人皮肤毛囊干细胞体外无血清扩增的相关研究

发布时间：2018-07-15 13:08

【摘要】：背景皮肤上皮干细胞作为种子细胞用于人造皮肤的制造和伤口修复已有多年历史,但通常所用的干细胞为单一分化潜能的表皮干细胞,无法重建任何皮肤附属器官,实现皮肤的全部功能。研究表明毛囊干细胞具有修复表皮,分化为毛发、皮脂腺、汗腺的多分化潜能,是伤口修复和人造皮肤的理想种子细胞。然而人类正常皮肤组织中,毛囊干细胞的比例极低,一根毛囊仅能提取200-400个毛囊干细胞,而临床治疗所需的毛囊干细胞通常需要百万数量级以上,原代毛囊干细胞数量远不能满足需求。而毛囊干细胞传统体外培养需要滋养层细胞及血清培养,但这样的细胞产品不能用于临床治疗,因此急需建立支持人类毛囊干细胞体外无血清无滋养层细胞的扩增技术。运用小分子抑制剂可以精确对信号通路进行激活或抑制,在细胞培养研究领域已经取得一些进展。小分子抑制剂Y-27632为Rock的抑制剂,可以促使人胚胎干细胞、角质上皮细胞增殖,抑制细胞的凋亡;A83-01可以抑制TGF-βI型受体ALK5的转录活性,协同CHIR-99021可将成熟肝细胞逆转成干细胞,A83-01协同DMH-1、CHIR-99021、Y-27632在无血清无滋养层细胞的条件下能够在体外大量扩增某些上皮细胞。以上这些结果表明在细胞培养中利用小分子抑制剂可以一定程度上弥补无血清培养营养不足导致的细胞生物学性状改变,开拓干细胞培养的新局面。目前,A83-01、DMH-1、CHIR-99021、Y-27632在毛囊干细胞体外无血清扩增的作用尚未见相关报道。本课题拟建立符合本实验室及贴近临床运用的人毛囊干细胞采集纯化保存的规范化流程,在此基础上研究小分子抑制剂A83-01、DMH-1、CHIR-99021、Y-27632对人毛囊干细胞体外无血清扩增的影响,为人毛囊干细胞临床应用及研究提供一定的理论基础和技术支持。目的1.建立人毛囊干细胞库及细胞采集纯化保存的规范化流程,并行鉴定;2.探索体外无血清培养人毛囊干细胞方法,优化扩增人毛囊干细胞及维持干性的条件。方法1.人毛囊外根鞘细胞获取:采用Dispase II初步消化带毛囊组织,显微镜下拔取毛囊,剔除皮脂腺,离断毛乳头,0.05%Trypsin-EDTA消化获得人毛囊外根鞘细胞单细胞悬液。免疫荧光染色观察处理前后干细胞变化。2.差速贴壁富集毛囊干细胞及原代细胞培养:利用含人I型胶原的COATING MATRIX KIT进行包被培养皿,15min差速贴壁分离毛囊干细胞,利用CNT-PR原代上皮培养液添加Y-27632进行原代细胞培养。流式分析及免疫荧光观察纯化结果。3.人毛囊干细胞的培养条件探索:分组培养毛囊干细胞,对照组培养基(CNT组)、实验组1(A83-01组)、实验组2(DMH-1组)、实验组3(A83-01+DMH-1组)、实验组4(CHIR-99021)、实验组5(A83-01+DMH-1+CHIR-99021组),CCK-8行细胞分组条件下短期培养细胞增殖速度,传代培养时,每代记录接种细胞及收细胞数目,免疫荧光染色K15细胞比例,计算细胞扩增总量及K15阳性细胞扩增情况。结果1、免疫荧光染色定位毛囊干细胞于毛囊的中段,显微外科除去毛乳头及皮脂腺,初步提高了毛囊干细胞纯化效果;2、以含人I型胶原的COATING MATRIX KIT进行差速贴壁分离毛囊干细胞,有效富集CD200+/CD49+毛囊干细胞(P0.05),同时避免传统利用小鼠IV型胶原差速贴壁带来的异种蛋白侵入危险;3、成功在无血清、无滋养层、培养基成分限定的条件下进行原代干细胞体外培养16±3天,每根毛囊可取得4.5±0.7×104 K15阳性细胞;4、添加A83-01、Y-27632或者添加A83-01、DMH-1、Y-27632于CNT-PR培养基较单纯添加Y-27632于CNT-PR培养基或其他分组,能提高总体细胞及K15阳性细胞增殖速度,缩短扩增时间(P0.05);5、本实验流程可在无血清、无滋养层、培养基成分限定的条件下进行毛囊干细胞的培养扩增,利于实验研究的分析和临床应用。结论:我们在避免异种蛋白或基因干扰的条件下,利用差速贴壁法纯化毛囊干细胞,在体外无血清扩增,6周内1根毛囊扩增出的毛囊干细胞可达百万数量级,贴近临床运用,并发现A83-01及DMH-1可以提高毛囊干细胞扩增效率及干性维持。
[Abstract]:Background skin epithelial stem cells have been used as seed cells for the manufacture and repair of artificial skin for many years. However, the stem cells used as a single differentiation potential stem cells can not reconstruct any skin accessory organs to achieve all the functions of the skin. The multiple differentiation potential of sebaceous glands and sweat glands is the ideal seed cell for wound repair and artificial skin. However, in human normal skin tissue, the proportion of hair follicle stem cells is very low. One hair follicle can only extract 200-400 hair follicle stem cells, and the hair follicle stem cells needed for clinical treatment usually require more than a million orders of grade, original hair follicle stem cells. The traditional culture of hair follicle stem cells in vitro requires the culture of trophoblast cells and serum, but this kind of cell product can not be used for clinical treatment. Therefore, it is urgent to establish the amplification technology that supports human hair follicle stem cells without serum-free trophoblast cells in vitro. In the field of cell culture, some progress has been made in the field of cell culture. Small molecular inhibitor Y-27632 is an inhibitor of Rock, which can promote the proliferation of human embryonic stem cells, keratinocytes and inhibit cell apoptosis; A83-01 can inhibit the transcriptional activity of TGF- beta I receptor ALK5 and reverse the maturation of mature hepatocytes into dry cells with CHIR-99021. Cells, A83-01 and DMH-1, CHIR-99021, and Y-27632 can amplify a large number of epithelial cells in vitro under the conditions of serum-free and non trophoblastic cells. These results suggest that the use of small molecular inhibitors in cell culture can partly compensate for the changes in cell biological properties caused by no serum-free culture, and to open up dry cells. At present, the role of A83-01, DMH-1, CHIR-99021 and Y-27632 in the serum-free expansion of hair follicle stem cells in vitro has not been reported. This topic intends to establish a standardized process for the collection, purification and preservation of human hair follicle stem cells, which is in accordance with this laboratory and close to clinical application. On this basis, the small molecule inhibitors, A83-01, DMH-1, are studied. The effect of CHIR-99021 and Y-27632 on the serum-free expansion of human hair follicle stem cells in vitro provides a theoretical basis and technical support for the clinical application and research of human hair follicle stem cells. Objective 1. to establish a standardized process for human hair follicle stem cell bank and cell collection and purification. 