天冬氨酸—丝氨酸—丝氨酸重复多肽促进毛发生长作用及其机制研究

发布时间：2018-05-30 23:05

本文选题：8DSS + 毛乳头细胞　；参考：《第三军医大学》2015年博士论文

【摘要】：研究背景毛发具有防御、保护及增加信息交流和美观的作用。毛发由毛囊生成,毛囊是一种结构复杂的皮肤附属器官,持续经历着生长期、退行期和休止期的周期循环性生长。毛囊周期一旦失调,将会影响毛囊正常生长从而导致脱发疾病的发生。随着社会发展,人们对具有保护与美观作用的毛发越来越重视,但临床上疗效确切的促毛发生长药物仍十分匮乏。目前,仅美国FDA批准的口服药物非那雄胺和外用药物米诺地尔具有较肯定的疗效。因此,对促进毛囊再生、毛发生长药物的研究逐渐成为皮肤学领域的研究热点与难点。毛乳头(DP)是毛囊的主要间充质成分,毛乳头细胞是构成毛乳头的主要间质细胞,具有维持诱导上皮细胞增殖分化形成毛囊,促进毛发生长发育和调控毛囊周期循环的能力。鉴于毛乳头细胞具有诱导非毛发皮肤毛囊形成的能力,毛乳头细胞可以作为应于脱发疾病的治疗的靶细胞。毛乳头细胞能分泌多种细胞因子如干细胞生长因子(SCF)、血管内皮细胞生长因子(VEGF)、肝细胞生长因子(HGF)、胰岛素样生长因子(IGF)、转化生长因子β(TGF-β)、骨形成蛋白(BMP)等生长因子和信号转导分子刺激上皮细胞增殖、分化,启动毛囊进入生长期,促进毛囊的生长发育。启动休止期毛囊激活的机制仍不清楚,但是认为来源于毛乳头的信号发挥了至关重要的作用,阻断毛乳头细胞与Wnt信号通路应答的能力将使毛乳头细胞分泌诱导上皮细胞增殖分化的细胞因子的能力下降,导致毛囊周期失调。在调控毛乳头细胞相关的复杂信号通路网络研究中,细胞信号传导(cell signal transduction)日益受到人们重视。经典信号通路Wnt/β-catenin信号对于毛乳头细胞的增殖、细胞因子分泌及诱导毛囊再生至关重要,是调控毛囊发育必须的、最早期和最重要的信号之一。糖原合成激酶3β(GSK-3β)为Wnt/β-catenin经典信号通路的关键分子,GSK-3β激酶失活可激活Wnt/β-catenin信号通路,维持毛乳头细胞诱导上皮细胞增殖、分化及调节细胞周期的能力,从而促进毛囊生长发育。GSK-3β不仅是β-catenin通路激活的关键分子,同时也是PI3K/Akt信号通路重要的下游靶基因,激活PI3K/Akt通路,磷酸化gsk-3β,从而抑制gsk-3β活性可促进细胞增殖,阻碍细胞的凋亡。天冬氨酸-丝氨酸-丝氨酸(dss)是牙本质磷蛋白(dpp)中一个氨基酸重复序列。8dss是一种人工合成的由8个dss重复序列组成的多肽。8dss有很强的结合钙离子的能力,并在牙齿基质的沉积以及矿化过程起到重要作用。研究发现8dss外用于balb/c小鼠脱毛区域后可促进该部位毛发再生(pub.no.us2010/0239503a1)。据此我们推测8dss极可能通过对pi3k/akt/gsk-3β通路的影响从而调控毛囊生长周期进而实现对毛发生长的促进作用。为了验证这一猜想,我们以毛囊生长周期为切入点,围绕wnt/β-catenin和pi3k/akt信号通路,采用real-timepcr,westernblot等方法从细胞及分子水平探讨了8dss对毛乳头细胞诱导功能及wnt/β-catenin和pi3k/akt信号通路的影响及其作用机制,并在脱发小鼠模型上进行了功能验证,以期揭示8dss在毛发生长中的作用及其作用机制。本研究将可能为脱发性疾病提供新的治疗靶点和防治策略。目的1、验证8dss能否促进c57bl/6脱毛小鼠毛发生长及其对毛囊生长周期的影响2、探讨8dss是否具有调控毛乳头细胞增殖、分泌细胞因子、信号分子的作用;3、探讨8dss是否通过调控pi3k/akt/gsk-3β信号通路参与调控毛乳头细胞分泌功能从而促进毛囊的生长发育。材料与方法1、将荧光1%8dss涂抹于处于生长期小鼠背部脱毛区域,观察8dss渗透途径。在小鼠脱毛区域涂抹1%8dss后,于不同时间点观察鼠背皮肤颜色变化及毛发生长情况;取用药7天后小鼠背部皮肤,利用he染色观察镜下毛囊形态变化情况;利用免疫组化检测毛囊中β-catenin表达情况;2、采用“二步酶消化法”提取毛乳头,进行毛乳头细胞培养;利用免疫荧光鉴定毛乳头细胞。3、利用cck8法检测8dss对毛乳头细胞增殖的影响;利用rt-pcr检测8dss对毛乳头细胞分泌细胞因子、信号分子的影响;4、利用westernblot检测p-gsk-3β及p-akt、bcl-2及核内β-catenin表达水平。5、利用特异性抑制剂ly294002阻断pi3k/akt信号通路后,利用westernblot检测p-gsk-3β及p-akt水平的表达。结果1、8dss能够沿毛囊皮脂腺途径渗透荧光8dss局部涂抹脱毛部位皮肤后1小时观察到绿色荧光主要分布在表皮,2小时后8dss逐渐沿毛囊外毛根鞘部位渗透到达整个毛囊,说明8dss能够作用于毛囊部位。2、8dss能够诱导毛囊周期性再生8dss溶液局部涂抹于小鼠脱毛部位后,观察到小鼠背部皮肤颜色粉红色到黑色的转变时间早于阴性对照组,说明8dss能够使毛囊从休止期提前进入生长期,从而促进毛发生长。he染色后显微镜下见8dss处理组毛囊生长期呈iv期表现变,而阴性对照组呈ii期表现。免疫组化显示8dss处理组毛囊显著表达β-catenin。3、8dss能够诱导毛乳头细胞分泌信号分子cck8检测结果显示8dss在不能促进毛乳头细胞增殖,低浓度8dss对毛乳头细胞增殖无明显影响,随着浓度升高对毛乳头细胞反有抑制作用;但是通过rt-pcr分析发现,低浓度8dss处理的毛乳头细胞内wnt信号通路相关基因和转录因子如wnt10b,lef-1和c-myc的表达上调,提示8dss虽不能促进毛乳头细胞增殖,但能促进毛乳头细胞的诱导上皮细胞增殖、分化的能力。4、8dss可诱导核内β-catenin蛋白激活wnt/β-catenin信号8dss处理毛乳头细胞后1h、24h后用westernblot检测结果显示核内β-catenin蛋白表达逐渐增强,并呈时间依赖性,提示8dss可显著增加wnt/β-catenin信号通路在毛乳头细胞内的活性,激活wnt/β-catenin信号通路。5、8dss能激活毛乳头细胞内pi3k/akt信号通路8dss处理后5min、15min、30min、60min用westernblot检测结果显示:抗凋亡因子bcl-2、p-gsk-3β及p-akt表达显著增强并呈时间依赖性,提示8dss可激活毛乳头细胞内pi3k/akt信号通路,并促进其下游靶基因bcl-2的表达抗细胞凋亡,使gsk-3β蛋白磷酸化而失活。6、8dss经由pi3k/akt/gsk-3β信号发挥诱导毛囊周期性再生作用ly294002阻断pi3k/akt信号通路后8dss不能增强gsk-3β及akt磷酸化水平,提示8dss增加wnt/β-catenin信号通路在毛乳头细胞内的活性可能是通过激活pi3k/akt/gsk-3β信号通路实现。结论1、8dss能调控毛囊周期性再生,具有促进毛发生长的作用。这为临床提供新的脱发疾病治疗药物提供了有意义的实验资料。2、8dss能够促进毛乳头细胞分泌wnt相关基因及增强核内β-catenin蛋白表达,PI3K/Akt/GSK-3β信号通路是8DSS调控毛乳头细胞这种功能的关键信号通路,这为8DSS以后用于临床上治疗脱发疾病提供了重要的理论参考。
