本文選題：胎鼠真皮細(xì)胞 + 毛發(fā)形成能力��； 參考：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文

【摘要】：研究背景及意義 毛發(fā)缺失給患者的心理造成了極大的困擾。毛發(fā)移植術(shù)是目前公認(rèn)治療毛發(fā)缺失最有效的手段,但同時也存在不足,如供區(qū)有限及手術(shù)的二次損傷等。毛囊重建是具有毛囊再生能力的細(xì)胞在特定環(huán)境中再生出新的毛發(fā),為毛發(fā)缺失的治療提供了新的思路。種子細(xì)胞是毛囊再生的關(guān)鍵元素,真皮和表皮兩種來源細(xì)胞間的相互作用是毛囊再生的關(guān)鍵。目前研究認(rèn)為真皮來源細(xì)胞具有誘導(dǎo)毛發(fā)形成能力,在毛發(fā)再生中發(fā)揮更為關(guān)鍵的作用。因此,分析毛發(fā)再生過程中真皮來源細(xì)胞影響毛發(fā)再生的相關(guān)機(jī)制,對毛發(fā)的再生研究具有十分重要的意義,也為未來構(gòu)建帶毛發(fā)的組織工程皮膚奠定了基礎(chǔ)。 研究目的： 1、掌握胎鼠真皮細(xì)胞的分離及體外培養(yǎng)技術(shù)；通過將不同胎齡胎鼠原代真皮細(xì)胞與表皮細(xì)胞混合注射入裸鼠皮下,比較不同胎齡胎鼠真皮細(xì)胞誘導(dǎo)毛發(fā)形成能力,為分析真皮細(xì)胞中毛發(fā)再生能力相關(guān)因素提供依據(jù)。 2、通過比較不同胎齡胎鼠真皮細(xì)胞的多向分化能力、表面標(biāo)志物表達(dá)差異、以及真皮組織中毛發(fā)再生相關(guān)信號通路基因表達(dá)的變化,探討真皮細(xì)胞影響毛發(fā)再生的相關(guān)機(jī)制。 3、掌握人毛囊干細(xì)胞的分離培養(yǎng)技術(shù),探討胎鼠真皮細(xì)胞與人毛囊干細(xì)胞構(gòu)建毛發(fā)再生研究模型的可行性；利用毛囊干細(xì)胞進(jìn)行組織工程皮膚的構(gòu)建,為構(gòu)建帶有毛發(fā)的組織工程皮膚奠定基礎(chǔ)。 研究方法 1、通過不同胎齡胎鼠皮膚HE染色,觀察胎鼠毛囊的發(fā)育情況；通過中性蛋白酶與Ⅰ型膠原酶二步酶消化法對不同胎齡胎鼠真皮細(xì)胞進(jìn)行體外分離培養(yǎng)。 2、采用胎鼠真皮細(xì)胞與胎鼠表皮細(xì)胞混合皮下注射,2周后計數(shù)毛發(fā)形成數(shù)量,驗證不同胎齡胎鼠真皮細(xì)胞的誘導(dǎo)毛發(fā)形成能力。 3、將不同胎齡胎鼠真皮細(xì)胞分別向成骨、成脂、成神經(jīng)、成皮脂腺、成表皮方向進(jìn)行體外誘導(dǎo)培養(yǎng),比較胎鼠真皮細(xì)胞多向分化能力；通過流式細(xì)胞分析與RealTime PCR檢測的方法,觀察毛囊細(xì)胞常見表面標(biāo)志以及毛發(fā)再生相關(guān)信號通路中重要分子在不同胎齡胎鼠真皮細(xì)胞中的表達(dá)情況,分析與胎鼠真皮細(xì)胞毛發(fā)形成能力相關(guān)的影響因素,并采用siRNA干擾的方法對毛發(fā)再生相關(guān)的Wnt信號通路分子進(jìn)行分析。 4、利用顯微分離加差速貼壁法對人毛囊干細(xì)胞進(jìn)行分離培養(yǎng),并驗證胎鼠真皮細(xì)胞混合人毛囊干細(xì)胞后的毛發(fā)形成能力,進(jìn)而利用毛囊干細(xì)胞嘗試構(gòu)建組織工程皮膚。 實驗結(jié)果 1、胎鼠皮膚毛囊發(fā)育和細(xì)胞分離培養(yǎng)：HE染色顯示,在E15天胎鼠皮膚開始出現(xiàn)毛囊的發(fā)育,E17天新生毛囊逐漸增多,E20天形成結(jié)構(gòu)比較完整的毛囊；二步酶消化法可成功分離胎鼠真皮細(xì)胞,并可在體外大量擴(kuò)增。 2、胎鼠真皮細(xì)胞毛發(fā)形成能力：E15天胎鼠真皮細(xì)胞無法形成毛發(fā),E16天胎鼠真皮細(xì)胞可形成少量毛發(fā),從E17天開始胎鼠真皮細(xì)胞形成毛發(fā)的數(shù)量明顯增加(P0.05),E18、E19、E20天胎鼠真皮細(xì)胞再生毛發(fā)的數(shù)量無明顯差異。 3、胎鼠真皮細(xì)胞毛發(fā)形成影響因素研究： ①誘導(dǎo)分化能力：E15、E17、E20天胎鼠真皮細(xì)胞經(jīng)誘導(dǎo)后,各類組織細(xì)胞分化相關(guān)基因表達(dá)均上調(diào)(P0.05)；但不同胎齡胎鼠真皮細(xì)胞誘導(dǎo)分化能力存在差異,隨胎齡增加,向表皮、神經(jīng)以及脂肪細(xì)胞方向誘導(dǎo)分化能力增強。 ②細(xì)胞標(biāo)志物表達(dá)：毛囊內(nèi)表皮細(xì)胞表面標(biāo)志物在不同胎齡胎鼠真皮細(xì)胞內(nèi)均有不同程度表達(dá),其中Sca-1的mRNA表達(dá)在E18天比E15天有非常顯著的增加(P0.01),流式檢測顯示其表達(dá)從E15天的(38.7%±4.87545)上升到E18天的(69.8333%±10.17912)。 ③信號通路：β-catenin和LHX2的表達(dá)分別在E17和E19天胎鼠真皮中表達(dá)明顯增加。RNA干擾實驗結(jié)果顯示β-catenin基因表達(dá)的抑制,伴隨著LHX2基因的同步顯著下調(diào)(P0.05)以及Sca-1基因的非常顯著的滯后下調(diào)(P0.01)；而LHX2基因表達(dá)的抑制也可伴隨Sca-1基因顯著下調(diào)(P0.05)。 4、人毛囊干細(xì)胞的相關(guān)研究：顯微分離加差速貼壁法分離的人毛囊干細(xì)胞可表達(dá)多種毛囊干細(xì)胞標(biāo)志物,其中以P3或P4代細(xì)胞表達(dá)最高,與E17胎鼠真皮細(xì)胞混合后可再生大量毛發(fā)；人毛囊干細(xì)胞與真皮成纖維細(xì)胞膠原凝膠構(gòu)建的組織工程皮膚細(xì)胞狀態(tài)良好,結(jié)構(gòu)類似正常皮膚,但機(jī)械強度差,容易收縮；與真皮成纖維細(xì)胞PGA構(gòu)建的組織工程皮膚形態(tài)穩(wěn)定,但細(xì)胞相容性稍差。 結(jié)論 1、E15-E20天胎鼠真皮細(xì)胞毛發(fā)形成能力隨胎齡增加而增加。E17、E18天開始胎鼠真皮細(xì)胞的毛發(fā)再生能力明顯增強,是毛發(fā)形成的關(guān)鍵時期。 2、E15、E17、E20天胎鼠真皮細(xì)胞均具有多向分化能力,E17、E20胎鼠真皮細(xì)胞具有更強的向表皮、神經(jīng)和脂肪細(xì)胞分化的能力,可能是毛發(fā)形成能力更強的因素；Sca-1的表達(dá)隨胎齡變化趨勢與胎鼠真皮細(xì)胞毛發(fā)形成能力變化趨勢基本一致,也可能與毛發(fā)形成能力密切相關(guān)。伴隨著毛發(fā)形成能力顯著增加,β-catenin和LHX2在胎鼠真皮內(nèi)的表達(dá)也相應(yīng)顯著增加,提示W(wǎng)NT信號通路是影響毛囊再生的重要因素。RNA干擾實驗進(jìn)一步提示β-catenin基因可以通過作用其下游的LHX2及其Sca-1基因在毛發(fā)形成中發(fā)揮重要調(diào)節(jié)作用。 3、人毛囊干細(xì)胞體外培養(yǎng)增殖能力強,可穩(wěn)定傳代,從標(biāo)志物檢測水平觀察發(fā)現(xiàn)以P3或P4代細(xì)胞純度高,可用于基于胎鼠真皮細(xì)胞的毛發(fā)再生模型；利用其與不同支架材料可構(gòu)建出類似正常形態(tài)的組織工程皮膚,為最終構(gòu)建出帶有毛發(fā)的組織工程皮膚奠定了基礎(chǔ)。
[Abstract]:Research background and significance
Hair loss caused great distress to the patient's psychological. Hair transplantation is currently recognized as the most effective means of treatment of hair loss, but it also has some disadvantages, such as limited donor area and two surgical injury. Hair follicle reconstruction is hair follicle regeneration ability of the cells to grow new hair in a specific environment. Provides a new idea for the treatment of hair loss. Seed cells are the key elements of hair follicle regeneration of the epidermis and dermis of two kinds of interactions between cells is a key source of hair follicle regeneration. The present study of dermis derived cells have the ability to induce hair shape, play a key role in hair regeneration. Therefore, analysis mechanism of effects of dermal cells during hair regeneration. The regeneration of hair, it is very important to research on the regeneration of hair with hair, also for the future construction of tissue engineering skin lay The foundation.
