Wnt10b在毛囊周期调控中的作用及机制研究

发布时间：2018-05-16 14:25

本文选题：Wnt10b + 毛囊　；参考：《第三军医大学》2011年博士论文

【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 ①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; ②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 ①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; ②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; ③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 ①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; ②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; ③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 ①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; ②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。
[Abstract]:background
Hair follicle is a special organ with periodic growth characteristics. The hair follicle cycle is divided into growth period, degenerative period and stationary phase. During the rest period, the follicle cells are stimulated by some factors and will enter the growth period again, so that the whole life of the mammalian is repeated around the whole life cycle. The hair follicles undergo growth, degenerative and static periods. The process of re entering the growth period after the ring is called the periodic regeneration of the hair follicle. As the hair follicle has the characteristics of periodic growth, it provides a very good model for the study of tissue regeneration. If any part of the hair follicle during the cycle transition occurs, it may affect the growth of the hair follicle and then the hair follicle related diseases, such as the hair follicle related diseases. Alopecia areata and alopecia areata. There are still many problems in mechanism research and clinical research of alopecia areata, such as recurrence.
Therefore, the regulation of hair follicle cycle is very important. Hair follicle cycle is mediated by hair follicle stem cells. The activation and resting of hair follicle stem cells are regulated by a variety of signal pathways, including the Wnt signaling pathway, BMP signaling pathway, FGF signaling pathway, Shh signaling pathway and other.Wnt signaling pathways, which are divided into classical Wnt signaling pathways and non Classical Wnt signaling pathway plays an important role in the development and periodic regeneration of hair follicles. The classical Wnt signaling pathway has been widely studied, which can seriously affect the formation of hair follicles in the important links of the classic Wnt signaling pathway. There are 19 kinds of Wnt egg white activating the Wnt signaling pathway, but which species in the periodic regeneration of the hair follicle The author finds that Wnt10b has the most obvious restrictive expression during the development of the skin attachment, which is specifically expressed in the early development of the hair follicle, while the periodic regeneration process of the hair follicle is similar to the hair follicle development. Therefore, the author speculates that the Wnt10b is in the hair follicle cycle. It plays an important role in regulation and control.
Purpose and significance
The purpose of this study is to study the specific role of Wnt10b in the regulation of hair follicle cycle, and to explore its mechanism of action, providing a basis for the study of drugs based on Wnt protein for the treatment of periodic regenerative disorders related to hair follicles.
Materials and methods
The expression of 1. Wnt10b in the hair follicle cycle
(1) immunohistochemical method was used to observe the expression of Wnt10b protein in the normal hair follicle of mice after birth, and the expression of Wnt10b in the whole layer of the skin was semi quantitative analyzed by immunoblotting.
(2) the Wnt10b RNA probe labeled with digoxin was prepared to observe the expression of Wnt10bmRNA in the normal hair follicles of the mice after birth, and the mRNA expression in the whole layer of the skin of the mice was semi quantitatively analyzed by reverse transcriptase PCR.
Study on the effect of 2. Wnt10b on hair follicle
(1) using HEK-293 cell amplification and CsCl ultracentrifugation to purify AdWnt10b, the adenovirus mediated biological activity of the target gene was verified.
2. The tentacle hair follicles were separated by anatomical microscope, and the model of organ culture was established. The model was evaluated by fluorescence staining. Then the hair follicles were treated with AdWnt10b, and the effects of Wnt10b on the hair follicle were observed in vitro.
(3) the adenovirus was injected into the skin of the mouse's back, and the animal models were evaluated by direct observation of fluorescence, X-gal staining and other methods. Then AdWnt10b was injected at different phase points, and the effects of Wnt10b on the hair follicle were observed in vivo.
Study on signal pathway of 3. Wnt10b
(1) the protein expression of the key molecules Frizzled, Lef1, TCF3 in the classical Wnt signal pathway was detected by immunohistochemistry, and the expression of mRNA and protein in situ of beta -catenin was detected by in situ hybridization and immunohistochemistry. The expression of Wnt10b was compared with the expression of Wnt10b, and PCR array technique was used to detect the hair follicle. The expression difference between the Wnt signal molecules of the same period;