[Abstract]:Background hair has the function of defending, protecting and increasing information exchange and beauty. Hair is produced by hair follicle. Hair follicle is a complex structure of skin accessory, which continues to undergo cycle cycle growth during the period of growth, degenerative period and rest period. Once the hair follicle cycle is in disorder, it will affect the normal growth of the hair follicle and lead to hair loss. With the development of society, people pay more and more attention to the hair which has the role of protection and beauty, but the clinically effective medicine for promoting hair growth is still very scarce. At present, only the American FDA approved oral drug, finasteride and external drug minoxidil, has a more positive effect. Therefore, it is necessary to promote hair follicle regeneration and hair growth. The study of drugs has gradually become a hot and difficult point in the field of dermatology. DP is the main interstitial component of hair follicles. The dermal papilla cells are the main interstitial cells of hair nipples, which have the ability to maintain the proliferation and differentiation of epithelial cells to form hair follicles, promote the growth and development of hair and regulate the cycle cycle of hair follicles. The head cells have the ability to induce the formation of non hairy skin follicles, and dermal papilla cells can be used as target cells for the treatment of hair loss. The dermal papilla cells can secrete a variety of cytokines such as stem cell growth factor (SCF), vascular endothelial growth factor (VEGF), liver cell growth factor (HGF), and insulin like growth factor (IGF). Growth factor beta (TGF- beta), bone morphogenetic protein (BMP) and other growth factors and signal transduction molecules stimulate epithelial cell proliferation, differentiation, initiation of hair follicles entering the growth period, promoting the growth and development of hair follicles. The mechanism of activating the hair follicle at the end of the rest period is still unclear, but it is considered that the signal from the hair nipple has played a vital role in blocking the hair milk. The ability of the head cells to respond to the Wnt signaling pathway will decrease the ability of the dermal papilla cells to induce the proliferation and differentiation of epithelial cells and lead to the dysregulation of hair follicles. In the study of the complex signaling pathway related to the regulation of dermal papilla cells, cell signal transduction is becoming more and more important. The signal pathway Wnt/ beta -catenin signal is very important for the proliferation of dermal papilla cells, cytokine secretion and induction of hair follicle regeneration. It is one of the most important signals to regulate the development of hair follicles. The glycogen kinase 3 beta (GSK-3 beta) is the key molecule of the classical signaling pathway of Wnt/ beta -catenin, and the inactivation of GSK-3 beta kinase activates Wnt/ beta. Catenin signaling pathway, which maintains the ability of dermal papilla cells to induce epithelial cell proliferation, differentiation and regulation of cell cycle, thus promotes the growth and development of.GSK-3 beta not only a key molecule in the activation of the beta -catenin pathway, but also an important downstream target gene of the PI3K/Akt signaling pathway, activating the PI3K/Akt pathway, phosphorylating GSK-3 beta, and thus inhibiting the gsk- 3 beta activity can promote cell proliferation and inhibit cell apoptosis. Aspartic acid serine