人成体干细胞诱导分化成视网膜感光细胞样细胞的实验研究

发布时间：2018-11-25 21:25

【摘要】：【目的】比较人眼眶脂肪组织来源干细胞与牙周韧带来源干细胞定向分化至视网膜感光细胞样细胞的能力。探索通过组织块培养法从人眼眶脂肪组织原代培养间充质干细胞的方案及技术。比较通过组织块培养法和酶消化法获取的人眼眶脂肪组织来源干细胞向视网膜感光细胞样细胞定向分化的能力。【方法】扩增培养通过酶消化法获取的人眼眶脂肪组织来源干细胞株和牙周韧带来源干细胞细胞株，检测其细胞表面的干细胞标记。然后使用多种培养基对两种不同组织来源的间充质干细胞进行定向诱导，使其向视网膜感光细胞分化，在不同时间点收获细胞，检测视网膜感光细胞特异性基因和蛋白表达。比较这两种细胞向视网膜感光细胞定向分化的能力。继而通过组织块培养法和酶消化法从人眼眶脂肪组织中分离脂肪组织来源干细胞，，并通过免疫荧光染色检测其干细胞标记物。对通过不同方法获得的脂肪组织来源干细胞进行培养，诱导其向视网膜感光细胞分化，在不同时间点收获细胞，检测细胞中视网膜感光细胞特异性基因和蛋白表达，比较两种细胞的分化能力。【结果】通过酶消化法获得的人眼眶脂肪组织来源干细胞株和牙周韧带来源干细胞株接受定向诱导分化后，前者表现出类似视网膜感光细胞外段样结构；后者的细胞形态无显著改变。牙周韧带来源干细胞表达Pax6基因早于脂肪组织来源干细胞，但是表达Rhodopsin基因及视紫红质蛋白晚于脂肪组织来源干细胞。本研究成功地通过组织块培养法从人眼眶脂肪组织中原代培养出脂肪组织来源干细胞。通过组织块培养法和酶消化法获取的人眼眶脂肪组织来源干细胞接受诱导分化后，均表现出类似视杆细胞外段样结构。通过组织块培养法获得的细胞较早表达Pax6基因、Rhodopsin基因及视紫红质蛋白。【结论】眼眶脂肪组织来源干细胞与牙周韧带来源干细胞具有定向分化至类似视网膜感光细胞样细胞的潜能，前者的分化能力强于后者。通过组织块培养法可以从人眼眶脂肪组织中原代培养出脂肪组织来源干细胞，该细胞具有定向分化至类似视网膜感光细胞样细胞的潜能，并且分化能力强于通过酶消化法分离获取的细胞。
[Abstract]:To compare the ability of adipose stem cells derived from human orbital adipose tissue and periodontal ligament derived stem cells to differentiate into photoreceptor cells of the retina. To explore the scheme and technique of primary culture of mesenchymal stem cells from human orbital adipose tissue by tissue mass culture. The ability of stem cells derived from human orbital adipose tissue to differentiate into retinal photoreceptor cells was compared by tissue mass culture and enzyme digestion. [methods] Stem cell lines derived from adipose tissue and periodontal ligament derived from human orbital adipose tissue were amplified by enzyme digestion and the stem cell markers on the surface of the cells were detected. Then two kinds of mesenchymal stem cells from different tissues were induced to differentiate into retinal photoreceptor cells. The specific gene and protein expression of retinal photoreceptor cells were detected at different time points. The ability of these two cells to differentiate into retinal photoreceptor cells was compared. Stem cells derived from adipose tissue were isolated from human orbital adipose tissue by tissue mass culture and enzyme digestion, and the stem cell markers were detected by immunofluorescence staining. Stem cells derived from adipose tissue obtained by different methods were cultured and induced to differentiate into retinal photoreceptor cells. The specific genes and proteins of retinal photoreceptor cells were detected at different time points. The differentiation ability of two kinds of cells was compared. [results] the stem cell lines derived from adipose tissue of human orbit and periodontal ligament derived stem cells were induced and differentiated by enzyme digestion, the former showed a structure similar to the outer segment of retinal photoreceptor cells. There was no significant change in cell morphology in the latter. Periodontal ligament derived stem cells expressed Pax6 gene earlier than adipose tissue derived stem cells, but expressed Rhodopsin gene and rhodopsin later than adipose tissue derived stem cells. In this study, adipose tissue derived stem cells were successfully cultured from human orbital adipose tissue by tissue mass culture. The stem cells derived from human orbital adipose tissue obtained by tissue mass culture and enzyme digestion showed similar extracellular segmental structure after induction and differentiation. The cells obtained by tissue mass culture expressed Pax6 gene, Rhodopsin gene and rhodopsin earlier. [conclusion] Orbital adipose tissue derived stem cells and periodontal ligament derived stem cells have the potential to differentiate into retinal photocell-like cells, and the former is stronger than the latter. Stem cells derived from adipose tissue can be cultured from human orbital adipose tissue by tissue mass culture, which has the potential to differentiate into photoreceptor cells like retinal cells. And the ability of differentiation was stronger than that of the cells isolated by enzyme digestion.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1

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