本文選題：Wnt10b + 毛囊��； 參考：《第三軍醫(yī)大學(xué)》2011年博士論文

【摘要】：背景 毛囊是具有周期性生長特性的一個特殊器官,毛囊周期分為生長期、退化期和靜止期。在靜止期,毛囊細胞受到某些因素的刺激,會再次進入生長期,如此周而復(fù)始在哺乳動物的整個生命期間循環(huán)往復(fù)。毛囊經(jīng)歷生長期、退化期、靜止期循環(huán)后再次進入生長期的過程稱為毛囊周期性再生。由于毛囊具有周期性生長的特性,為組織再生研究提供了一個非常好的模型。如果毛囊各周期轉(zhuǎn)換中的任何一個環(huán)節(jié)出現(xiàn)問題,就可能會影響毛囊的生長,進而出現(xiàn)毛囊相關(guān)疾病,例如脫發(fā)、斑禿等。而斑禿等的治療在機制研究和臨床研究上還存在諸多問題,如容易復(fù)發(fā)等。 因此,毛囊周期的調(diào)控就顯得至關(guān)重要,毛囊周期由毛囊干細胞介導(dǎo)發(fā)生。毛囊干細胞的激活和靜息受到多種信號通路的調(diào)控,包括Wnt信號通路、BMP信號通路、FGF信號通路、Shh信號通路等。Wnt信號通路廣受關(guān)注,它分為經(jīng)典Wnt信號通路和非經(jīng)典Wnt信號通路,在毛囊的發(fā)育和周期性再生中發(fā)揮著重要的調(diào)控作用。經(jīng)典Wnt信號通路研究較多,在經(jīng)典Wnt信號通路的各個重要環(huán)節(jié)進行阻斷,均會嚴(yán)重影響毛囊的生成。激活Wnt信號通路的Wnt蛋白有19種,但在毛囊周期性再生中,哪種或哪幾種發(fā)揮關(guān)鍵作用,還不得而知。作者通過文獻篩選,發(fā)現(xiàn)Wnt10b在皮膚附件發(fā)育時具有最明顯的限制性表達,即特異性地表達于毛囊發(fā)育早期的基板。而毛囊的周期性再生過程與毛囊發(fā)育具有相似性,因此,作者推測Wnt10b在毛囊周期調(diào)控中發(fā)揮著重要作用。 目的與意義 本課題擬研究Wnt10b在毛囊周期調(diào)控中發(fā)揮的具體作用,并探討其作用機制。為研究基于Wnt蛋白的治療毛囊周期性再生障礙相關(guān)疾病的藥物提供基礎(chǔ)。 材料與方法 1. Wnt10b在毛囊周期中的表達觀察 ①用免疫組化的方法觀察Wnt10b蛋白在小鼠生后正常周期毛囊中的表達特性,并采用免疫印跡方法對小鼠各周期中皮膚全層Wnt10b的表達進行半定量分析; ②制備地高辛標(biāo)記的Wnt10b RNA探針,觀察Wnt10bmRNA在小鼠生后正常周期毛囊中的表達狀況,并采用逆轉(zhuǎn)錄PCR的方法對小鼠各周期中皮膚全層Wnt10b的mRNA表達進行半定量分析。 2. Wnt10b對毛囊的作用研究 ①利用HEK-293細胞擴增,CsCl超速離心純化AdWnt10b,驗證腺病毒介導(dǎo)目的基因的生物學(xué)活性; ②解剖顯微鏡分離觸須毛囊,建立器官培養(yǎng)模型,并通過熒光染色對模型進行評價,然后用AdWnt10b處理培養(yǎng)毛囊,離體觀察Wnt10b對毛囊的影響; ③在小鼠背部皮內(nèi)注射腺病毒,并通過直接觀察熒光、X-gal染色等方法對動物模型進行評價,然后在不同時相點注射AdWnt10b,在體觀察Wnt10b對毛囊的影響。 3. Wnt10b的信號通路研究 ①用免疫組化檢測正常毛囊周期中經(jīng)典Wnt信號通路中的關(guān)鍵分子Frizzled、Lef1、TCF3的蛋白表達,并分別通過原位雜交和免疫組化對β-catenin的原位mRNA和蛋白表達進行檢測,與Wnt10b的表達情況進行對比,進一步利用PCR array技術(shù),檢測毛囊不同周期Wnt信號分子間的表達差異; ②在毛囊器官培養(yǎng)模型上,利用腺病毒載體從細胞膜受體水平、細胞漿水平和細胞核轉(zhuǎn)錄水平分別阻斷經(jīng)典Wnt信號通路的關(guān)鍵分子后,觀察其對AdWnt10b誘導(dǎo)毛干生長作用的影響; ③靜止期小鼠拔毛誘導(dǎo)毛囊生長,然后利用腺病毒載體從細胞膜受體水平、細胞漿水平和細胞核轉(zhuǎn)錄水平分別阻斷經(jīng)典Wnt信號通路的關(guān)鍵分子,觀察毛囊生長情況;在以上各在體動物注射模型上,利用腺病毒介導(dǎo)的β-catenin干擾序列干擾經(jīng)典Wnt信號通路后,觀察其對Wnt10b誘導(dǎo)毛囊周期性再生作用的影響。 