serine (DSS) is an amino acid repeat sequence.8dss in dentin phosphate (DPP), a synthetic polypeptide composed of 8 DSS repeats, which has a strong ability to bind calcium ions, and in the deposition and mineralization of dental matrix. The study found that 8dss was used to promote hair regeneration (pub.no.us2010/0239503a1) in the balb/c mice after being used in hair removal (pub.no.us2010/0239503a1). Therefore, we speculate that the 8dss may regulate the growth cycle of the hair follicle by regulating the growth cycle of the follicle to promote the growth of hair. In order to verify this guess, With the growth cycle of hair follicles as a breakthrough point, we studied the effects and mechanism of 8dss on the induction of dermal papilla cells and the wnt/ beta -catenin and pi3k/akt signaling pathways from the cellular and molecular levels around wnt/ beta -catenin and pi3k/akt signaling pathways, using real-timepcr, Westernblot and other methods, and the function of 8dss in the hair loss mouse model was carried out. The purpose of this study is to reveal the role of 8dss in hair growth and its mechanism of action. This study may provide new therapeutic targets and prevention strategies for hair loss. 1, the purpose of this study is to verify whether 8dss can promote hair growth and the growth cycle of hair follicles in c57bl/6 hairy mice, and to explore whether 8dss can regulate the proliferation of dermal papilla cells. The role of secreting cell factors and signal molecules; 3, to explore whether 8dss promotes the growth and development of hair follicles by regulating the pi3k/akt/gsk-3 beta signaling pathway to regulate the secretory function of dermal papilla cells. Materials and methods 1, smear fluorescent 1%8dss on the back depilatory region of the growth stage of mice and observe the 8dss infiltration pathway. After applying 1%8dss, the skin color changes and hair growth were observed at different time points. After taking medication for 7 days, the morphological changes of the hair follicles were observed by HE staining, and the expression of beta -catenin in the hair follicles was detected by immunohistochemistry; 2, the papilla cell culture was extracted by the "two step enzyme digestion", and the dermal papilla cells were cultured. .3 was used to detect the effect of 8dss on the proliferation of dermal papilla cells by CCK8, and the effect of 8dss on the secretion of cytokines and signal molecules by 8dss in dermal papilla cells. 4, Westernblot was used to detect p-gsk-3 beta and p-Akt, Bcl-2 and the expression level of beta -catenin was.5, and specific inhibitor LY294002 was used. After blocking the pi3k/akt signaling pathway, Westernblot was used to detect the expression of p-gsk-3 beta and p-Akt. Results 1,8dss was able to observe the green fluorescence in the epidermis for 1 hours along the sebaceous gland pathway of the hair follicle, and observed that the green fluorescence was mainly in the epidermis. After 2 hours, 8dss gradually penetrated into the whole hair follicle along the outer hair root sheath of the hair follicle. It is indicated that 8dss can act on the hair follicle site.2,8dss to induce the periodic regeneration of the hair follicle by applying the 8dss solution to the depilatory part of the mouse. The time of the color pink to black change of the mouse's back skin