人汗腺发生与修复过程中基因表达特征的系列研究
发布时间：2019-01-10 16:14
【摘要】：目的:1.研究皮肤及汗腺附属器官在胚胎不同发育阶段的基因表达特征,探讨汗腺发育在分子基因水平上的调控机制;2.研究汗腺细胞(SGCs)与骨髓间充质干细胞(MSCs)共培养和MSCs向SGCs诱导培养过程中细胞表型和基因表达的变化规律,探讨干细胞的可塑性在皮肤创面修复和汗腺组织再生方面的治疗可行性。 方法:1.采用HE方法检测不同发育阶段人胎儿皮肤组织的生理结构和特征,其次采用免疫组化、RT-PCR和基因芯片筛选等方法全面分析人类胎儿皮肤及汗腺附属器生理发育过程中的基因表达规律。2.体外分离培养成人MSCs和SGCs;将达70%融合的SGCs热休克损伤后与(1-2)×10~5MSCs共培养;观测细胞表型变化并采用免疫细胞化学、RT-PCR和Western blotting等方法检测融合细胞相比较于SGCs、骨髓MSCs及成纤维细胞的基因及蛋白表达特征。3.通过设立对照组、汗腺组织匀浆组和不同浓度的EGF组,比较在不同的诱导培养介质下骨髓MSCs向SGCs横向分化的潜能,并采用流式细胞术和Western blotting等分子检测方法加以鉴定。 结果:1.不同发育阶段胎儿皮肤组织结构的变化:光镜下可见不同发育阶段的胎儿皮肤具有典型的组织学结构。在E12周,表皮仅由1-2层细胞组成,表皮嵴较明显,出现局灶增殖相间分布细胞团;E13-14周皮肤表皮细胞层数增多,排列无极性,偶见汗腺原基;E16周后周皮细胞开始脱落,表皮和真皮均增厚,原基部汗腺细胞呈索状向真皮深层生长;在E20周可见管状汗腺结构;E23-24周,表皮细胞4-5层,规律排列,汗腺结构初步形成;E25-32周,汗腺结构和数量稳定。2.基因芯片筛选、免疫组化和RT-PCR结果发现胎儿皮肤及汗腺的形成过程是不同基因如EGF、FGF10等在细胞向空间分化时优势表达的过程。3.汗腺的微分离技术不仅有助于其生理学的研究,还有助于汗腺细胞功能学方面的研究。4.SGCs与骨髓MSCs共培养后,与自发融合相比产生了更多的融合细胞,并在细胞表型上出现扁平状上皮样改变和在分子基因水平上出现SGCs所具有的特异标记。5.骨髓MSCs在用不同的汗
[Abstract]:Objective: 1. To study the gene expression characteristics of skin and sweat gland accessory organs at different stages of embryonic development and to explore the regulation mechanism of sweat gland development at molecular gene level. 2. To study the changes of cell phenotype and gene expression during the co-culture of sweat gland cell (SGCs) and bone marrow mesenchymal stem cells (MSCs) and the induction of MSCs to SGCs. To explore the feasibility of the plasticity of stem cells in the treatment of skin wound repair and sweat gland regeneration. Methods: 1. HE method was used to detect the physiological structure and characteristics of human fetal skin at different stages of development, followed by immunohistochemistry. RT-PCR and gene chip screening were used to analyze the gene expression in the physiological development of human fetal skin and sweat gland appendages. 2. Adult MSCs and SGCs; were co-cultured with (1-2) 脳 10~5MSCs after heat shock injury of 70% fusion. The phenotypic changes of the cells were observed and the expression characteristics of genes and proteins in fusion cells compared with SGCs, bone marrow MSCs and fibroblasts were detected by immunocytochemistry, RT-PCR and Western blotting. 3. By setting up control group, sweat gland homogenate group and different concentration EGF group, the potential of bone marrow MSCs to differentiate into SGCs was compared under different induction culture medium, and was identified by flow cytometry and Western blotting. Results: 1. The changes of fetal skin tissue structure in different developmental stages: under the light microscope, the fetal skin of different developmental stages has typical histological structure. At the 12th week of E12, the epidermis was composed of only 1-2 layers of cells, the epidermal ridge was obvious, and the focal proliferative phase distributed cell mass appeared in the epidermis, and at 13-14 weeks, the number of the layers of epidermal cells increased, the arrangement of the epidermis was not polar, and occasionally the sweat gland primordium was found. After 16 weeks of E16 weeks, the pericarp cells began to fall off, both the epidermis and the dermis thickened, the original basal sweat gland cells grew into the deep layer of the dermis, and the tubular sweat gland structure was observed at the 20th week of E16 weeks. E23-24 weeks, epidermal cells 4-5 layers, regular arrangement, sweat gland structure preliminary formation, E25-32 weeks, sweat gland structure and number of stable. 2. The results of gene chip screening, immunohistochemistry and RT-PCR showed that the formation of fetal skin and sweat glands was the dominant expression process of different genes, such as EGF,FGF10, when the cells differentiated into space. 3. The microseparation of sweat glands is not only helpful to the study of physiology, but also to the study of the function of sweat gland cells. After co-culture of 4.SGCs and bone marrow MSCs, more fusion cells are produced than spontaneous fusion. There were flattened epithelioid changes in cell phenotype and specific markers of SGCs at molecular gene level. 5. 5. Bone marrow MSCs is using different sweats
