚期，毛球下端的上皮細(xì)胞凋亡，毛囊逐漸縮短；同時(shí)黑素細(xì)胞退化，主要黑素合成酶(TYR/TRP1)活性明顯下降，黑素生成減少，部分分化的黑素細(xì)胞凋亡。在退化期和靜止期，毛囊干細(xì)胞和黑素干細(xì)胞主要處于靜息狀態(tài)，但在靜止期末，它們又被重新激活并觸發(fā)下一個(gè)毛囊周期，開始新一輪增殖、分化和遷移的單向路徑，并再次完成毛發(fā)生長、黑素生成和傳遞。 Wnt3a是Wnt家族的成員之一，通過經(jīng)典Wnt/β-catenin途徑起作用。已有的研究表明，Wnt3a對黑素細(xì)胞發(fā)育有重要作用。靶向敲除Wnt1和Wnt3a基因的小鼠出生后沒有黑素細(xì)胞。體外研究發(fā)現(xiàn)，神經(jīng)嵴細(xì)胞在無Wnt1和Wnt3a條件情況下，分化為神經(jīng)元細(xì)胞而非黑素細(xì)胞；當(dāng)用Wnt3a處理后，可誘導(dǎo)其分化為黑素細(xì)胞而非神經(jīng)細(xì)胞。Wnt3a也可促進(jìn)神經(jīng)嵴來源細(xì)胞或胚胎干細(xì)胞向黑素母細(xì)胞分化，并進(jìn)一步分化為黑素細(xì)胞。 雖然目前對Wnt3a在黑素細(xì)胞發(fā)育過程的重要作用已有一定了解，但Wnt3a對生后的黑素細(xì)胞譜系是否也有重要作用仍不清楚。 研究目的： 本課題擬研究Wnt3a在生后小鼠毛囊周期中的時(shí)空表達(dá)情況，以及Wnt3a在黑素細(xì)胞譜系中的表達(dá)情況，并進(jìn)一步探討體內(nèi)、體外情況下過表達(dá)Wnt3a對黑素細(xì)胞譜系(成熟黑素細(xì)胞、未成熟黑素細(xì)胞——黑素干細(xì)胞和TA細(xì)胞)黑素合成的影響，并探索可能的機(jī)制。 方法： 1. Wnt3a在小鼠黑素細(xì)胞譜系中的表達(dá)研究 (1)本課題以Dct-LacZ轉(zhuǎn)基因小鼠為動物模型，通過X-gal染色技術(shù)觀察小鼠黑素細(xì)胞譜系在生后小鼠皮膚中的分布情況，并采用RT-PCR和Western blot方法對小鼠各周期中皮膚全層Wnt3a的mRNA和蛋白表達(dá)進(jìn)行半定量分析； (2)采用X-gal染色結(jié)合免疫熒光技術(shù)，免疫熒光雙標(biāo)技術(shù)，觀察Wnt3a在小鼠黑素細(xì)胞譜系中的表達(dá)情況。 (3)運(yùn)用X-gal染色結(jié)合免疫組織化學(xué)技術(shù)、免疫熒光技術(shù)，檢測Wnt/β-catenin信號途徑在生長期毛囊黑素細(xì)胞譜系中是否存在。 2. Wnt3a對小鼠黑素細(xì)胞譜系黑素生成的體外研究 (1) HEK293細(xì)胞擴(kuò)增AdWnt3a，AdGFP和AdSimMITF，并通過氯化銫梯度離心純化病毒。 (2)選取melan-a黑素細(xì)胞為成熟黑素細(xì)胞模型，通過免疫細(xì)胞化學(xué)、Western blot方法檢測腺病毒介導(dǎo)下Wnt3a在細(xì)胞中的表達(dá)情況； TOP/FOP-flash雙熒光素酶報(bào)告基因方法分析AdWnt3a感染后細(xì)胞中Wnt/β-catenin信號的激活情況；MTT、細(xì)胞計(jì)數(shù)、BrdU以及流式檢測細(xì)胞周期方法檢測Wnt3a對黑素細(xì)胞增殖的影響；黑素含量測定、酪氨酸酶活性分析、RT-PCR以及Western blot方法檢測Wnt3a對黑色合成的影響；并通過AdSimMITF阻斷內(nèi)源性MITF，分析Wnt3a對黑素細(xì)胞作用的可能機(jī)制。 (3)選取iMC23黑素細(xì)胞前體細(xì)胞為未成熟黑素細(xì)胞模型，通過形態(tài)學(xué)觀察、酪氨酸酶活性分析、Western blot方法檢測Wnt3a對未成熟黑素細(xì)胞分化以及黑素合成的影響。 (4)培養(yǎng)小鼠皮膚原代黑素細(xì)胞，通過形態(tài)學(xué)觀察分析Wnt3a對原代黑素細(xì)胞的影響。 3. Wnt3a對小鼠黑素細(xì)胞譜系黑素生成的體內(nèi)研究 (1)通過小鼠靜止期皮膚在體注射腺病毒模型，皮內(nèi)注射AdWnt3a，觀察皮膚表型及組織學(xué)觀察。 (2)通過小鼠拔毛誘導(dǎo)同步化模型，皮內(nèi)注射AdSimMITF、聯(lián)合注射AdSimMITF+AdWnt3，觀察表型改變及組織學(xué)觀察。 