本文選題：毛囊干細(xì)胞 + 氯化鋰�。� 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：背景和目的 毛囊干細(xì)胞(Hair follicle stem cells，HFSCs)被選為種子細(xì)胞用于構(gòu)建組織工程皮膚以修復(fù)臨床燒傷、創(chuàng)傷、慢性潰瘍等皮膚缺失已成為醫(yī)學(xué)研究的熱點。毛囊是皮膚的重要附屬器官，從內(nèi)向外依次由毛干、內(nèi)根鞘和外根鞘組成。毛囊隆突部位于外根鞘近表皮端，皮脂腺下方豎毛肌附著處，此處的細(xì)胞體外培養(yǎng)時表現(xiàn)出干細(xì)胞的特性，即可自我復(fù)制、慢周期性和具有多向分化潛能。目前已經(jīng)得到證實，隆突部的這一群特殊的細(xì)胞為毛囊干細(xì)胞。毛囊干細(xì)胞具有多向分化遷移能力，不僅能向上分化并遷移參與表皮更新和皮脂腺維持，，而且能向下端毛球部分化并遷移參與形成毛囊。毛囊干細(xì)胞分化傾向的不同是由多種因素共同決定的，不僅有細(xì)胞內(nèi)部分子及基因的調(diào)控，也有外來信號及微環(huán)境壁龕的協(xié)同作用，更有多條信號通路傳導(dǎo)分化“命令”。在眾多科研工作者的共同努力下發(fā)現(xiàn)，毛囊干細(xì)胞Wnt信號通路(Wnt signaling pathway)在調(diào)控其增殖、分化中起到重要作用。 Wnt/β-catenin信號通路在全身幾乎所有的細(xì)胞中都存在，細(xì)胞受到刺激后釋放Wnt蛋白，激活下游一系列大分子信號活性物質(zhì)，最終導(dǎo)致基因表達(dá)改變，是誘導(dǎo)細(xì)胞分化和增殖的一條分子級聯(lián)通路，在胚胎發(fā)育和腫瘤發(fā)生中，這條通路也起到關(guān)鍵性的作用。在HFSCs中，Wnt/β-catenin信號通路激活后會引起β-catenin在胞漿內(nèi)的堆積，隨之轉(zhuǎn)入到細(xì)胞核中，與核內(nèi)轉(zhuǎn)錄因子Tcf3/Lefl結(jié)合，激活下游靶基因c-myc、cyclin D1等的轉(zhuǎn)錄，促進(jìn)毛囊干細(xì)胞定向分化。近年來有研究發(fā)現(xiàn)細(xì)胞外來干預(yù)因子氯化鋰(LiCl)可抑制Wnt信號通路中β-catenin的降解，使之在細(xì)胞質(zhì)中聚集，激活下游通路，影響毛囊干細(xì)胞分化的表型，但對于LiCl調(diào)節(jié)HFSCs分化的分子機制研究較少，且局限于在細(xì)胞質(zhì)中作用的研究。本課題選用了氯化鋰激活Wnt信號通路，探討Wnt/β-catenin信號通路在人HFSCs向毛囊形成細(xì)胞或表皮細(xì)胞定向分化中的作用及與其他信號因子的相互關(guān)系，尤其是細(xì)胞核內(nèi)的分子水平改變，以及藥物控釋系統(tǒng)的建立和在組織工程皮膚中的應(yīng)用。 方法 1.頭皮組織采用改進(jìn)的毛囊隆突部獲取方法及兩步酶消化后，將帶有隆突部的毛發(fā)剪成泥狀，Ⅳ型膠原差速貼壁法獲取毛囊干細(xì)胞。使用免疫熒光染色法對毛囊干細(xì)胞進(jìn)行鑒定。以500個/孔(2.5×10~3/m1)、1000個/孔(5×10~3/m1)、2000個/孔(1×10~4/m1)、3000個/孔(1.5×10~4/m1)、5000個/孔(2.5×10~4/m1)接種于96孔培養(yǎng)板中，觀察細(xì)胞生長情況，采用MTT法描繪其生長曲線，觀察各密度培養(yǎng)的HFSCs在不同時間點的增殖效應(yīng)，篩選出體外最佳的HFSCs傳代培養(yǎng)密度。 2.選用0、0.1、1、5、10、20、40、100mM/l的氯化鋰誘導(dǎo)毛囊干細(xì)胞分化，觀察毛囊干細(xì)胞的生存狀態(tài)，確定氯化鋰的毒性作用范圍；選用0、5、10mM/l氯化鋰作用于毛囊干細(xì)胞，MTT法對比分析各組對細(xì)胞增殖效應(yīng)；LiCl終濃度分別為0、0.1、1、10、50、100、200、400μg/ml作用于毛囊干細(xì)胞，檢測氯化鋰的毒性，并探索促HFSCs分化的最佳LiCl濃度；采用real-time PCR技術(shù)定量檢測0、5、10mmol/ml LiCl干預(yù)毛囊干細(xì)胞5d后Wnt信號通路中生物大分子：β-catenin、GSK-3β、Tcf3、c-myc和cyclin D1的mRNA水平，探討LiCl誘導(dǎo)HFSCs分化過程中，LiCl的不同濃度對Wnt/β-catenin信號途徑及其相關(guān)信號因子的表達(dá)的影響和相互作用。 3.LiCl-PLGA納米微球的制備：選用聚乳酸-羥基乙酸(PLGA)作為微囊敷料，荷載適當(dāng)濃度的氯化鋰，采用復(fù)乳-溶劑揮發(fā)法制備載有氯化鋰的納米微囊。完全培養(yǎng)基培養(yǎng)人成纖維細(xì)胞，選用異體脫細(xì)胞真皮(ADM)為支架材料，以擴增培養(yǎng)的HFSCs為種子細(xì)胞接種于ADM表皮面，成纖維細(xì)胞接種于ADM真皮面，構(gòu)建組織工程皮膚。采用5mM/l的LiCl干預(yù)構(gòu)建的組織工程皮膚，培養(yǎng)5d后切片行HE染色、免疫熒光染色和免疫組織化學(xué)染色，觀察種子細(xì)胞在ADM上的生長及分化情況，為動物實驗奠定良好基礎(chǔ)。 結(jié)果 1.從人頭皮成功分離培養(yǎng)出HFSCs，在體外經(jīng)多次傳代后仍具有很強的增殖能力和多向分化潛能，當(dāng)HFSCs接種密度為1x10~4ml時，細(xì)胞增殖速度最快，擴展能力強，其生長曲線呈典型的S型。 2.隨LiCl濃度升高，細(xì)胞增殖效應(yīng)減弱。含有LiCl的K-SFM條件培養(yǎng)基中HFSCs形態(tài)改變明顯，各組間有明顯差別，LiCl10mmol/L時分化比例高。