(2) on the model of hair follicle organ culture, the effects of adenovirus vector on the key molecules of Wnt signaling pathway were observed from the level of cell membrane receptor, plasma level and nuclear transcription level respectively, and the effect of adenovirus vector on AdWnt10b induced hairy stem growth was observed.
(3) the growth of hair follicles was induced by Pullout in the stationary phase of mice, and then the key molecules of the classic Wnt signaling pathway were blocked by adenovirus vector from the level of cell membrane receptor, cytoplasm level and nuclear transcription level, and the growth of hair follicles was observed. The adenovirus mediated beta -catenin interference sequence was used in the above injection models. After classical Wnt signaling pathway was disturbed, the effect of Wnt10b on the regeneration of hair follicles was observed.
Study on the mechanism of 4.Wnt10b in hair follicle cycle
(1) the proliferation of skin and hair follicle cells after Wnt10b induction was detected by BrdU markers.
(2) immunofluorescence and Western blotting were used to study the effect of Wnt10b overexpression on cell differentiation in JB6 cell line.
Results and discussion
1. Wnt10b expression has spatio-temporal correlation with the hair follicle cycle. Wnt10b protein and mRNA are not expressed at the stationary phase. There is a weak expression in the internal and external root sheath at the proximal end of the degenerative phase (near the dermis). In the growing period, the hair mother is strongly expressed, the internal and external root sheath and the precursor cells of the growth period are also apparent. It shows that the Wnt10b is closely related to the hair follicle cycle and may be in the hair. Cystic regeneration plays an important regulatory role.
2. Wnt10b can induce periodic regeneration of hair follicle
The adenovirus amplified in this experiment has biological activity. It was found that the hair follicle of the organ cultured by AdWnt10b was faster than the control group. This in vitro showed that Wnt10b could promote the growth of hair follicle. After AdWnt10b injection of the skin, the green fluorescence could be observed at the injection site, indicating that the adenovirus can be used. Enough for intradermal injection; after injection of AdWnt10b, there is no change in the follicles at the growth stage and the degenerative period, while the stationary hair follicles enter the growth period in advance. This indicates that the Wnt10b can induce the periodic regeneration of the hair follicles.
3. Wnt10b plays a role in inducing periodic regeneration of hair follicle through classical Wnt signaling pathway.
The protein expression of Frizzled, Lef1, TCF3, the key molecules of the classical Wnt signal pathway in the normal hair follicle cycle, was detected, and the expression of mRNA and protein in situ of beta -catenin was detected. It was found that they were associated with the expression of Wnt10b. Further, the classical Wnt signal molecules in the growth period were also found to be significantly higher than the stationary phase and the degenerative phase by PCR array analysis. In the hair follicle organ culture model, blocking the key molecules of the classic Wnt signaling pathway can block the role of AdWnt10b to induce hairy stem growth. In vitro studies show that Wnt10b promotes hairy stem growth via the classic Wnt signaling pathway; in vivo animal injection model, after interfering with the classic Wnt signaling pathway key molecule beta -catenin, the AdWnt10 can block AdWnt10. The role of B in the induction of cyclical regeneration of hair follicles suggests that Wnt10b can induce cyclical regeneration of hair follicle through classical Wnt signaling pathway.
4. Wnt10b may initiate periodic regeneration of hair follicle by inducing proliferation and differentiation of epidermal basal layer cells.
The proliferation of 24h in the basal layer of the epidermis is the first proliferation after Wnt10b induction, and the proliferation of the epidermis and follicle cells in the epidermis of the epidermis is the first. It suggests that Wnt10b may play a role in inducing the periodic regeneration of the hair follicle by direct or indirect action on the basal layer cells of the epidermis. Wnt10b induces the epidermal basal layer cells to express the hair follicle node in the 24h. It is suggested that Wnt10b may induce cell proliferation by inducing epidermal basal layer cells and differentiation to hair follicle cells to induce the periodic regeneration of hair follicles. However, further research is needed if the inference is still possible.
conclusion
It is the first time that Wnt10b can induce the periodic regeneration of hair follicles, and it is proved that it plays a regulatory role through the classical Wnt signaling pathway, that is, Wnt10-Frizzled- beta -catenin-LEF/TCF signal chain. In overexpression, the epidermal basal layer cells may be the target cells of Wnt10b action, but further study is needed.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R751

【参考文献】