基于兔骨髓间充质干细胞构建预血管化细胞膜片的体外研究
发布时间：2018-10-13 17:21
【摘要】：本研究旨在探索基于兔骨髓间充质干细胞在体外构建预血管化的细胞膜片,为构建三维预血管化工程化组织提供新的思路和方法。方法：(1)体外对兔骨髓间充质干细胞(rBMSCs)进行分离、培养：全骨髓贴壁法体外分离兔的股骨骨髓间充质干细胞,观察其增殖、生长特性。(2)诱导rBMSCs向血管内皮样细胞ECs分化：将分离获得的rBMSCs在含血管内皮细胞生长因子(VEGF,10μg/L浓度)和碱性成纤维细胞生长因子(b-FGF,10μg/L浓度)的培养基中培养,在特定时间点(3天、7天、14天),对细胞形态改变给予观察,并进行von Willebrand factor (vWF). CD31免疫荧光染色,对诱导分化得到的ECs进行鉴定。(3)预血管化细胞膜片的构建：将分离获得的rBMSCs以9×104cells/cm2的密度在体外连续体外培养2周形成未分化细胞膜片,并在此细胞膜片上将诱导分化的ECs以5×104cells/cm2的密度接种并培养3天,7天,和14天。镜下进行细胞形貌及血管网络观察、免疫学及组织学检测。结果：(1)全骨髓贴壁法分离、培养的rBMSCs贴壁生长,形状大多呈长梭形、多角形,克隆样生长；MTT检测呈“S”型生长。(2)分离纯化的rBMSC在血管内皮细胞培养基诱导下,细胞呈“鹅卵样、铺路石样”内皮细胞样结构,细胞表面抗原CD31、假性血友病因子vWF免疫荧光染色阳性。(3)rBMSCs诱导分化获得的ECs按照一定密度接种至未分化的rBMSCs膜片上,3天后细胞发生了重排,可见空泡的形成；7天后,ECs相互连接,形成网状结构；14天后,网状结构更加明显。组织学的检测可见微管管腔样结构的形成。结论：(1)全骨髓贴壁法可获取骨髓间充质干细胞,取材方便简单,rBMSCs增殖能力较强。(2)rBMSCs在一定浓度的VEGF、b-FGF诱导作用下可向内皮细胞样ECs分化,得到具有内皮样结构的细胞。(3)将由rBMSCs诱导分化形成的内皮样细胞接种到未分化膜片上,可在体外形成具有血管网络结构的预血管化膜片,为工程化血管组织的构建提供了新的方法。
[Abstract]:The purpose of this study was to explore the construction of prevascularized membrane slices based on rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro, and to provide a new idea and method for the construction of 3D prevascularized tissue. Methods: (1) (rBMSCs) of rabbit bone marrow mesenchymal stem cells were isolated and cultured in vitro. (2) differentiation of rBMSCs into vascular endothelioid cells (ECs): the isolated rBMSCs was cultured in a medium containing vascular endothelial growth factor (VEGF,10 渭 g / L) and basic fibroblast growth factor (b-FGF10 渭 g / L). Observe cell morphological changes at specific time points (3 days, 7 days, 14 days) and perform von Willebrand factor (vWF). CD31 immunofluorescence staining was used to identify the ECs induced by differentiation. (3) Construction of prevascularized cell membrane: the isolated rBMSCs was cultured in vitro for 2 weeks with the density of 9 脳 104cells/cm2 to form undifferentiated cell membrane. The differentiated ECs was inoculated at the density of 5 脳 104cells/cm2 and cultured for 3 days, 7 days and 14 days. Cell morphology and vascular network were observed and immunological and histological examination were performed under microscope. Results: (1) the whole bone marrow adherent method was used to isolate the cultured rBMSCs, most of which were spindle-shaped, polygonal and clone like growth, and MTT assay showed "S" type growth. (2) the purified rBMSC was induced by vascular endothelial cell medium. The cells were "goose egg like, paving stone like" endothelial cell like structure, The cell surface antigen CD31, pseudophilic factor vWF immunofluorescence staining was positive. (3) ECs induced by rBMSCs was inoculated into undifferentiated rBMSCs membrane according to a certain density. After 3 days, the cells were rearranged and vacuoles were formed. After 7 days, ECs was connected to each other. After 14 days, the reticular structure was more obvious. Histologic examination showed the formation of microtubule lumen-like structure. Conclusion: (1) Bone marrow mesenchymal stem cells (BMSCs) can be obtained by whole bone marrow adherent method, which is convenient and simple to obtain. (2) rBMSCs can differentiate into endothelial cell-like ECs induced by certain concentration of VEGF,b-FGF. Endothelial cells with endothelial structure were obtained. (3) Endothelium-like cells induced by rBMSCs were inoculated into undifferentiated membranes to form prevascularized membranes with vascular network structure in vitro. It provides a new method for the construction of engineered vascular tissue.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783
本文编号：2269371
[Abstract]:The purpose of this study was to explore the construction of prevascularized membrane slices based on rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro, and to provide a new idea and method for the construction of 3D prevascularized tissue. Methods: (1) (rBMSCs) of rabbit bone marrow mesenchymal stem cells were isolated and cultured in vitro. (2) differentiation of rBMSCs into vascular endothelioid cells (ECs): the isolated rBMSCs was cultured in a medium containing vascular endothelial growth factor (VEGF,10 渭 g / L) and basic fibroblast growth factor (b-FGF10 渭 g / L). Observe cell morphological changes at specific time points (3 days, 7 days, 14 days) and perform von Willebrand factor (vWF). CD31 immunofluorescence staining was used to identify the ECs induced by differentiation. (3) Construction of prevascularized cell membrane: the isolated rBMSCs was cultured in vitro for 2 weeks with the density of 9 脳 104cells/cm2 to form undifferentiated cell membrane. The differentiated ECs was inoculated at the density of 5 脳 104cells/cm2 and cultured for 3 days, 7 days and 14 days. Cell morphology and vascular network were observed and immunological and histological examination were performed under microscope. Results: (1) the whole bone marrow adherent method was used to isolate the cultured rBMSCs, most of which were spindle-shaped, polygonal and clone like growth, and MTT assay showed "S" type growth. (2) the purified rBMSC was induced by vascular endothelial cell medium. The cells were "goose egg like, paving stone like" endothelial cell like structure, The cell surface antigen CD31, pseudophilic factor vWF immunofluorescence staining was positive. (3) ECs induced by rBMSCs was inoculated into undifferentiated rBMSCs membrane according to a certain density. After 3 days, the cells were rearranged and vacuoles were formed. After 7 days, ECs was connected to each other. After 14 days, the reticular structure was more obvious. Histologic examination showed the formation of microtubule lumen-like structure. Conclusion: (1) Bone marrow mesenchymal stem cells (BMSCs) can be obtained by whole bone marrow adherent method, which is convenient and simple to obtain. (2) rBMSCs can differentiate into endothelial cell-like ECs induced by certain concentration of VEGF,b-FGF. Endothelial cells with endothelial structure were obtained. (3) Endothelium-like cells induced by rBMSCs were inoculated into undifferentiated membranes to form prevascularized membranes with vascular network structure in vitro. It provides a new method for the construction of engineered vascular tissue.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783
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