LiCl誘導(dǎo)毛囊干細(xì)胞Wnt信號通路的同時還有其一定的細(xì)胞毒性，濃度大于200μg/ml(約5mmol/L)時明顯抑制細(xì)胞生長。實驗研究發(fā)現(xiàn)在HFSCs分化過程中，LiCl可激活Wnt/β-catenin信號通路，當(dāng)濃度為5mM/l時促使β-catenin及其拮抗物GSK3β、核內(nèi)轉(zhuǎn)錄因子Tcf3、下游基因c-myc、cyclin D1的轉(zhuǎn)錄上調(diào)，濃度為10mM/l時除cyclin D1轉(zhuǎn)錄仍上調(diào)外，其余基因轉(zhuǎn)錄下調(diào)。 3.掃描電鏡觀察細(xì)胞在PLGA-ADM材料上生長良好，分泌的胞外基質(zhì)豐富，并隨時間進(jìn)展，細(xì)胞逐漸融合，細(xì)胞外基質(zhì)分泌逐漸增多。HE染色觀察發(fā)現(xiàn)HFSCs生長狀態(tài)良好，單層排列分布于ADM表皮面上，LiCl干預(yù)組表皮面有毛囊樣小凹形成，成纖維細(xì)胞與ADM真皮面貼附緊密，能分泌胞外基質(zhì)形成連續(xù)細(xì)胞層包繞ADM。 結(jié)論 1.我們改進(jìn)的培養(yǎng)方法能簡便快捷的獲取人HFSCs，且得到的人HFSCs在體外有較強的增殖及傳代能力，1×10~4/ml接種密度是促進(jìn)HFSCs增殖的最佳密度。 2.LiCl促HFSCs分化明顯，對HFSCs的干預(yù)有臨界毒性濃度，當(dāng)濃度大于200μg/ml時，細(xì)胞生長增殖受到明顯抑制，同時這一濃度也是促HFSCs分化的最佳濃度。LiCl促HFSCs分化作用可能與激活Wnt/β-catenin信號通路、通路下游各因子轉(zhuǎn)錄上調(diào)相關(guān)，Tcf3在維持細(xì)胞未分化狀態(tài)中發(fā)揮了一定的作用，cyclinD1的轉(zhuǎn)錄與氯化鋰濃度作用呈正相關(guān)，在HFSCs的分化中起到重要作用。 3.復(fù)乳-溶劑揮發(fā)法能快速高效的制備出載有LiCl的納米微囊，其PLGA材料具有良好的細(xì)胞相容性，人HFSCs能在其上較好的粘附和生長。構(gòu)建載有人HFSCs的組織工程皮膚，在LiCl誘導(dǎo)下能夠促使種子細(xì)胞發(fā)生分子學(xué)改變形成毛囊樣小凹，使構(gòu)建帶有皮膚附屬器的組織工程替代物成為可能。
[Abstract]:Background and purpose
Hair follicle stem cells (HFSCs) is selected as seed cells to construct tissue engineered skin to repair clinical burns, trauma, chronic ulcers and other skin defects. The hair follicle is an important accessory organ of the skin, which consists of the hair stem, the inner root sheath and the outer root sheath from the inside to the outside. The hair follicle is located in the protuberance of the hair. The outer root sheath near the epidermis and the attachment of the erection muscle beneath the sebaceous glands. The cells here show the characteristics of stem cells in vitro, which can be self replicating, slow cycle and multidirectional differentiation potential. It has been confirmed that the special cells in the protuberance are hair follicle stem cells. The hair follicle stem cells have multiple differentiation and migration ability. Not only can it be differentiated and migrated to participate in epidermal renewal and the maintenance of sebaceous glands, but also can migrate to the lower end of the hair and migrate to form a hair follicle. The differentiation tendency of the hair follicle stem cells is determined by a variety of factors, not only the regulation of the internal molecules and genes of the cells, but also the synergy of the external signals and microenvironmental niches. There are multiple signaling pathways that differentiate "commands". With the joint efforts of many researchers, the Wnt signal pathway (Wnt signaling pathway) of hair follicle stem cells (Wnt) plays an important role in regulating its proliferation and differentiation.
