Wnt10b在毛囊周期调控中的作用及机制研究

Wnt10b在毛囊周期调控中的作用及机制研究第三军医大学博士学位论文Wnt10b在毛囊周期调控中的作用及机制研究姓名:李玉红申请学位级别:博士专业:细胞生物学指导教师:杨恬 2011-05 第三军医大学博士学位论文 Wnt10b 在毛囊周期调控中的作用及机制研究摘要背景毛囊是具有周期性生长特性的一个特殊器官，毛囊周期分为生长期、退化期和静止期。

在静止期，毛囊细胞受到某些因素的刺激，会再次进入生长期，如此周而复始在哺乳动物的整个生命期间循环往复。

毛囊经历生长期、退化期、静止期循环后再次进入生长期的过程称为毛囊周期性再生。

由于毛囊具有周期性生长的特性，为组织再生研究提供了一个非常好的模型。

如果毛囊各周期转换中的任何一个环节出现问题，就可能会影响毛囊的生长，进而出现毛囊相关疾病，例如脱发、斑秃等。

而斑秃等的治疗在机制研究和临床研究上还存在诸多问题，如容易复发等。

因此，毛囊周期的调控就显得至关重要，毛囊周期由毛囊干细胞介导发生。

毛囊干细胞的激活和静息受到多种信号通路的调控，包括 Wnt 信号通路、BMP 信号通路、FGF 信号通路、Shh 信号通路等。

Wnt 信号通路广受关注，它分为经典 Wnt 信号通路和非经典 Wnt 信号通路，在毛囊的发育和周期性再生中发挥着重要的调控作用。

经典Wnt 信号通路研究较多，在经典 Wnt 信号通路的各个重要环节进行阻断，均会严重影响毛囊的生成。

激活 Wnt 信号通路的 Wnt 蛋白有 19 种，但在毛囊周期性再生中，哪种或哪几种发挥关键作用，还不得而知。

作者通过文献筛选，发现Wnt10b 在皮肤附件发育时具有最明显的限制性表达，即特异性地表达于毛囊发育早期的基板。

而毛囊的周期性再生过程与毛囊发育具有相似性，因此，作者推测Wnt10b 在毛囊周期调控中发挥着重要作用。

目的与意义本课题拟研究Wnt10b在毛囊周期调控中发挥的具体作用，并探讨其作用机制。

为研究基于Wnt蛋白的治疗毛囊周期性再生障碍相关疾病的药物提供基础。

材料与方法 1. Wnt10b在毛囊周期中的表达观察 ?用免疫组化的方法观察Wnt10b蛋白在小鼠生后正常周期毛囊中的表达特性，并采用免疫印迹方法对小鼠各周期中皮肤全层Wnt10b的表达进行半定量分析; ?制备地高辛标记的Wnt10b RNA探针，观察Wnt10bmRNA在小鼠生后正常周期毛 7 第三军医大学博士学位论文囊中的表达状况，并采用逆转录PCR的方法对小鼠各周期中皮肤全层Wnt10b的mRNA表达进行半定量分析。

2. Wnt10b 对毛囊的作用研究 ?利用 HEK-293 细胞扩增，CsCl 超速离心纯化 AdWnt10b，验证腺病毒介导目的基因的生物学活性; ?解剖显微镜分离触须毛囊，建立器官培养模型，并通过荧光染色对模型进行评价，然后用 AdWnt10b 处理培养毛囊，离体观察Wnt10b 对毛囊的影响; ?在小鼠背部皮内注射腺病毒，并通过直接观察荧光、X-gal 染色等方法对动物模型进行评价，然后在不同时相点注射 AdWnt10b，在体观察 Wnt10b 对毛囊的影响。

3. Wnt10b 的信号通路研究 ?用免疫组化检测正常毛囊周期中经典 Wnt 信号通路中的关键分子 Frizzled、Lef1、TCF3 的蛋白表达，并分别通过原位杂交和免疫组化对β-catenin 的原位 mRNA和蛋白表达进行检测，与 Wnt10b 的表达情况进行对比，进一步利用 PCR array 技术，检测毛囊不同周期 Wnt 信号分子间的表达差异; ?在毛囊器官培养模型上，利用腺病毒载体从细胞膜受体水平、细胞浆水平和细胞核转录水平分别阻断经典 Wnt 信号通路的关键分子后，观察其对 AdWnt10b 诱导毛干生长作用的影响; ?静止期小鼠拔毛诱导毛囊生长，然后利用腺病毒载体从细胞膜受体水平、细胞浆水平和细胞核转录水平分别阻断经典 Wnt 信号通路的关键分子，观察毛囊生长情况;在以上各在体动物注射模型上，利用腺病毒介导的β-catenin 干扰序列干扰经典 Wnt信号通路后，观察其对Wnt10b 诱导毛囊周期性再生作用的影响。

