胎鼠真皮细胞诱导毛发形成的研究

发布时间：2018-04-16 07:38

本文选题：胎鼠真皮细胞 + 毛发形成能力　；参考：《北京协和医学院》2013年博士论文

【摘要】：研究背景及意义毛发缺失给患者的心理造成了极大的困扰。毛发移植术是目前公认治疗毛发缺失最有效的手段,但同时也存在不足,如供区有限及手术的二次损伤等。毛囊重建是具有毛囊再生能力的细胞在特定环境中再生出新的毛发,为毛发缺失的治疗提供了新的思路。种子细胞是毛囊再生的关键元素,真皮和表皮两种来源细胞间的相互作用是毛囊再生的关键。目前研究认为真皮来源细胞具有诱导毛发形成能力,在毛发再生中发挥更为关键的作用。因此,分析毛发再生过程中真皮来源细胞影响毛发再生的相关机制,对毛发的再生研究具有十分重要的意义,也为未来构建带毛发的组织工程皮肤奠定了基础。研究目的： 1、掌握胎鼠真皮细胞的分离及体外培养技术；通过将不同胎龄胎鼠原代真皮细胞与表皮细胞混合注射入裸鼠皮下,比较不同胎龄胎鼠真皮细胞诱导毛发形成能力,为分析真皮细胞中毛发再生能力相关因素提供依据。 2、通过比较不同胎龄胎鼠真皮细胞的多向分化能力、表面标志物表达差异、以及真皮组织中毛发再生相关信号通路基因表达的变化,探讨真皮细胞影响毛发再生的相关机制。 3、掌握人毛囊干细胞的分离培养技术,探讨胎鼠真皮细胞与人毛囊干细胞构建毛发再生研究模型的可行性；利用毛囊干细胞进行组织工程皮肤的构建,为构建带有毛发的组织工程皮肤奠定基础。研究方法 1、通过不同胎龄胎鼠皮肤HE染色,观察胎鼠毛囊的发育情况；通过中性蛋白酶与Ⅰ型胶原酶二步酶消化法对不同胎龄胎鼠真皮细胞进行体外分离培养。 2、采用胎鼠真皮细胞与胎鼠表皮细胞混合皮下注射,2周后计数毛发形成数量,验证不同胎龄胎鼠真皮细胞的诱导毛发形成能力。 3、将不同胎龄胎鼠真皮细胞分别向成骨、成脂、成神经、成皮脂腺、成表皮方向进行体外诱导培养,比较胎鼠真皮细胞多向分化能力；通过流式细胞分析与RealTime PCR检测的方法,观察毛囊细胞常见表面标志以及毛发再生相关信号通路中重要分子在不同胎龄胎鼠真皮细胞中的表达情况,分析与胎鼠真皮细胞毛发形成能力相关的影响因素,并采用siRNA干扰的方法对毛发再生相关的Wnt信号通路分子进行分析。 4、利用显微分离加差速贴壁法对人毛囊干细胞进行分离培养,并验证胎鼠真皮细胞混合人毛囊干细胞后的毛发形成能力,进而利用毛囊干细胞尝试构建组织工程皮肤。实验结果 1、胎鼠皮肤毛囊发育和细胞分离培养：HE染色显示,在E15天胎鼠皮肤开始出现毛囊的发育,E17天新生毛囊逐渐增多,E20天形成结构比较完整的毛囊；二步酶消化法可成功分离胎鼠真皮细胞,并可在体外大量扩增。 2、胎鼠真皮细胞毛发形成能力：E15天胎鼠真皮细胞无法形成毛发,E16天胎鼠真皮细胞可形成少量毛发,从E17天开始胎鼠真皮细胞形成毛发的数量明显增加(P0.05),E18、E19、E20天胎鼠真皮细胞再生毛发的数量无明显差异。 3、胎鼠真皮细胞毛发形成影响因素研究： ①诱导分化能力：E15、E17、E20天胎鼠真皮细胞经诱导后,各类组织细胞分化相关基因表达均上调(P0.05)；但不同胎龄胎鼠真皮细胞诱导分化能力存在差异,随胎龄增加,向表皮、神经以及脂肪细胞方向诱导分化能力增强。 ②细胞标志物表达：毛囊内表皮细胞表面标志物在不同胎龄胎鼠真皮细胞内均有不同程度表达,其中Sca-1的mRNA表达在E18天比E15天有非常显著的增加(P0.01),流式检测显示其表达从E15天的(38.7%±4.87545)上升到E18天的(69.8333%±10.17912)。 ③信号通路：β-catenin和LHX2的表达分别在E17和E19天胎鼠真皮中表达明显增加。RNA干扰实验结果显示β-catenin基因表达的抑制,伴随着LHX2基因的同步显著下调(P0.05)以及Sca-1基因的非常显著的滞后下调(P0.01)；而LHX2基因表达的抑制也可伴随Sca-1基因显著下调(P0.05)。 4、人毛囊干细胞的相关研究：显微分离加差速贴壁法分离的人毛囊干细胞可表达多种毛囊干细胞标志物,其中以P3或P4代细胞表达最高,与E17胎鼠真皮细胞混合后可再生大量毛发；人毛囊干细胞与真皮成纤维细胞胶原凝胶构建的组织工程皮肤细胞状态良好,结构类似正常皮肤,但机械强度差,容易收缩；与真皮成纤维细胞PGA构建的组织工程皮肤形态稳定,但细胞相容性稍差。结论 1、E15-E20天胎鼠真皮细胞毛发形成能力随胎龄增加而增加。E17、E18天开始胎鼠真皮细胞的毛发再生能力明显增强,是毛发形成的关键时期。 2、E15、E17、E20天胎鼠真皮细胞均具有多向分化能力,E17、E20胎鼠真皮细胞具有更强的向表皮、神经和脂肪细胞分化的能力,可能是毛发形成能力更强的因素；Sca-1的表达随胎龄变化趋势与胎鼠真皮细胞毛发形成能力变化趋势基本一致,也可能与毛发形成能力密切相关。伴随着毛发形成能力显著增加,β-catenin和LHX2在胎鼠真皮内的表达也相应显著增加,提示WNT信号通路是影响毛囊再生的重要因素。RNA干扰实验进一步提示β-catenin基因可以通过作用其下游的LHX2及其Sca-1基因在毛发形成中发挥重要调节作用。 3、人毛囊干细胞体外培养增殖能力强,可稳定传代,从标志物检测水平观察发现以P3或P4代细胞纯度高,可用于基于胎鼠真皮细胞的毛发再生模型；利用其与不同支架材料可构建出类似正常形态的组织工程皮肤,为最终构建出带有毛发的组织工程皮肤奠定了基础。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R758.71

【参考文献】