is earlier than that of the negative control group. It shows that the 8dss can advance the hair follicle from the rest period to the growth period, thus promoting the growth of hair.He. The growth period of the follicle in the 8dss treatment group was IV stage after staining, and the negative control group showed II stage performance. The immunohistochemical staining showed that the expression of beta -catenin.3,8dss in the hair follicle in the 8dss treatment group could induce the CCK8 detection of the secretory signal molecules of the dermal papilla cells, which showed that 8dss could not promote the proliferation of dermal papilla cells and the low concentration 8dss to hair milk. The proliferation of head cells had no obvious effect on the proliferation of dermal papilla cells. However, RT-PCR analysis showed that the expression of Wnt signaling pathway related genes and transcription factors such as Wnt10b, LEF-1 and c-myc in the dermal papilla cells treated with low concentration of 8dss increased, suggesting that 8dss could not promote the proliferation of dermal papilla cells, but could promote the proliferation of dermal papilla cells. The proliferation of epithelial cells induced by the dermal papilla cells, the ability of.4,8dss to induce beta -catenin protein to activate wnt/ beta -catenin signal 8dss to treat 1h in dermal papilla cells after 24h, and Westernblot detection results showed that the expression of beta -catenin protein in the nucleus increased gradually after 24h, and was dependent on the time dependence, suggesting that 8dss can significantly increase wnt/ beta -catenin signal. The activity of the pathway in the dermal papilla cells, activating the wnt/ beta -catenin signaling pathway.5,8dss can activate the pi3k/akt signaling pathway in the dermal papilla cells after 8dss treatment, 5min, 15min, 30min, and 60min using Westernblot detection results show that the anti apoptotic factor Bcl-2, p-gsk-3 beta and expression are significantly enhanced and time dependent, suggesting that the dermal papilla can be activated. The intracellular pi3k/akt signaling pathway and promoting the expression of the downstream target gene Bcl-2 against apoptosis, the GSK-3 beta protein phosphorylation and inactivation of.6,8dss via pi3k/akt/gsk-3 beta signal to induce the periodic regeneration of the hair follicle by LY294002 blocking the pi3k/akt signaling pathway, 8dss can not enhance the GSK-3 beta and Akt phosphorylation level, suggesting 8dss increase of wnt/ beta. The activity of -catenin signaling pathway in the dermal papilla cells may be realized by activating the pi3k/akt/gsk-3 beta signaling pathway. Conclusion 1,8dss can regulate the periodic regeneration of hair follicles and promote hair growth. This provides a meaningful experimental data for the clinical treatment of new hair loss diseases,.2,8dss can promote dermal papilla cells. Secreting Wnt related genes and enhancing the expression of beta -catenin protein in the nucleus, PI3K/Akt/GSK-3 beta signaling pathway is the key signal pathway for the function of 8DSS to regulate the function of dermal papilla cells, which provides an important theoretical reference for the clinical treatment of hair loss after 8DSS.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R339.1

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