【摘要】：【目的】比較人眼眶脂肪組織來源干細胞與牙周韌帶來源干細胞定向分化至視網(wǎng)膜感光細胞樣細胞的能力。探索通過組織塊培養(yǎng)法從人眼眶脂肪組織原代培養(yǎng)間充質干細胞的方案及技術。比較通過組織塊培養(yǎng)法和酶消化法獲取的人眼眶脂肪組織來源干細胞向視網(wǎng)膜感光細胞樣細胞定向分化的能力。 【方法】擴增培養(yǎng)通過酶消化法獲取的人眼眶脂肪組織來源干細胞株和牙周韌帶來源干細胞細胞株，檢測其細胞表面的干細胞標記。然后使用多種培養(yǎng)基對兩種不同組織來源的間充質干細胞進行定向誘導，使其向視網(wǎng)膜感光細胞分化，在不同時間點收獲細胞，檢測視網(wǎng)膜感光細胞特異性基因和蛋白表達。比較這兩種細胞向視網(wǎng)膜感光細胞定向分化的能力。繼而通過組織塊培養(yǎng)法和酶消化法從人眼眶脂肪組織中分離脂肪組織來源干細胞，，并通過免疫熒光染色檢測其干細胞標記物。對通過不同方法獲得的脂肪組織來源干細胞進行培養(yǎng)，誘導其向視網(wǎng)膜感光細胞分化，在不同時間點收獲細胞，檢測細胞中視網(wǎng)膜感光細胞特異性基因和蛋白表達，比較兩種細胞的分化能力。 【結果】通過酶消化法獲得的人眼眶脂肪組織來源干細胞株和牙周韌帶來源干細胞株接受定向誘導分化后，前者表現(xiàn)出類似視網(wǎng)膜感光細胞外段樣結構；后者的細胞形態(tài)無顯著改變。牙周韌帶來源干細胞表達Pax6基因早于脂肪組織來源干細胞，但是表達Rhodopsin基因及視紫紅質蛋白晚于脂肪組織來源干細胞。本研究成功地通過組織塊培養(yǎng)法從人眼眶脂肪組織中原代培養(yǎng)出脂肪組織來源干細胞。通過組織塊培養(yǎng)法和酶消化法獲取的人眼眶脂肪組織來源干細胞接受誘導分化后，均表現(xiàn)出類似視桿細胞外段樣結構。通過組織塊培養(yǎng)法獲得的細胞較早表達Pax6基因、Rhodopsin基因及視紫紅質蛋白。 【結論】眼眶脂肪組織來源干細胞與牙周韌帶來源干細胞具有定向分化至類似視網(wǎng)膜感光細胞樣細胞的潛能，前者的分化能力強于后者。通過組織塊培養(yǎng)法可以從人眼眶脂肪組織中原代培養(yǎng)出脂肪組織來源干細胞，該細胞具有定向分化至類似視網(wǎng)膜感光細胞樣細胞的潛能，并且分化能力強于通過酶消化法分離獲取的細胞。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R774.1

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1 王宏;田海濱;陳娟;沙紅英;陳建泉;成國祥;;Knockout血清替代品可提高C57BL/6J小鼠胚胎干細胞建系效率[J];細胞與分子免疫學雜志;2007年03期

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