人早胚间充质干细胞向毛囊上皮转变过程中JAM1、Tiam1与细胞极性形成的关系

发布时间：2018-04-28 10:25

本文选题：人早胚间充质干细胞 + 毛囊　；参考：《第二军医大学》2008年博士论文

【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。
[Abstract]:Aim: to establish a model of inducing human early embryonic mesenchymal stem cells (hMSCs) to differentiate into hair follicle epithelial cells (HSCs), and to preliminarily realize the formation of local skin hair in nude mice. To analyze the relationship between hMSC and polar protein 3PAR3 during the differentiation of hMSC into hair follicle epithelial cells, such as adhesion molecule 1, tumor cell migration factor Tiam1, atypical phosphorylated protein kinase 味 -p-PKC 味 and polar protein 3PAR3, and the relationship between JAM1Tiam1 and the formation of cell polarity. The relationship between JAM1 and hair follicle growth direction and cell polarity formation was analyzed. Method 1: 1. HMSC and epidermal stem cells were resuscitated for biological characteristics and related safety tests, followed by large-scale culture and amplification. 2. 2. Isolation and culture of human dermal papilla cells (DPN) and identification of. 3. The expression of DP cell specific molecules SCFtoET-1 and bFGF in hMSC were detected by RT-PCR and Western-blotting respectively. 4. DP cells induced epidermal stem cells were detected by RT-PCR and Western-blotting. Changes of uPA in cells. A certain amount of hMSC was transplanted into the skin tissue of nude mice by subcutaneous or intradermal injection to observe the hair follicle formation at different time points, and the expression of HLA-1 in the newborn hair follicles of nude mice at different time points was detected by immunohistochemistry. The expression of keratin K) and SM(smooth muscle)-a-actin in newborn hair follicles of nude mice was analyzed by immunohistochemistry. The expression of JAM1 in hMSC was inhibited by RNA interference, and the interfering hMSC was transplanted into the skin of nude mice. Laser confocal and Western-Blotting methods were used to detect the expression of JAM1Tiam1 p-PKC 味 and PAR3 in the hair follicles of nude mice at different time points after intradermal hMSC transplantation. Results 1.hMSC transplanted into skin tissue of nude mice by intradermal injection could induce local hair formation in nude mice. The results of laser confocal analysis showed that the expression of PAR3 and p-PKC 味 in hair follicles showed a strong directionality. Western-blotting results showed that the expressions of PAR3p-PKC 味 and Tiam1 increased gradually. Inhibition of JAM1 expression in hMSC decreased its ability to form hair follicles in nude mice. In mature hair follicles, JAM1PAR3 and p-PKC 味 were distributed in the root sheath of hair follicles. 4. DP cells could induce epidermal stem cells to differentiate into hair follicles, induce hMSC to express specific molecules of DP cells and express the specific agglutinating growth of DP cells. ConclusionTwo h MSC can induce and participate in hair formation in nude mice. The distribution and location of PAR3 p-PKC 味 during hair formation are closely related to the direction and volume of hair follicle formation. It showed obvious directionality. P-PKC 味 mediated the reaction between PAR3 and Tiam1. JAM1 sensed the information in local microenvironment, which induced the formation of hair follicles and expanded Tiam1 as a reaction platform of polarity related molecules to maintain the polarity of hair follicle epithelial cells.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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