Wnt/ beta -catenin signaling pathway exists in almost all the cells in the whole body. The cells are stimulated to release the Wnt protein and activate a series of large molecular signal active substances downstream, which eventually leads to the change of gene expression. It is a molecular link to induce cell differentiation and proliferation. In embryo development and tumorigenesis, this pathway also acts as a pathway. In HFSCs, the activation of Wnt/ beta -catenin signaling pathway causes the accumulation of beta -catenin in the cytoplasm, and then into the nucleus, combined with the nuclear transcription factor Tcf3/Lefl, activates the transcription of the downstream target gene c-myc, cyclin D1 and so on, and promotes the directional differentiation of the hair follicle stem cells. In recent years, there has been a study on the external intervention of cells. Factor lithium chloride (LiCl) can inhibit the degradation of beta -catenin in the Wnt signal pathway, make it accumulate in the cytoplasm, activate the downstream pathway and affect the phenotype of hair follicle stem cell differentiation, but few studies on the molecular mechanism of LiCl regulation of HFSCs differentiation, and limited to the study in cytoplasm. This topic selects lithium chloride to activate Wnt signal. To explore the role of Wnt/ beta -catenin signaling pathway in the direction differentiation of human HFSCs to hair follicle forming cells or epidermal cells and the relationship with other signal factors, especially the molecular level changes in the nucleus, the establishment of drug controlled-release system and the application in tissue engineering skin.
Method
1. scalp tissue was used to obtain the improved hair follicle protuberance method and two step enzyme digestion. The hair follicle was shears with the hair of the protuberance, and the hair follicle stem cells were obtained by the type IV collagen differential adhesion method. The hair follicle stem cells were identified by immunofluorescence staining. 500 / 2.5 x 10~3/ M1, 1000 / 5 (5 x 10~3/m1), 2000 / 1 x 10~4/m1 (1 x 10~4/m1) 3000 / holes (1.5 x 10~4/m1) and 5000 / holes (2.5 x 10~4/m1) were inoculated in 96 hole culture plates. The growth of cells was observed. The growth curve of the cells was described by MTT method. The proliferation effect of HFSCs at different time points was observed and the best density of HFSCs in vitro was screened out.
