本文選題：表皮干細(xì)胞 + 分化�。� 參考：《福建醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:探討表皮干細(xì)胞向神經(jīng)細(xì)胞分化的影響因素。 方法: 1.分離3-4天的SD大鼠表皮基底層細(xì)胞,利用10分鐘貼壁法分離出表皮干細(xì)胞。2,用免疫組織化學(xué)法測定K15、β1整合素、CD34和巢蛋白(nestin)來鑒定培養(yǎng)出的表皮干細(xì)胞。3.加入不同的培養(yǎng)基:K-SFM、NEUROBASALTM-A和DMEM培養(yǎng)分離出的表皮干細(xì)胞,并觀察不同培養(yǎng)基組細(xì)胞形態(tài)的變化。4.通過調(diào)整不同濃度的bFGF,觀察表皮干細(xì)胞的形態(tài)變化。5.用K-SFM為培養(yǎng)基培養(yǎng)表皮干細(xì)胞,以表皮干細(xì)胞的細(xì)胞密度分組,分為0.5×10~7/ml組、0.3×10~7/ml組、0.1×10~7/ml組、0.5×105/ml,每組均加入20ng/ml的bFGF,觀察其細(xì)胞形態(tài)發(fā)生以及細(xì)胞標(biāo)記物的改變。 結(jié)果: 1.利用十分鐘貼壁法成功分離出表皮干細(xì)胞。分離出來表皮干細(xì)胞細(xì)胞密度大,細(xì)胞呈圓形或多角形,細(xì)胞形態(tài)清晰,胞質(zhì)均勻,可見有核分裂細(xì)胞,核質(zhì)比例大,其特征性標(biāo)記分子K15及β整合素表達(dá)陽性。 2.表皮干細(xì)胞在NEUROBASALTM-A和DMEM培養(yǎng)基中均出現(xiàn)細(xì)胞體積增大,細(xì)胞膜邊界不清,細(xì)胞核溶解、核碎裂、細(xì)胞死亡。 3.表皮干細(xì)胞在K-SFM培養(yǎng)基中,加入bFGF的濃度為0ng/ ml、5ng/ml、10ng/ml、15ng/ml時(shí),細(xì)胞形態(tài)沒有明顯改變,呈圓形或多角形,細(xì)胞形態(tài)清晰,胞質(zhì)均勻,可見有核分裂細(xì)胞,核質(zhì)比例大;當(dāng)加入的bFGF濃度為20ng/ml時(shí), 24h即可見到細(xì)胞開始伸展,呈多角形、圓形或短棒樣,分布比較均勻,第3-4天可看到部分細(xì)胞呈鋪路石樣,細(xì)胞體積增大,核質(zhì)比例變小,可見有分裂核細(xì)胞,極少部分細(xì)胞呈現(xiàn)雙極,多極細(xì)胞改變,約一周后,雙極、多極細(xì)胞消失,部分鋪路石樣細(xì)胞形成克隆,細(xì)胞90%融合。 4.在0.3×10~7/ml組和0.1×10~7/ml組, 24h-48h可見到細(xì)胞開始伸展,呈多角形、圓形或短棒樣,分布比較均勻,第5-6天可看到部分細(xì)胞呈鋪路石樣改變,細(xì)胞體積大,核質(zhì)比例變小,可見有分裂核細(xì)胞,部分細(xì)胞呈出現(xiàn)雙極,多極細(xì)胞改變,約一周后,雙極、多極細(xì)胞增多。免疫組織化學(xué)nestin、NSE染色陽性,β1整合素、CD34染色陰性; 0.5×10~6/ml組,約3-4天后可見到散在的伸展,呈多角形、圓形或短棒樣的細(xì)胞,大部分細(xì)胞一直未伸展; 0.5×10~7/ml組, 24h即可見到細(xì)胞開始伸展,呈多角形、圓形或短棒樣,分布比較均勻,第3-4天可看到部分細(xì)胞呈鋪路石樣,細(xì)胞體積增大,核質(zhì)比例變小,可見有分裂核細(xì)胞,極少部分細(xì)胞呈現(xiàn)雙極,多極細(xì)胞改變,約一周后,雙極、多極細(xì)胞消失,部分鋪路石樣細(xì)胞形成克隆,細(xì)胞90%融合。 結(jié)論 1.10分鐘快速貼壁法可以培養(yǎng)出表皮干細(xì)胞,表皮干細(xì)胞的特征性標(biāo)記分子K15及β1整合素表達(dá)陽性,CD34、nestin表達(dá)陰性。 3.培養(yǎng)表皮干細(xì)胞細(xì)胞的最佳培養(yǎng)基為K-SFM。 4.通過bFGF可誘導(dǎo)表皮干細(xì)胞向神經(jīng)系分化,bFGF對(duì)表皮干細(xì)胞的誘導(dǎo)具有細(xì)胞密度依賴性。
[Abstract]:Objective: to investigate the factors influencing the differentiation of epidermal stem cells into neural cells. Methods: 1. Epidermal stem cells (ESCs) were isolated from SD rat epidermal basal layer cells for 3-4 days. The epidermal stem cells (ESCs) were isolated by 10-minute adherent method. K15, 尾 1-integrin CD34 and nestin were determined by immunohistochemistry to identify the cultured epidermal stem cells. The epidermal stem cells (ESCs) were cultured in different culture medium: K- K SFMN NEUROBASALTM-A and DMEM, and the morphological changes of ESCs in different culture medium groups were observed. 4. Morphological changes of epidermal stem cells were observed by adjusting different concentrations of bFGF.5. Epidermal stem cells were cultured on K-SFM medium. The epidermal stem cells were divided into 0.5 脳 10~7/ml group (0. 3 脳 10~7/ml group) and 0. 1 脳 10 ~ 5 脳 10~7/ml group (0. 