4.Wnt10b 在毛囊周期中的作用机制研究 ?通过BrdU 标记检测 Wnt10b 诱导后不同时相点皮肤及毛囊细胞的增殖情况; ?通过免疫荧光和免疫印迹研究 Wnt10b 在 JB6 细胞系中过表达后对细胞分化的影响。

结果与讨论 1. Wnt10b表达与毛囊周期具有时空相关性 Wnt10b蛋白和mRNA在静止期无表达，在退化期的近心端(靠近真皮一端)内外根鞘有弱表达，在生长期的毛母质强表达，生长期的内外根鞘及前体细胞也有表达。

说明Wnt10b与毛囊周期密切相关，可能在毛囊的周期性再生中发挥重要的调控作用。

2. Wnt10b 能够诱导毛囊周期性再生 8 第三军医大学博士学位论文本实验所扩增的腺病毒具有生物学活性;研究发现经 AdWnt10b 处理的器官培养毛囊，其毛干生长的速度明显比对照组快，此离体说明 Wnt10b 对毛囊生长有促进作用;用 AdWnt10b 注射小鼠背皮后，能够在注射部位观察到绿色荧光，说明该腺病毒能够用于在体皮内注射;注射AdWnt10b 后，生长期和退化期的毛囊无变化，而静止期毛囊提前进入生长期，此在体说明 Wnt10b 能诱导毛囊周期性再生。

3. Wnt10b 经由经典 Wnt 信号通路发挥诱导毛囊周期性再生作用检测正常毛囊周期中经典 Wnt 信号通路中关键分子Frizzled、Lef1、TCF3 的蛋白表达，并对β-catenin 的原位 mRNA 和蛋白表达进行检测，发现它们与 Wnt10b 的表达相关联。

进一步通过 PCR array 分析也发现，生长期的经典 Wnt 信号分子显著高于静止期和退化期;在毛囊器官培养模型上，阻断经典 Wnt 信号通路关键分子后，能够阻断 AdWnt10b 诱导毛干生长的作用，此离体研究说明 Wnt10b 经由经典 Wnt 信号通路促进毛干生长;在体动物注射模型上，干扰经典 Wnt 信号通路关键分子β-catenin 后，能够阻断 AdWnt10b 诱导毛囊周期性再生的作用，此在体研究说明 Wnt10b 经由经典Wnt 信号通路诱导毛囊的周期性再生。

4. Wnt10b 可能通过诱导表皮基底层细胞增殖、分化而启动毛囊周期性再生 BrdU 标记检测增殖发现 Wnt10b 诱导后 24h，表皮基底层细胞最先发生增殖，然后全层皮肤和毛囊细胞发生增殖。

提示 Wnt10b 可能通过直接或间接作用于表皮基底层细胞，来发挥诱导毛囊周期性再生的作用; Wnt10b 诱导表皮基底层细胞表达毛囊结构的标记物，说明 Wnt10b 可能通过诱导表皮基底层细胞增殖，并向毛囊细胞分化而诱导毛囊周期性再生，但推论是否成立还需要后续实验进一步研究。

结论本研究首次发现 Wnt10b 能够诱导毛囊周期性再生，并证实其通过经典Wnt 信号通路，即 Wnt10-Frizzled- β-catenin-LEF/TCF 信号链来发挥调控作用;在过表达的情况下，表皮基底层细胞可能是 Wnt10b 作用的靶细胞，但还需要进一步研究。