The purpose of the study is:
1, master separation and dermal fetal rats in vitro; the fetal rats primary epidermal cells and epidermal cells injected into nude mice, comparing different dermal cells of fetal rats induced by hair formation ability, for the analysis of hair dermal cell regeneration can provide a basis for stress related factors.
2, by comparing the multidifferentiation ability of dermal cells in different gestational age, the difference of surface markers, and the expression of genes related to hair regeneration in dermal tissue, we explored the mechanism of dermal cell influence on hair regeneration.
3, we should master the isolation and culture technology of human hair follicle stem cells, explore the feasibility of constructing hair regeneration models from fetal rat dermal cells and human hair follicle stem cells, construct dermal tissue engineering skin by hair follicle stem cells, and lay the foundation for constructing tissue-engineered skin with hair.
research method
1, the hair follicle development of fetal rats was observed by HE staining of fetal skin at different gestational ages. The dermal cells from fetal rats were isolated and cultured in vitro by neutral protease and type I collagenase two step enzyme digestion.
2, the fetal rat dermal cells were mixed with fetal rat epidermal cells to subcutaneous injection. After 2 weeks, the number of hair formation was counted, and the hair forming ability of dermal cells of fetal rats at different gestational ages was verified.
3, the fetal rats dermal cells were osteogenic, adipogenic, nerve, into the sebaceous gland, were induced in vitro into epidermal direction, comparison of fetal rat dermal cells differentiation; by means of flow cytometry and RealTime PCR detection, observation of hair follicle cells and expression of surface markers of common hair regeneration the important molecular signaling pathway in epidermal cells in fetal rats, influence factors related to the ability of analysis and dermal cells of fetal rat hair formation, and method of using siRNA interference analysis of the regeneration of Wnt signaling molecules related to the hair.