2. to explore the serum-free culture of human hair follicle stem cells in vitro Methods to optimize the expansion of human hair follicle stem cells and maintain the dry condition. Methods 1. human hair follicle outer root sheath cells were obtained: using Dispase II preliminary digestive tract hair follicle tissue, extracting hair follicles, removing sebaceous glands, breaking off hair nipples, and digesting human hair follicle outer root sheath cells single cell suspension. Immunofluorescence staining observation place. Pre and post stem cell changes.2. differential adherent enrichment of hair follicle stem cells and primary cell culture: using COATING MATRIX KIT containing human I collagen to pack culture dish, 15min differential adherent separation of hair follicle stem cells, primary cultured cell culture with Y-27632 in CNT-PR primary culture medium. Flow analysis and immunofluorescence observation and purification Fruit culture conditions of.3. human hair follicle stem cells: group culture hair follicle stem cells, control group (group CNT), experimental group 1 (group A83-01), experimental group 2 (DMH-1 group), experimental group 3 (A83-01+DMH-1 group), experimental group 4 (CHIR-99021), experimental group 5 (A83-01+DMH-1+ CHIR-99021 group), CCK-8 row cell group conditions for short-term culture of cell proliferation speed, transmission, transmission of cells. At the time of culture, the inoculation cells and the number of cells collected, the proportion of immunofluorescent staining K15 cells, the total amount of cell amplification and the amplification of K15 positive cells were calculated. Results 1, immunofluorescence staining was used to locate the hair follicle stem cells in the middle part of the hair follicle, microsurgical removal of dermal papilla and skin fat gland, initially raising the purification effect of hair follicle stem cells; 2, COATING MATRIX KIT containing human type I collagen was used for differential adhesion separation of hair follicle stem cells, effectively enriching CD200+/CD49+ hair follicle stem cells (P0.05), and avoiding the invasion of xenogeneic proteins caused by the differential adhesion of IV collagen in mice. 3, the primary stem cells were successfully carried out under the condition of serum-free, no trophoblastic layer and limited culture medium. For 16 + 3 days in vitro culture, 4.5 + 0.7 x 104 K15 positive cells could be obtained in each hair follicle. 4, adding A83-01, Y-27632 or adding A83-01, DMH-1, Y-27632 on CNT-PR culture medium could increase the proliferation rate and shorten the amplification time (P0.05) of the whole cell and K15 positive cells (P0.05); 5. It is beneficial to the analysis and clinical application of hair follicle stem cells in the condition of no serum-free, no trophoblast and culture medium. Conclusion: we have purified hair follicle stem cells with differential adherent method under the condition of avoiding heterogenous protein or gene interference, and expanded the hair follicle without serum in vitro, and amplified 1 hair follicles within 6 weeks. Hair follicle stem cells can reach one million orders of magnitude, which is close to clinical application. It is found that A83-01 and DMH-1 can enhance the amplification efficiency and dry maintenance of hair follicle stem cells.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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