关键词:Wnt10b;毛囊;再生;Wnt 信号通路 9 第三军医大学博士学位论文 The Mechanisms of Hair Follicle Cycle Regulated by Wnt10b AbstractBackgrounds Hair follicle is a specific organ with the characteristic of periodic growth whichconsists of anagen catagen and telogen. The hair follicle can reenter anagen under somestimuli in telogen thus continue the hair follicle cycle. The process of hair follicle reenteranagen is named regeneration. Because of its characteristic of periodic growth hair follicleprovides a nice model for tissue regeneration research. If the hair follicle cycle wasinterrupted hair follicle related disease such as hair loss and alopecia areata would occur.There are still some problems calling for solve in the therapies for alopecia areata such asrecurrence. Above all the research for hair follicle cycle regulation is very important. Hair folliclecycleis mediated by hair follicle stem cell which is regulated by many signalingpathwaysuch as Wnt BMP FGF Shh. Wnt signaling pathway plays an important role in hairfollicle morphogenesis and regeneration. Usually it is divided into canonical pathway andnon-canonical pathway. The canonical pathway is paid more attention and its signal chain isclearer. The hair follicle morphogenesis and regeneration would be impacted seriously ifany molecule in the canonical signal chain was blocked. Wnt gene family consists of 19members but which one or ones take effects on hair follicle cycle is still unknown.Literatures revealed that Wnt10b was highly and specifically expressed in the placode ofepidermis in hair follicle morphogenesis. It can be inferred that Wnt10b may also playdistinguish role in hair follicle regeneration in that there are many similarities between hairfollicle regeneration and hair follicle morphogenesis.Objectives This study focuses on the practical roles that Wnt10b plays in the regulation of hair follicle cycle and the mechanisms underlying it. It may provide a basement for the exploring and developing of drugs for the therapy of diseases related with hairfollicle 3 第三军医大学博士学位论文 regeneration dysfunction.Materials and Methods 1. Research on the expression pattern of Wnt10b in hair follicle cycle ? The protein expression pattern ofWnt10b was observed by immunohistochemistryin the slides of skin in hair follicle cycle. The protein expression quantity of Wnt10bwasobserved by Western Blot in the whole skin in hair follicle cycle. ? RNA probe of Wnt10b was labeled by digoxin and wasused for the observation ofWnt10b mRNA in the slides of skin in hair follicle cycle. The mRNA expression quantity ofWnt10b was observed by reverse transcription polymerase chain reaction in the whole skinin hair follicle cycle. 2. Research on the effects that Wnt10btakes on hair follicle cycle ? AdWnt10b was amplified in HEK 293 cells and purified by centrifuging in CsClgradient. The bioactivity ofinterest gene was validated by the infection of JB6 cells. ? The whisker follicles of C57 mice were dissected underanatomical microscope andcultured in culture medium. This model was assessed by observation of fluorescence. Thelength of cultured whisker follicles was assessed after being treated by AdWnt10b. ? Recombinant adenovirus were injected intradermicallyinto the back skin of mice.This model was evaluated through fluorescence microscope and X-gal staining. ThenAdWnt10b was injected at different stages of hair follicle cycle and the statuses of hairfollicle were assessed. 3. Research on the signaling pathway that Wnt10btransduce through ? The expression patterns of some key molecules in canonical Wnt signaling pathwayFrizzled Lef1 and TCF3 were determined by immunohistochemistry. The expressionpattern of the central molecule β-catenin wasdetermined by both immunohistochemistryand in situ hybridization. They were compared with the expression pattern of Wnt10b.Thenthe Wntsignaling pathway molecules in different hair follilcle stages were also determinedby PCR array. ? The Wnt signalingpathway was blocked separately at the cell membrane receptor levelcell plasm level and cell nucleus transcription level in the cultured whisker follicle by adenovirus mediated gene expression. The AdWnt10b was added into the culture medium on this base and the lengths of hair stems were assessed. 