4.Wnt10b在毛囊周期中的作用機制研究 ①通過BrdU標(biāo)記檢測Wnt10b誘導(dǎo)后不同時相點皮膚及毛囊細胞的增殖情況; ②通過免疫熒光和免疫印跡研究Wnt10b在JB6細胞系中過表達后對細胞分化的影響。 結(jié)果與討論 1. Wnt10b表達與毛囊周期具有時空相關(guān)性Wnt10b蛋白和mRNA在靜止期無表達,在退化期的近心端(靠近真皮一端)內(nèi)外根鞘有弱表達,在生長期的毛母質(zhì)強表達,生長期的內(nèi)外根鞘及前體細胞也有表達。說明Wnt10b與毛囊周期密切相關(guān),可能在毛囊的周期性再生中發(fā)揮重要的調(diào)控作用。 2. Wnt10b能夠誘導(dǎo)毛囊周期性再生 本實驗所擴增的腺病毒具有生物學(xué)活性;研究發(fā)現(xiàn)經(jīng)AdWnt10b處理的器官培養(yǎng)毛囊,其毛干生長的速度明顯比對照組快,此離體說明Wnt10b對毛囊生長有促進作用;用AdWnt10b注射小鼠背皮后,能夠在注射部位觀察到綠色熒光,說明該腺病毒能夠用于在體皮內(nèi)注射;注射AdWnt10b后,生長期和退化期的毛囊無變化,而靜止期毛囊提前進入生長期,此在體說明Wnt10b能誘導(dǎo)毛囊周期性再生。 3. Wnt10b經(jīng)由經(jīng)典Wnt信號通路發(fā)揮誘導(dǎo)毛囊周期性再生作用 檢測正常毛囊周期中經(jīng)典Wnt信號通路中關(guān)鍵分子Frizzled、Lef1、TCF3的蛋白表達,并對β-catenin的原位mRNA和蛋白表達進行檢測,發(fā)現(xiàn)它們與Wnt10b的表達相關(guān)聯(lián)。進一步通過PCR array分析也發(fā)現(xiàn),生長期的經(jīng)典Wnt信號分子顯著高于靜止期和退化期;在毛囊器官培養(yǎng)模型上,阻斷經(jīng)典Wnt信號通路關(guān)鍵分子后,能夠阻斷AdWnt10b誘導(dǎo)毛干生長的作用,此離體研究說明Wnt10b經(jīng)由經(jīng)典Wnt信號通路促進毛干生長;在體動物注射模型上,干擾經(jīng)典Wnt信號通路關(guān)鍵分子β-catenin后,能夠阻斷AdWnt10b誘導(dǎo)毛囊周期性再生的作用,此在體研究說明Wnt10b經(jīng)由經(jīng)典Wnt信號通路誘導(dǎo)毛囊的周期性再生。 4. Wnt10b可能通過誘導(dǎo)表皮基底層細胞增殖、分化而啟動毛囊周期性再生 BrdU標(biāo)記檢測增殖發(fā)現(xiàn)Wnt10b誘導(dǎo)后24h,表皮基底層細胞最先發(fā)生增殖,然后全層皮膚和毛囊細胞發(fā)生增殖。提示W(wǎng)nt10b可能通過直接或間接作用于表皮基底層細胞,來發(fā)揮誘導(dǎo)毛囊周期性再生的作用; Wnt10b誘導(dǎo)表皮基底層細胞表達毛囊結(jié)構(gòu)的標(biāo)記物,說明Wnt10b可能通過誘導(dǎo)表皮基底層細胞增殖,并向毛囊細胞分化而誘導(dǎo)毛囊周期性再生,但推論是否成立還需要后續(xù)實驗進一步研究。 結(jié)論 本研究首次發(fā)現(xiàn)Wnt10b能夠誘導(dǎo)毛囊周期性再生,并證實其通過經(jīng)典Wnt信號通路,即Wnt10-Frizzled-β-catenin-LEF/TCF信號鏈來發(fā)揮調(diào)控作用;在過表達的情況下,表皮基底層細胞可能是Wnt10b作用的靶細胞,但還需要進一步研究。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R751

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