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346
本文编号：2406522
[Abstract]:Objective: 1. To study the gene expression characteristics of skin and sweat gland accessory organs at different stages of embryonic development and to explore the regulation mechanism of sweat gland development at molecular gene level. 2. To study the changes of cell phenotype and gene expression during the co-culture of sweat gland cell (SGCs) and bone marrow mesenchymal stem cells (MSCs) and the induction of MSCs to SGCs. To explore the feasibility of the plasticity of stem cells in the treatment of skin wound repair and sweat gland regeneration. Methods: 1. HE method was used to detect the physiological structure and characteristics of human fetal skin at different stages of development, followed by immunohistochemistry. RT-PCR and gene chip screening were used to analyze the gene expression in the physiological development of human fetal skin and sweat gland appendages. 2. Adult MSCs and SGCs; were co-cultured with (1-2) 脳 10~5MSCs after heat shock injury of 70% fusion. The phenotypic changes of the cells were observed and the expression characteristics of genes and proteins in fusion cells compared with SGCs, bone marrow MSCs and fibroblasts were detected by immunocytochemistry, RT-PCR and Western blotting. 3. By setting up control group, sweat gland homogenate group and different concentration EGF group, the potential of bone marrow MSCs to differentiate into SGCs was compared under different induction culture medium, and was identified by flow cytometry and Western blotting. Results: 1. The changes of fetal skin tissue structure in different developmental stages: under the light microscope, the fetal skin of different developmental stages has typical histological structure. At the 12th week of E12, the epidermis was composed of only 1-2 layers of cells, the epidermal ridge was obvious, and the focal proliferative phase distributed cell mass appeared in the epidermis, and at 13-14 weeks, the number of the layers of epidermal cells increased, the arrangement of the epidermis was not polar, and occasionally the sweat gland primordium was found. After 16 weeks of E16 weeks, the pericarp cells began to fall off, both the epidermis and the dermis thickened, the original basal sweat gland cells grew into the deep layer of the dermis, and the tubular sweat gland structure was observed at the 20th week of E16 weeks. E23-24 weeks, epidermal cells 4-5 layers, regular arrangement, sweat gland structure preliminary formation, E25-32 weeks, sweat gland structure and number of stable. 2. The results of gene chip screening, immunohistochemistry and RT-PCR showed that the formation of fetal skin and sweat glands was the dominant expression process of different genes, such as EGF,FGF10, when the cells differentiated into space. 3. The microseparation of sweat glands is not only helpful to the study of physiology, but also to the study of the function of sweat gland cells. After co-culture of 4.SGCs and bone marrow MSCs, more fusion cells are produced than spontaneous fusion. There were flattened epithelioid changes in cell phenotype and specific markers of SGCs at molecular gene level. 5. 5. Bone marrow MSCs is using different sweats
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346
【参考文献】
相关期刊论文 前2条
1 付小兵,方利君,王玉新,孙同柱,程飚;骨髓间充质干细胞自体移植提高猪皮肤创面修复质量的初步研究[J];中华医学杂志;2004年11期
2 付小兵,程飚;成体干细胞研究及其可能的临床应用[J];中华医学杂志;2005年27期
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