2. the lithium chloride of 0,0.1,1,5,10,20,40100mM/l was used to induce the differentiation of hair follicle stem cells. The survival state of hair follicle stem cells was observed and the toxicity range of lithium chloride was determined. The effect of 0,5,10mM/l lithium chloride on hair follicle stem cells was selected and the proliferation effect of each group was compared and analyzed by MTT method; the final concentration of LiCl was 0,0.1,1,10,50100200400 mu g/, respectively. Ml was used in hair follicle stem cells to detect the toxicity of lithium chloride, and to explore the optimal LiCl concentration for the differentiation of HFSCs. The real-time PCR technique was used to detect the 0,5,10mmol/ml LiCl in the Wnt signal pathway of the follicle stem cell 5D. The influence and interaction of different concentrations of LiCl on the expression of Wnt/ beta -catenin signaling pathway and its related signal factors were also discussed.
Preparation of 3.LiCl-PLGA nanospheres: using poly lactic acid hydroxy acetic acid (PLGA) as microcapsule dressing, loaded lithium chloride with proper concentration, the nanoscale containing lithium chloride was prepared by compound emulsion solvent evaporation method. Human fibroblasts were cultured in complete medium and allogenic decellular dermis (ADM) was used as scaffold material, and the cultured HFSCs was amplified. The seed cells were inoculated on the surface of the ADM surface, and the fibroblasts were inoculated on the true skin surface of ADM. The tissue engineering skin was constructed. The tissue engineering skin was constructed by the LiCl intervention of 5mM/l. After 5D, HE staining, immunofluorescence staining and immunohistochemical staining were used to observe the growth and differentiation of seed cells on ADM, which lay a good foundation for animal experiments. Good foundation.
Result
1. HFSCs was successfully isolated and cultured from the human scalp. After multiple passages in vitro, it still had strong proliferation ability and multidirectional differentiation potential. When the density of HFSCs was 1x10~4ml, the cell proliferation rate was the fastest and the expansion ability was strong, and its growth curve was typical S type.
2. the proliferation effect of cell proliferation decreased with the increase of LiCl concentration. The morphologic changes of HFSCs in the K-SFM conditioned medium containing LiCl were obviously different. The high.LiCl induced Wnt signaling pathway in hair follicle stem cells at the time of LiCl10mmol/L, and also had certain cytotoxicity, and the concentration was obviously inhibited when the concentration was greater than 200 u g/ml (about 5mmol/L). Growth. The experimental study found that in the process of HFSCs differentiation, LiCl activates the Wnt/ beta -catenin signaling pathway. When the concentration is 5mM/l, the beta -catenin and its antagonist GSK3 beta, the internal transcription factor Tcf3, the downstream gene c-myc, cyclin D1 are up regulated, and the concentration is still up regulation while the concentration is still up, and the other genes are down regulated.
3. scanning electron microscopy showed that the cells grew well on PLGA-ADM, and the secreted extracellular matrix was rich, and the cells gradually fused with time, and the secretion of extracellular matrix was gradually increased by.HE staining. The growth state of HFSCs was good and the single layer was arranged on the surface of ADM surface. The skin surface of the LiCl intervention group was formed by the follicle like fovea, and fibrous thin. The cells are tightly attached to ADM, which can secrete extracellular matrix and form a continuous cell layer wrapped around ADM..
conclusion
1. the improved culture method can easily and quickly obtain human HFSCs, and the obtained human HFSCs has strong proliferation and generation ability in vitro. The density of 1 x 10~4/ml inoculation is the best density to promote the proliferation of HFSCs.
2.LiCl promotes HFSCs differentiation obviously, and there is a critical toxicity concentration for HFSCs intervention. When the concentration is more than 200 g/ml, the cell growth and proliferation are obviously inhibited. At the same time, this concentration is also the best concentration of HFSCs to promote HFSCs differentiation, which may be associated with the activation of Wnt/ beta -catenin signal, and the transcription regulation of various factors downstream of the pathway is associated with Tcf3 in the downstream. It plays a certain role in maintaining cell undifferentiated state. Transcription of cyclinD1 is positively correlated with the concentration of lithium chloride, and plays an important role in the differentiation of HFSCs.
The 3. compound milk solvent evaporation method can quickly and efficiently prepare the nano microcapsules carrying LiCl. The PLGA material has good cellular compatibility. Human HFSCs can adhere and grow on it. Construction of tissue engineered skin with human HFSCs can induce seed cell differentiation to form hair follicle like fovea. It is possible to construct tissue engineering substitutes with skin appendages.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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