5 脳 10 ~ 5 / ml). The cell morphogenesis and the changes of cell markers were observed in each group. Results: 1. Epidermal stem cells were successfully isolated by ten minutes adherence method. The epidermal stem cells showed high density, round or polygonal shape, clear morphology, homogeneous cytoplasm, mitotic cells, large nuclear / cytoplasm ratio and positive expression of K15 and 尾 integrin. 2. The epidermal stem cells in both NEUROBASALTM-A and DMEM medium had increased cell volume, unclear cell membrane boundaries, nuclear dissolution, nuclear fragmentation and cell death. 3. When epidermal stem cells were added to K-SFM medium at the concentration of 0ng/ 5ng / ml 10 ng / ml 10 ng / ml 15ng / ml, the cell morphology was round or polygonal, the cell morphology was clear, the cytoplasm was uniform, mitotic cells were found, and the ratio of nucleus to cytoplasm was large. When the concentration of bFGF added was 20ng/ml, the cells began to stretch out at 24 hours. The cells were polygonal, round or short stick, and the distribution was even. On the 3-4 days, some cells showed paving stone, the cell volume increased, and the ratio of nucleus and cytoplasm became smaller. There were mitotic nuclear cells, very few cells showed bipolar, multipolar cell changes, about a week later, bipolar, multipolar cells disappeared, some paving stone like cells formed clone, 90% of the cells fused. 4. In 0. 3 脳 10~7/ml and 0. 1 脳 10~7/ml groups, 24h-48h showed that the cells began to stretch, were polygonal, round or short rod, and distributed evenly. On the 5th to 6th day, some of the cells showed paving stone changes, the cells were large, the ratio of nucleus and cytoplasm became smaller, and mitotic cells could be seen. Some cells showed bipolar, multipolar cell changes, about a week later, bipolar, multipolar cells increased. In the 0.5 脳 10~6/ml group, scattered, polygonal, round or short stick like cells could be seen after 3 to 4 days. In 0. 5 脳 10~7/ml group, the cells began to extend at 24 hours, which were polygonal, round or short stick, and distributed evenly. On the 3-4 days, some cells were found to be paving stone, the cell volume was increasing, and the ratio of nucleus and cytoplasm was smaller. There were mitotic nuclear cells, very few cells showed bipolar, multipolar cell changes, about a week later, bipolar, multipolar cells disappeared, some paving stone like cells formed clone, 90% of the cells fused. Conclusion 1. Epidermal stem cells could be cultured by rapid adherence for 10 minutes. The characteristic marker molecules K15 and 尾 1 integrin were positive and CD34 nestin expression was negative in epidermal stem cells. 3. The best culture medium for epidermal stem cells was K-SFM. 4. BFGF can induce the differentiation of epidermal stem cells into neural system.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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