4 第三军医大学博士学位论文 ?The Wntsignaling pathway was blocked separately at the cell membrane receptorlevel cell plasm level and cell nucleus transcription level in the back skin of C57 mice thatafter the depilating of hair. And the statuses of hair and skin were observed daily. The Wntsignaling pathway was blocked in the back skin of C57 mice by adenovirus mediatedsiRNAgen e of β-catenin. Then the AdWnt10b was injected and the hair follicle statuseswere assessed. 4. Research on the mechanism of Wnt10b impacting hair follicle cycle ? The proliferative cells of skin and hair follicle at different time point after Wnt10b induction were determined by BrdU labeling. ? The cell differentiationstatuses of JB6 cells after Wnt10b induction weredetermined by immune fluorescence and Western Blot.Results and Discussions 1. The expression pattern of Wnt10b is temporally and spatially related with hairfollicle cycle The telogen hair follicle does not express proteinand mRNA of Wnt10b. The proximalpart of catagen hair follicle inner root sheath and outer root sheath weakly expressedWnt10b. The matrix cells of anagen hair follicle strongly expressed Wnt10b and the innerroot sheathouter root sheath and precortex of anagen hair follicle also normally expressWnt10b. This implies that Wnt10b is temporally and spatially related with hair follicle cycleand it may regulate the hair follicle regeneration. 2. Wnt10b induces hair follicle regeneration The adenoviruses we amplified have bioactivity. Hair stem of AdWnt10b treated hairfollicle grow faster than control suggesting that Wnt10b promotes the growth of hairfollicle in vitro. There are green fluoresces in the injection locus of AdWnt10b treated backskin indicating that the adenovirus can be used in vivo. After AdWnt10b injection theanagen hair follicle and catagen hair follicle are not influenced the telogen hair follicleenters anagen ahead of period time this supplies that Wnt110b can induce hair follicleregeneration in vivo. 3. Wnt10b induces hair follicle regeneration via a canonical Wnt signaling pathway. The expression patterns of Frizzled β-catenin Lef1 and TCF3 were similar to theexpressionpattern of Wnt10b suggesting some relationship between canonical Wnt 5 第三军医大学博士学位论文signaling pathway and Wnt10b. PCR array also reveals that the mRNA quantity ofcanonical Wnt signaling pathway molecules in anagen hair follicle was higher than that incatagen hair follicle and telogen hair follicle. The promote effect of AdWnt10b on hair stemgrowth was abrogated by AdFrzB AdSimBC and AddnTCF4 suggesting thatWnt10btransduce through canonical Wnt signaling pathway in vitro. The induction effect ofdepilating was abrogated by AdSimBC but not abrogatedby AdFrzB and AddnTCF4. Inthe injection model AdSimBC can abrogate the induction effect of AdWnt10b suggestingthat Wnt10b transduce through canonical Wnt signaling pathway in vivo.4. Wnt10b may induce the basal layer of epidermis cells to proliferate and differentiate thusinitiate the hair follicle regeneration. Through BrdU labeling we found thatafter the injection of Wnt10b for 24 hours thebasal layer of epidermis cells proliferated first then the whole skin and hair folliclesuggesting that Wnt10b may take effects on the basal layer of epidermis cells directly orindirectly. The Wnt10b treated JB6 cells expresse markers of hair follicle structure furthersupports that Wnt10b may promote the differentiation of the basal layer of epidermis cells.Conclusions Wnt10b can induce hair follicle regeneration through Wnt10-Frizzled- β-catenin-LEF/TCF signal chain. Itmay promote the proliferation and differentiation of basal layer ofepidermis cells to take this effect but it requires further research. Key words:Wnt10b hair follicle regenerationWnt signaling pathway 6 第三军医大学博士学位论文英文缩略词对照表英文缩写英文名称中文名称Ad Adenovirus Vector 腺病毒载体Amp Ampicillin 氨苄青霉素BMP bone morphogenetic protein 骨形成蛋白bp base pair 碱基对BrdU 5-bromodeoxyuridine 5-溴脱氧尿嘧啶BSA bovine serum albumin 牛血清白蛋白d Day天DAB diaminobenzidine 二氨基联苯胺ddH2O double distilled water 双蒸水DEPCDiethyl pyrocarbonate 焦炭酸二乙酯DIG digoxin 地高辛DMEM dulbecco modifiedEagle medium 达尔伯克改良伊格尔培养基DMSO Dimethylsulfoxide 二甲基亚砜DNA Deoxyribonuclease 脱氧核糖核酸dnTCF dominant negative TCF 显性负相 T 细胞因子DP dermal papilla 毛乳头DTT dithiothreitol 二硫苏糖醇EB ethidium bromide 溴化乙啶EDTA ethylene diaminetetraacetin acid 乙二胺四乙酸EP Eppendorf 微量离心管FGF fibroblast growth factor 成纤维细胞生长因子g Gravity 重力h Hour 小时HE hematoxylin and eosin 苏木精和伊红HG hair germ 毛芽HRP Horseradish peroxidase 辣根过氧化物酶IHC immunohistochemistry 免疫组化实验 1 第三.。