【作者】 仵敏娟；

【导师】 刘厚奇；

【作者基本信息】 第二军医大学 ， 人体解剖与组织胚胎学， 2008， 博士

【摘要】 目的:建立诱导人早胚间充质干细胞(hMSC)向毛囊上皮细胞分化的模型,初步实现裸鼠局部皮肤毛发的生成。分析hMSC向毛囊上皮细胞分化过程中连接黏附分子1(JAM1)、肿瘤细胞迁移因子(Tiam1)、非典型性磷酸化的蛋白激酶ζ(p-PKCζ)与极性蛋白3(PAR3)的关系,分析JAM1、Tiam1与细胞极性形成的关系。分析JAM1与毛囊生长方向和细胞极性形成的关系。方法:1.复苏hMSC和表皮干细胞,进行生物学特性和相关的安全性检测,随后进行大规模培养扩增。2.分离培养人毛乳头细胞(DP)并进行鉴定。3.DP细胞诱导hMSC,RT-PCR方法检测诱导后hMSC中DP细胞特异性分子SCF、ET-1和bFGF表达的变化。4.DP细胞诱导表皮干细胞,RT-PCR和Western-blotting方法检测诱导后表皮干细胞中uPA的变化。5.将一定数量的hMSC以皮下或皮内注射的方式移植到裸鼠皮肤组织中,H.E观察不同时间点毛囊生成情况,免疫组化检测HLA-1在不同时间点裸鼠新生毛囊中的表达,免疫组化分析角蛋白(K)和SM(smooth muscle)-a-actin在裸鼠新生毛囊中的表达。采用RNA干扰的方式抑制JAM1在hMSC中的表达,将干扰后的hMSC移植到裸鼠皮内,后续实验方法同正常hMSC移植裸鼠。6.激光共聚焦和Western-Blotting方法检测hMSC皮内移植后不同时间点裸鼠新生毛囊中JAM1、Tiam1、p-PKCζ和PAR3的表达并初步分析这些分子表达的位置关系和时间顺序。结果:1.hMSC以皮内注射的方式移植到裸鼠皮肤组织,可引起裸鼠皮肤局部毛发的生成。2.激光共聚焦结果显示在毛囊生成过程中,JAM1、PAR3和p-PKCζ的表达有很强的方向性,Tiam1表达均匀。Western-blotting结果显示,JAM1、PAR3、p-PKCζ和Tiam1的表达量逐渐增加。抑制JAM1在hMSC中的表达,其在裸鼠体内形成毛囊的能力下降。3.在成熟毛囊中,JAM1、PAR3和p-PKCζ分布在毛囊的根鞘处。4.DP细胞可诱导表皮干细胞往毛囊方向分化,可诱导hMSC表达DP细胞的特异性分子并表现DP细胞特有的凝集样生长。结论:hMSC可诱导并参与裸鼠毛发的生成。在毛发生成过程中,JAM1、PAR3、p-PKCζ的分布和位置关系与毛囊的生成方向和体积增加有密切关系,体现明显的方向性。p-PKCζ介导PAR3与Tiam1的反应。JAM1感受局部微环境中的信息而引发毛囊的生成和拓展,Tiam1作为极性相关分子的反应平台而维持毛囊上皮细胞的极性。更多还原

【Abstract】 Objection: To construct a model of hMSC differentiate into epithelial cells from hair follicle (HF) and induce local skin hair follicle reformation. To detect the roles of JAM1; Tiam1; p-PKCζand PAR3 in the process of hMSC differentiate into epithelial cells. To find the relationships between JAM1; Tiam1 and cell polarition; hair follicle oritention.Methods: 1. Thawing hMSC, undertake cells biological character and pathogen detection, then large scale cultivation of cells for next experiment. 2. Isolation, culture and identification of human dermal papilla cells. 3. Using real-time polymerase chain reaction to detect the uPA expression in epidermal cells after human dermal papilla cells induced. 4. Xenografts were initiated by intracutaneous injection of 2×10~4 hMSC or hMSC with JAM1 RNAi. Haematoxylin & Eosin staining was used to detect hair follicle information in different time. 5. Observation of JAM1, PAR3, p-PKCζand Tiam1 by double immunofluorescense of different stage HF using laser scanning confocal microscopy. Western-blotting was used to detect the expression of JAM1, PAR3, p-PKCζand Tiam1 in different stage HF.Results: 1. Hair follicle formation was detected in local nude mice skin after human mesenchymal cell suspensions intracutaneous injection. 2. In the process of HF formation, it’s showed that Tiam1 was expressed in all around cells of HF, however, JAM1, PAR3 and p-PKCζwere selectively expressed and enriched on the top of different development stages of HF. 3. JAM1, PAR3 and p-PKCζwere selectively expressed in the root sheath of mature haif follicle. 4. Dermal papilla cells can induce epidermal cells differentiate into hair follicle.Conclusion: hMSC can induce and participate in the HF formation in nude mice. In the process of HF formation, the expression of JAM1, PAR3 and p-PKCζwere selective. The selective expressions of JAM1, PAR3 and p-PKCζhave close relationship to the growth direction and volume increases of HF. p-PKCζare the regulator of PAR3 and Tiam1. JAM1 acceptance the messengers form the microenvironment of nude mice skin tissue and induce the hair follicle development and growth, Tiam1 is the platform of polarity molecular which play key role in the maintenance of hair follicle epithelial cell polarity.更多还原