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人尿源干细胞用于组织工程构建的可行性研究报告DOC

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目的通过对尿源干细胞与尿源诱导多潜能干细胞进行多向诱导分化比较, 并利用诱导后的平滑肌样细胞进行复层细胞膜片构建,探讨两种干细胞用于组织 工程构建的可行性。 方法将尿源干细胞分别向平滑肌、内皮、神经细胞方向诱导分化,通过实 时定量-聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR )、细胞免疫荧光染色鉴定多向诱导后的特异性mRNA及蛋白表达情况。 将诱导后的平滑肌样细胞转移入温度敏感型培养皿(NuncUpcell)中,通过降低 温度获得单层细胞膜片,并将单层细胞膜片进行多次叠加,来获得复层平滑肌细 胞膜片。同时对前期实验获得的尿源诱导多潜能干细胞进行多向诱导分化,并进 行qRT-PCR、细胞免疫荧光染色鉴定多向诱导后的特异性mRNA及蛋白表达情 况。对两种细胞的诱导效果进行比较,探讨其用于组织工程构建的可行性。 结果⑴尿源干细胞向平滑肌、内皮细胞方向诱导的14天过程中,0ct4、 Sox2、Nanog等多潜能基因的相对表达量下降,而平滑肌、内皮细胞特异性标志 物的表达明显增多。⑵尿源干细胞在向神经细胞方向诱导14天后,除Nesting 因表达增高,其它基因表达及细胞形态未见明显变化。⑶诱导后的平滑肌样细胞 结合温度敏感型培养皿可获得复层平滑肌细胞膜片。⑷尿源诱导多潜能干细胞在 平滑肌、内皮细胞方向诱导效果均优于尿源干细胞,而神经细胞方向诱导后可形 成神经祖细胞特有的“玫瑰花环”样结构,在神经细胞特异性基因的表达明显增 高的同时,Sox2基因表达也维持在较高水平。 结论本研宄发现尿源干细胞向平滑肌和内皮细胞方向诱导效果较好,而神 经细胞方向诱导效果欠佳。结合温度敏感型培养皿可获得诱导后平滑肌复层细胞 膜片。尿源干细胞与诱导多潜能干细胞分别向平滑肌、内皮和神经细胞方向进行 诱导分化比较后发现,诱导多潜能干细胞具有多向分化潜能,相比尿源干细胞分 上海交通大学硕士学位报告 化能力更好,在组织工程方面具有更好的应用前景。 [关键词]组织工程;尿源干细胞;诱导多潜能干细胞;诱导分化;复层细胞膜 片 上海交通大学硕士学位报告 The Feasibi I ity Study of Tissue Engineer ing with Human Urine-derived Stem Cells Abstract Objectives To compare the capacity of induced differentiation between urine-derived stem cells and urine-derived induced-pluripotent stem cells, the feasibility of two sorts of cells applying in the tissue engineering was discussed. Smooth muscle cells derived from urine stem cells were used to get the multi-layer cell sheet with the use of Nunc Upcell dishes. Methods Urine-derived stem cells was induced towards three different directions of smooth muscle cells, endothelial cells and nerve cells, and was tested the expression of specific mRNA and proteins with the use of qRT-PCR and immunofluorescent staining. Transferring the induced smooth muscle cells into Nunc Upcell dishes, we can acquired the single-layer cell sheet. And then multi-layer cell sheet was gained by overlaying single-layer cell sheet several times. Urine-derived induced-pluripotent stem cells was also induced into three different directions, and was tested the expression of specific mRNA and proteins with the use of qRT-PCR and immunofluorescent staining. By comparing the induction effects, the feasibility of two sorts of cells applying in the tissue engineering was discussed. Results (1) During the process of smooth muscle cells and endothelial cells induction of urine-derived stem cells, the expression of pluripotent gene was declining relatively, while the expression of specific markers of smooth muscle cells and endothelial cells was increasing clearly. (2)During the process of nerve cells induction of urine-derived stem cells, the expression of gene was not fluctuated fiercely except in 上海交通大学硕士学位报告 Nestin. (3)The multi-layer cell sheet was gained by using USC-SMC combining with Nunc Upcell dishes. (4)The induced potency of iPSCs is better than USCs in different directions. Neural progenitor cell specific structure rose set can be seen in the iPSC-NC. The expression of neural specific gene was increasing during the neural induced process, meanwhile the expression of Sox2 gene maintained in a high level. Conclusions This research identified the fact that the induction effect of USCs to SMC and EC is better than NC. The multi-layer cell sheet was gained by using USC-SMC combining with Nunc Upcell dishes. By comparing the induction effects between USCs and iPSCs, this research identified the fact that iPSCs had a better induced potency and had a better prospect in tissue engineering. [Key Words] Tissue engineering; Urine-derived stem cells; Induced-pluripotent stem cells; Induced differentiation; Multi-layer cell sheet IV 上海交通大学硕士学位报告 目录 英文縮略语和单位说明 VI 谢仓 1 第一部分:尿源干细胞诱导分化和诱导后平滑肌复层细胞膜片构建 3 1. 1实验材料 3 1.2实验方法 6 1.2.1尿源干细胞的培养、扩增 6 1.2.2尿源干细胞多向诱导分化 7 1.2.3诱导前后细胞鉴定 8 1.2.4诱导后平滑肌复层细胞I旲片构建 11 1.3实验结果 11 1. 3. 1尿源干细胞向平滑肌细胞方向诱导分化结果 11 1. 3. 2尿源干细胞向内皮细胞方向诱导分化结果 13 1. 3. 3尿源干细胞向神经细胞方向诱导分化结果 13 1.3.4构建复层平滑肌细胞膜片 17 1.4讨论 17 第二部分:尿源干细胞与诱导多潜能干细胞多向诱导分化比较 20 2. 1实验材料 20 2.2实验方法 23 2.2.1 iPS细胞的培养 23 2. 2. 2 i PS细胞多向诱导分化 23 2. 2. 3诱导前后细胞鉴定 24 2.3实验结果 27 2. 3. 1 i PS细胞与尿源干细胞向平滑肌细胞方向诱导结果比较 27 2. 3. 2 i PS细胞与尿源干细胞向内皮细胞方向诱导结果比较 29 2. 3. 3 i PS细胞与尿源干细胞向神经细胞方向诱导结果比较 31 2. 4讨论 33