4, we used microisolation and differential adherence method to isolate and culture human hair follicle stem cells, and verify the hair formation ability of fetal rat dermal cells mixed with human hair follicle stem cells, and then use hair follicle stem cells to construct tissue-engineered skin.
experimental result
1, cultured skin follicle development and cell separation: HE staining showed that in E15 days of fetal rat skin began to hair follicle development, hair follicle E17 days gradually increased, E20 days to form relatively complete structure of hair follicle; successful fetal rat dermal cells can be separated two step enzymatic digestion, and can be expanded in vitro.
2, the hair of fetal rat dermal cells forming ability: E15 day fetal rat dermal cells to form hair, E16 day fetal rat dermal cells can form a small amount of hair, the number of days from E17 fetal rat dermal hair increased significantly (P0.05), E18, E19, no significant difference between the number of days of fetal rat dermal cells and E20 hairy.
3, study on the factors affecting the formation of dermal cell hair in fetal rat:
Differentiation capacity: E15, E17, E20 day fetal rat dermal cells after induction of cell differentiation related genes were up-regulated (P0.05); but the dermal cells of fetal rats induced differentiation ability differences, with the increase of gestational age, to the skin, nerves and fat cell differentiation ability.
The cell marker expression of epidermal cell surface markers of hair follicles found in fetal rats dermal cells during different expression, Sca-1 expression of mRNA in E18 days than E15 days increased significantly (P0.01), flow cytometry showed that the expression of E15 from day (38.7% + 4.87545) to rise E18 day (69.8333% + 10.17912).
The signal pathway: the expression of beta -catenin and LHX2 were expressed in E17 and E19 days were significantly increased in the fetal rat dermal.RNA interference experiment showed that the inhibition of the expression of beta -catenin gene, with the synchronous LHX2 gene significantly decreased (P0.05) and Sca-1 gene significantly delayed transfer (P0.01); and the inhibition of LHX2 gene the expression of Sca-1 can also be accompanied by significant downregulation of genes (P0.05).
4, the human hair follicle stem cells: isolation of cells can express a variety of hair follicle stem cell markers and differential centrifugation separation of human hair follicle stem microstructure, which P3 or P4 cells was the highest, and the mixed epidermal cells of E17 rat after a large number of renewable hair cells and dermal fibroblasts; collagen gel construct the tissue engineering skin cells in good condition of human hair follicle stem structure similar to normal skin, but poor mechanical strength, easy to shrink; fibroblast PGA tissue engineering skin morphology stability and biocompatibility of leather, but slightly worse.
conclusion
1, the hair forming ability of E15-E20 day dermis cells increased with the increase of gestational age,.E17, E18 days, the hair regeneration ability of fetal dermis cells was significantly enhanced, which is the key period of hair formation.
2, E15, E17, E20 day fetal rat dermal cells have the ability of multi-directional differentiation, E17, dermal cells of E20 rat has stronger epidermotropism, ability of neural and fat cell differentiation, may be a factor for hair formation ability is stronger; the expression of Sca-1 changes with gestational age and the trend of hair dermal cells of mouse embryo formation the ability is basically the same trend. It may be closely related to hair formation ability. With the hair forming ability increased significantly, the expression of -catenin and LHX2 beta in the fetal rat dermis also increased significantly, suggesting that WNT signaling pathway is one of the important factors affecting hair follicle regeneration.RNA interference experiments further suggest that beta -catenin gene may play an important role in the formation of hair by LHX2 Sca-1 gene and its downstream effects.
3, the proliferation ability of human hair follicle stem cells in vitro can stably, from marker detection level was observed in P3 or P4 cells of high purity, can be used for the regeneration of fetal rat model of dermal cells of hair based on; with the different scaffolds to construct tissue engineering skin similar to the normal form, laid the foundation for the final construction of a tissue engineering skin with hair.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R758.71

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 邵勇;倪振洪;李玉紅;;Wnt信號通路與毛囊干細(xì)胞[J];生物醫(yī)學(xué)工程學(xué)雜志;2010年04期

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本文編號：1757886