結(jié)果與討論： 1. Wnt3a在小鼠黑素細(xì)胞譜系中的表達(dá)研究 (1)在生長期，Wnt3a蛋白的分布較為廣泛，在表皮、外根鞘、內(nèi)根鞘以及毛球部均有表達(dá)；在退化期，Wnt3a蛋白僅在外根鞘有表達(dá)；在靜止期，幾乎檢測不到Wnt3a蛋白的表達(dá)。Wnt3a mRNA相對表達(dá)情況與免疫組化結(jié)果一致，在生長期達(dá)到峰值，退化期減弱，靜止期最弱。實(shí)驗(yàn)結(jié)果證明了Wnt3a在毛囊周期中的動態(tài)表達(dá)情況，提示W(wǎng)nt3a可能對毛囊生長、毛囊中多種組織/細(xì)胞的生物學(xué)活性起到一定的作用。 (2)在生長期毛囊中，毛囊外根鞘的黑素干細(xì)胞/TA細(xì)胞、毛球部的黑素細(xì)胞均有Wnt3a蛋白表達(dá)。在退化期毛囊中，毛囊中黑素細(xì)胞凋亡，雖然Wnt3a在外根鞘有微弱表達(dá)，但在黑素干細(xì)胞中未觀察到Wnt3a蛋白。在靜止期，無黑素細(xì)胞存在，黑素干細(xì)胞不表達(dá)Wnt3a蛋白。當(dāng)毛囊進(jìn)入下一個(gè)生長期時(shí)，Wnt3a蛋白再次表達(dá)于黑素細(xì)胞譜系。Wnt3a蛋白在黑素細(xì)胞譜系中的周期性表達(dá)，提示W(wǎng)nt3a可能對調(diào)節(jié)黑素細(xì)胞譜系的生物學(xué)活性相關(guān)。 (3)在生長期毛囊黑素細(xì)胞中，β-catenin和Lef-1均呈細(xì)胞核表達(dá)，黑素干細(xì)胞中未檢測到β-catenin細(xì)胞核表達(dá)與Lef-1的表達(dá)。實(shí)驗(yàn)結(jié)果證明生長期黑素細(xì)胞中Wnt/β-catenin信號途徑處于活化狀態(tài)，提示W(wǎng)nt/β-catenin信號途徑可能對黑素細(xì)胞的增殖、分化或黑素合成起到一定作用。 2. Wnt3a對小鼠黑素細(xì)胞譜系黑素生成的體外研究 (1)通過HEK293成功擴(kuò)增了高滴度的AdWnt3a、AdGFP和AdSimMITF。 (2) Wnt3a對小鼠成熟黑素細(xì)胞(melan-a細(xì)胞)的體外研究結(jié)果：AdWnt3a可有效感染黑素細(xì)胞，成功表達(dá)Wnt3a蛋白，并且能夠激活細(xì)胞內(nèi)Wnt/β-catenin信號途徑；Wnt3a對黑素細(xì)胞的增殖具有抑制作用，同時(shí)伴隨著S期細(xì)胞減少、G1期細(xì)胞增加現(xiàn)象；Wnt3a可通過上調(diào)MITF以及下游靶基因TYR和TRP1的表達(dá)，從而促進(jìn)黑素合成。 (3) Wnt3a對小鼠未成熟黑素細(xì)胞(iMC23細(xì)胞)的體外研究結(jié)果：Wnt3a可促進(jìn)未成熟黑素細(xì)胞前體細(xì)胞分化并產(chǎn)生黑素，在此過程中MITF可作為Wnt3a下游分子參與分化過程。 (4) Wnt3a對小鼠原代黑素細(xì)胞的體外研究結(jié)果：Wnt3a可促進(jìn)原代黑素細(xì)胞分化并產(chǎn)生黑素。 3.Wnt3a對小鼠黑素細(xì)胞譜系黑素生成的體內(nèi)研究 (1)小鼠靜止期皮膚在體注射模型結(jié)果：Wnt3a可促進(jìn)毛囊干細(xì)胞激活，同時(shí)也促進(jìn)黑素干細(xì)胞激活，分化生成黑素細(xì)胞并產(chǎn)生黑素。 (2)小鼠拔毛誘導(dǎo)同步化注射模型結(jié)果：阻斷體內(nèi)毛囊黑素細(xì)胞中MITF，可抑制黑素合成，但在Wnt3a存在的情況下，Wnt3a可挽回SimMITF對黑素合成的抑制作用，說明MITF是Wnt3a的直接靶基因。 結(jié)論： 本課題首次證明了Wnt3a在小鼠毛囊周期中的動態(tài)表達(dá)模式以及在黑素細(xì)胞譜系表達(dá)情況，并且從體外和體內(nèi)兩個(gè)方面證明了Wnt3a具有促進(jìn)未成熟黑素細(xì)胞分化以及黑素合成、促進(jìn)成熟黑素細(xì)胞黑素合成的作用。本課題著重于Wnt3a促進(jìn)毛囊黑素干細(xì)胞分化以及促進(jìn)黑素合成的作用以及相關(guān)的信號轉(zhuǎn)導(dǎo)途徑，研究結(jié)果能夠進(jìn)一步闡明黑素細(xì)胞譜系的功能并為各種色素疾患的臨床治療提供重要的理論依據(jù)。
[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錛

本文編號：1807813