Wnt信号通路介导的基因调控毛囊干细胞定向分化的研究

发布时间：2018-06-24 11:04

本文选题：毛囊干细胞 + 氯化锂　；参考：《广州医学院》2012年硕士论文

【摘要】：背景和目的毛囊干细胞(Hair follicle stem cells，HFSCs)被选为种子细胞用于构建组织工程皮肤以修复临床烧伤、创伤、慢性溃疡等皮肤缺失已成为医学研究的热点。毛囊是皮肤的重要附属器官，从内向外依次由毛干、内根鞘和外根鞘组成。毛囊隆突部位于外根鞘近表皮端，皮脂腺下方竖毛肌附着处，此处的细胞体外培养时表现出干细胞的特性，即可自我复制、慢周期性和具有多向分化潜能。目前已经得到证实，隆突部的这一群特殊的细胞为毛囊干细胞。毛囊干细胞具有多向分化迁移能力，不仅能向上分化并迁移参与表皮更新和皮脂腺维持，，而且能向下端毛球部分化并迁移参与形成毛囊。毛囊干细胞分化倾向的不同是由多种因素共同决定的，不仅有细胞内部分子及基因的调控，也有外来信号及微环境壁龛的协同作用，更有多条信号通路传导分化“命令”。在众多科研工作者的共同努力下发现，毛囊干细胞Wnt信号通路(Wnt signaling pathway)在调控其增殖、分化中起到重要作用。 Wnt/β-catenin信号通路在全身几乎所有的细胞中都存在，细胞受到刺激后释放Wnt蛋白，激活下游一系列大分子信号活性物质，最终导致基因表达改变，是诱导细胞分化和增殖的一条分子级联通路，在胚胎发育和肿瘤发生中，这条通路也起到关键性的作用。在HFSCs中，Wnt/β-catenin信号通路激活后会引起β-catenin在胞浆内的堆积，随之转入到细胞核中，与核内转录因子Tcf3/Lefl结合，激活下游靶基因c-myc、cyclin D1等的转录，促进毛囊干细胞定向分化。近年来有研究发现细胞外来干预因子氯化锂(LiCl)可抑制Wnt信号通路中β-catenin的降解，使之在细胞质中聚集，激活下游通路，影响毛囊干细胞分化的表型，但对于LiCl调节HFSCs分化的分子机制研究较少，且局限于在细胞质中作用的研究。本课题选用了氯化锂激活Wnt信号通路，探讨Wnt/β-catenin信号通路在人HFSCs向毛囊形成细胞或表皮细胞定向分化中的作用及与其他信号因子的相互关系，尤其是细胞核内的分子水平改变，以及药物控释系统的建立和在组织工程皮肤中的应用。方法 1.头皮组织采用改进的毛囊隆突部获取方法及两步酶消化后，将带有隆突部的毛发剪成泥状，Ⅳ型胶原差速贴壁法获取毛囊干细胞。使用免疫荧光染色法对毛囊干细胞进行鉴定。以500个/孔(2.5×10~3/m1)、1000个/孔(5×10~3/m1)、2000个/孔(1×10~4/m1)、3000个/孔(1.5×10~4/m1)、5000个/孔(2.5×10~4/m1)接种于96孔培养板中，观察细胞生长情况，采用MTT法描绘其生长曲线，观察各密度培养的HFSCs在不同时间点的增殖效应，筛选出体外最佳的HFSCs传代培养密度。 2.选用0、0.1、1、5、10、20、40、100mM/l的氯化锂诱导毛囊干细胞分化，观察毛囊干细胞的生存状态，确定氯化锂的毒性作用范围；选用0、5、10mM/l氯化锂作用于毛囊干细胞，MTT法对比分析各组对细胞增殖效应；LiCl终浓度分别为0、0.1、1、10、50、100、200、400μg/ml作用于毛囊干细胞，检测氯化锂的毒性，并探索促HFSCs分化的最佳LiCl浓度；采用real-time PCR技术定量检测0、5、10mmol/ml LiCl干预毛囊干细胞5d后Wnt信号通路中生物大分子：β-catenin、GSK-3β、Tcf3、c-myc和cyclin D1的mRNA水平，探讨LiCl诱导HFSCs分化过程中，LiCl的不同浓度对Wnt/β-catenin信号途径及其相关信号因子的表达的影响和相互作用。 3.LiCl-PLGA纳米微球的制备：选用聚乳酸-羟基乙酸(PLGA)作为微囊敷料，荷载适当浓度的氯化锂，采用复乳-溶剂挥发法制备载有氯化锂的纳米微囊。完全培养基培养人成纤维细胞，选用异体脱细胞真皮(ADM)为支架材料，以扩增培养的HFSCs为种子细胞接种于ADM表皮面，成纤维细胞接种于ADM真皮面，构建组织工程皮肤。采用5mM/l的LiCl干预构建的组织工程皮肤，培养5d后切片行HE染色、免疫荧光染色和免疫组织化学染色，观察种子细胞在ADM上的生长及分化情况，为动物实验奠定良好基础。结果 1.从人头皮成功分离培养出HFSCs，在体外经多次传代后仍具有很强的增殖能力和多向分化潜能，当HFSCs接种密度为1x10~4ml时，细胞增殖速度最快，扩展能力强，其生长曲线呈典型的S型。 2.随LiCl浓度升高，细胞增殖效应减弱。含有LiCl的K-SFM条件培养基中HFSCs形态改变明显，各组间有明显差别，LiCl10mmol/L时分化比例高。LiCl诱导毛囊干细胞Wnt信号通路的同时还有其一定的细胞毒性，浓度大于200μg/ml(约5mmol/L)时明显抑制细胞生长。实验研究发现在HFSCs分化过程中，LiCl可激活Wnt/β-catenin信号通路，当浓度为5mM/l时促使β-catenin及其拮抗物GSK3β、核内转录因子Tcf3、下游基因c-myc、cyclin D1的转录上调，浓度为10mM/l时除cyclin D1转录仍上调外，其余基因转录下调。 3.扫描电镜观察细胞在PLGA-ADM材料上生长良好，分泌的胞外基质丰富，并随时间进展，细胞逐渐融合，细胞外基质分泌逐渐增多。HE染色观察发现HFSCs生长状态良好，单层排列分布于ADM表皮面上，LiCl干预组表皮面有毛囊样小凹形成，成纤维细胞与ADM真皮面贴附紧密，能分泌胞外基质形成连续细胞层包绕ADM。结论 1.我们改进的培养方法能简便快捷的获取人HFSCs，且得到的人HFSCs在体外有较强的增殖及传代能力，1×10~4/ml接种密度是促进HFSCs增殖的最佳密度。 2.LiCl促HFSCs分化明显，对HFSCs的干预有临界毒性浓度，当浓度大于200μg/ml时，细胞生长增殖受到明显抑制，同时这一浓度也是促HFSCs分化的最佳浓度。LiCl促HFSCs分化作用可能与激活Wnt/β-catenin信号通路、通路下游各因子转录上调相关，Tcf3在维持细胞未分化状态中发挥了一定的作用，cyclinD1的转录与氯化锂浓度作用呈正相关，在HFSCs的分化中起到重要作用。 3.复乳-溶剂挥发法能快速高效的制备出载有LiCl的纳米微囊，其PLGA材料具有良好的细胞相容性，人HFSCs能在其上较好的粘附和生长。构建载有人HFSCs的组织工程皮肤，在LiCl诱导下能够促使种子细胞发生分子学改变形成毛囊样小凹，使构建带有皮肤附属器的组织工程替代物成为可能。
[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.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】