【摘要】：本研究旨在探索基于兔骨髓間充質(zhì)干細(xì)胞在體外構(gòu)建預(yù)血管化的細(xì)胞膜片,為構(gòu)建三維預(yù)血管化工程化組織提供新的思路和方法。方法：(1)體外對(duì)兔骨髓間充質(zhì)干細(xì)胞(rBMSCs)進(jìn)行分離、培養(yǎng)：全骨髓貼壁法體外分離兔的股骨骨髓間充質(zhì)干細(xì)胞,觀察其增殖、生長(zhǎng)特性。(2)誘導(dǎo)rBMSCs向血管內(nèi)皮樣細(xì)胞ECs分化：將分離獲得的rBMSCs在含血管內(nèi)皮細(xì)胞生長(zhǎng)因子(VEGF,10μg/L濃度)和堿性成纖維細(xì)胞生長(zhǎng)因子(b-FGF,10μg/L濃度)的培養(yǎng)基中培養(yǎng),在特定時(shí)間點(diǎn)(3天、7天、14天),對(duì)細(xì)胞形態(tài)改變給予觀察,并進(jìn)行von Willebrand factor (vWF). CD31免疫熒光染色,對(duì)誘導(dǎo)分化得到的ECs進(jìn)行鑒定。(3)預(yù)血管化細(xì)胞膜片的構(gòu)建：將分離獲得的rBMSCs以9×104cells/cm2的密度在體外連續(xù)體外培養(yǎng)2周形成未分化細(xì)胞膜片,并在此細(xì)胞膜片上將誘導(dǎo)分化的ECs以5×104cells/cm2的密度接種并培養(yǎng)3天,7天,和14天。鏡下進(jìn)行細(xì)胞形貌及血管網(wǎng)絡(luò)觀察、免疫學(xué)及組織學(xué)檢測(cè)。結(jié)果：(1)全骨髓貼壁法分離、培養(yǎng)的rBMSCs貼壁生長(zhǎng),形狀大多呈長(zhǎng)梭形、多角形,克隆樣生長(zhǎng)；MTT檢測(cè)呈“S”型生長(zhǎng)。(2)分離純化的rBMSC在血管內(nèi)皮細(xì)胞培養(yǎng)基誘導(dǎo)下,細(xì)胞呈“鵝卵樣、鋪路石樣”內(nèi)皮細(xì)胞樣結(jié)構(gòu),細(xì)胞表面抗原CD31、假性血友病因子vWF免疫熒光染色陽(yáng)性。(3)rBMSCs誘導(dǎo)分化獲得的ECs按照一定密度接種至未分化的rBMSCs膜片上,3天后細(xì)胞發(fā)生了重排,可見空泡的形成；7天后,ECs相互連接,形成網(wǎng)狀結(jié)構(gòu)；14天后,網(wǎng)狀結(jié)構(gòu)更加明顯。組織學(xué)的檢測(cè)可見微管管腔樣結(jié)構(gòu)的形成。結(jié)論：(1)全骨髓貼壁法可獲取骨髓間充質(zhì)干細(xì)胞,取材方便簡(jiǎn)單,rBMSCs增殖能力較強(qiáng)。(2)rBMSCs在一定濃度的VEGF、b-FGF誘導(dǎo)作用下可向內(nèi)皮細(xì)胞樣ECs分化,得到具有內(nèi)皮樣結(jié)構(gòu)的細(xì)胞。(3)將由rBMSCs誘導(dǎo)分化形成的內(nèi)皮樣細(xì)胞接種到未分化膜片上,可在體外形成具有血管網(wǎng)絡(luò)結(jié)構(gòu)的預(yù)血管化膜片,為工程化血管組織的構(gòu)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R783

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