碱性成纤维细胞生长因子对胚胎干细胞来源暴式集落形成细胞形成的作用及其机制研究

发布时间：2018-01-01 18:09

本文关键词：碱性成纤维细胞生长因子对胚胎干细胞来源暴式集落形成细胞形成的作用及其机制研究　出处：《滨州医学院》2010年硕士论文　论文类型：学位论文

【摘要】：目的:在拟胚体培养阶段施加碱性成纤维细胞生长因子,验证碱性成纤维细胞生长因子对暴式集落形成细胞产生的调控作用及探讨其作用机制。方法:1.购买小鼠胚胎干细胞D3细胞系,取第3-5代小鼠原代胚胎成纤维细胞,加入新配制含丝裂霉素C的DMEM生长培养基孵育2.5 h,使饲养层细胞失去增殖能力;加入胰酶消化适度,离心后制成单细胞悬液,以10×10~4个/cm~2的密度种至用明胶包被的培养瓶中,放入孵箱内培养24 h之后使用。复苏胚胎干细胞D3细胞并将其接种于饲养层细胞之上。培养至一定时期后传代并及时冻存。 2.在拟胚体培养阶段按培养基成分不同分为4组:对照组(标准培养基+血管内皮生长因子+干细胞因子组)、5μg/L bFGF组(标准培养基+血管内皮生长因子+干细胞因子+5μg/L bFGF组)、10μg/L bFGF组(标准培养基+血管内皮生长因子+干细胞因子+10μg/L bFGF组)、15μg/L bFGF组(标准培养基+血管内皮生长因子+干细胞因子+15μg/L bFGF组)。各组于培养3、4、5、6d时分别计数克隆形成数,通过免疫荧光法及IMAGE-PRO PLUS图像分析系统检测集落形成细胞Flk-1的表达情况,以此来筛选其最佳作用浓度。3.通过免疫荧光法检测集落形成细胞相关表面标志(Flk-1、CD133、CD34)的表达情况。4.通过免疫荧光法检测相关通路的受体表达情况,通过RT-PCR法检测相关信号通路特异性靶基因(Axin2、Gli)的表达情况,从而来进一步探讨bFGF发挥作用的相关机制。结果:1.成功制备小鼠的胚胎成纤维细胞;2.扩增并冻存大量的胚胎干细胞,并证明扩增的胚胎干细胞具有全能性;3.培养体系中加入bFGF后,有大量克隆形成,克隆Flk-1呈阳性表达,不同的浓度梯度克隆形成数量不同,且Flk-1的表达强度不同,其中10μg/L bFGF组克隆形成数量最多且Flk-1的表达最强(P 0.01);3.在BL-CFC生成过程中,Flk-1逐渐减弱,而CD133,CD34的表达逐渐增强;4.在BL-CFC形成过程中,Wnt, Shh信号通路的表面受体表达逐渐增强,其Wnt通路的特异性靶基因(Axin2)的表达也逐渐增强。结论:1.成功制备小鼠的胚胎成纤维细胞,扩增并冻存了大量的胚胎干细胞; 2. bFGF促进了BL-CFC的形成,其最佳浓度为10μg/L; 3.形成的BL-CFC能够向造血干/祖细胞方向分化; 4.在BL-CFC形成过程中,Wnt, Shh信号通路的表面受体表达逐渐增强,Wnt通路的特异性靶基因(Axin2)的表达也逐渐增强,初步证明bFGF促进了BL-CFC的形成的作用机制与Wnt通路的激活相关。
[Abstract]:Objective : To investigate the effect of basic fibroblast growth factor ( bFGF ) on the formation of cells and explore its mechanism of action by applying basic fibroblast growth factor ( bFGF ) to the culture stage . Methods : 1 . Mouse embryonic stem cell D3 cell line was purchased , and the 3 - 5 mouse primary embryonic fibroblasts were cultured . The cells were cultured for 2.5 hours in DMEM medium supplemented with mitomycin C . 2 . The culture medium was divided into 4 groups according to the culture medium composition : control group ( standard medium + vascular endothelial growth factor + stem cell factor group ) , 5 渭g / L bFGF group ( standard medium + vascular endothelial growth factor + stem cell factor + 5 渭g / L bFGF group ) , 10 渭g / L bFGF group ( standard medium + vascular endothelial growth factor + stem cell factor + 10 渭g / L bFGF group ) , 15 渭g / L bFGF group ( standard medium + vascular endothelial growth factor + stem cell factor + 15 渭g / L bFGF group ) . The expression of Flk - 1 , CD133 , CD34 was detected by immunofluorescence and IMAGE - PRO PLUS image analysis system . The expression of Flk - 1 , CD133 , CD34 was detected by immunofluorescence assay . Results : 1 . The mouse embryonic fibroblasts were successfully prepared ; 2 . Large amount of embryonic stem cells were amplified and frozen , and the expression intensity of Flk - 1 was higher than that of Flk - 1 . The expression intensity of Flk - 1 was different , and the expression intensity of Flk - 1 was significantly increased in the formation of BL - CFC . Conclusion : 1 . Mouse embryonic fibroblasts were successfully prepared , and a large number of embryonic stem cells were amplified and frozen . 2 . bFGF promoted BL - CFC formation , and the optimal concentration was 10 渭g / L . 3 . BL - CFC can differentiate into hematopoietic stem / progenitor cells . 4 . During the formation of BL - CFC , the expression of Wnt signaling pathway and Wnt signaling pathway gradually increased , and the expression of Wnt signaling pathway was gradually enhanced . The mechanism of bFGF promoting the formation of BL - CFC was associated with the activation of Wnt pathway .

【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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