【摘要】：目的:1.研究皮膚及汗腺附屬器官在胚胎不同發(fā)育階段的基因表達(dá)特征,探討汗腺發(fā)育在分子基因水平上的調(diào)控機(jī)制;2.研究汗腺細(xì)胞(SGCs)與骨髓間充質(zhì)干細(xì)胞(MSCs)共培養(yǎng)和MSCs向SGCs誘導(dǎo)培養(yǎng)過程中細(xì)胞表型和基因表達(dá)的變化規(guī)律,探討干細(xì)胞的可塑性在皮膚創(chuàng)面修復(fù)和汗腺組織再生方面的治療可行性。 方法:1.采用HE方法檢測(cè)不同發(fā)育階段人胎兒皮膚組織的生理結(jié)構(gòu)和特征,其次采用免疫組化、RT-PCR和基因芯片篩選等方法全面分析人類胎兒皮膚及汗腺附屬器生理發(fā)育過程中的基因表達(dá)規(guī)律。2.體外分離培養(yǎng)成人MSCs和SGCs;將達(dá)70%融合的SGCs熱休克損傷后與(1-2)×10~5MSCs共培養(yǎng);觀測(cè)細(xì)胞表型變化并采用免疫細(xì)胞化學(xué)、RT-PCR和Western blotting等方法檢測(cè)融合細(xì)胞相比較于SGCs、骨髓MSCs及成纖維細(xì)胞的基因及蛋白表達(dá)特征。3.通過設(shè)立對(duì)照組、汗腺組織勻漿組和不同濃度的EGF組,比較在不同的誘導(dǎo)培養(yǎng)介質(zhì)下骨髓MSCs向SGCs橫向分化的潛能,并采用流式細(xì)胞術(shù)和Western blotting等分子檢測(cè)方法加以鑒定。 結(jié)果:1.不同發(fā)育階段胎兒皮膚組織結(jié)構(gòu)的變化:光鏡下可見不同發(fā)育階段的胎兒皮膚具有典型的組織學(xué)結(jié)構(gòu)。在E12周,表皮僅由1-2層細(xì)胞組成,表皮嵴較明顯,出現(xiàn)局灶增殖相間分布細(xì)胞團(tuán);E13-14周皮膚表皮細(xì)胞層數(shù)增多,排列無極性,偶見汗腺原基;E16周后周皮細(xì)胞開始脫落,表皮和真皮均增厚,原基部汗腺細(xì)胞呈索狀向真皮深層生長(zhǎng);在E20周可見管狀汗腺結(jié)構(gòu);E23-24周,表皮細(xì)胞4-5層,規(guī)律排列,汗腺結(jié)構(gòu)初步形成;E25-32周,汗腺結(jié)構(gòu)和數(shù)量穩(wěn)定。2.基因芯片篩選、免疫組化和RT-PCR結(jié)果發(fā)現(xiàn)胎兒皮膚及汗腺的形成過程是不同基因如EGF、FGF10等在細(xì)胞向空間分化時(shí)優(yōu)勢(shì)表達(dá)的過程。3.汗腺的微分離技術(shù)不僅有助于其生理學(xué)的研究,還有助于汗腺細(xì)胞功能學(xué)方面的研究。4.SGCs與骨髓MSCs共培養(yǎng)后,與自發(fā)融合相比產(chǎn)生了更多的融合細(xì)胞,并在細(xì)胞表型上出現(xiàn)扁平狀上皮樣改變和在分子基因水平上出現(xiàn)SGCs所具有的特異標(biāo)記。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
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

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本文編號(hào)：2406522