碱性成纤维细胞生长因子对胚胎干细胞来源集落形成细胞的作用*

背景：有研究表明，在卵黄囊造血、胎肝造血和在胚胎干细胞向造血干细胞分化过程中，碱性成纤维细胞生长因子可强烈表达。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集落形成细胞产生的调控作用。方法：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10×104个/cm2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。在拟胚体培养阶段按培养基成分不同分为2组：对照组(标准培养基+血管内皮生长因子+干细胞因子组)、实验组(标准培养基+血管内皮生长因子+干细胞因子+碱性成纤维细胞生长因子组)。各组于培养3，6d时分别计数克隆形成数，通过免疫荧光法检测Flk-1+细胞表达情况，并用IMAGE-PRO PLUS图像分析系统统计阳性细胞数及平均吸光度值。结果与结论：与对照组比较，在拟胚体培养阶段加入碱性成纤维细胞生长因子可显著增加集落形成细胞的数量(P < 0.01), Flk-1阳性细胞数及平均吸光度值也显著增加(P < 0.01)。证明碱性成纤维细胞生长因子能够有效地促进胚胎体的扩增以及成血管血液干细胞的产生与增殖。

The role of bFGF on the formation of BL-CFC derived from embryonic stem cells

BACKGROUND: Some studies show that basic fibroblast growth factor (bFGF) strongly expresses during the process of embryonic stem cells differentiation into hematopoietic stem cell, yolk sac blooding, and fetal liver hematopoiesis.OBJECTIVE: To study the regulation of bFGF on the blast-colony-forming cell (BL-CFC) by adding bFGF in the medium of embryoid body generation.METHODS: The third to fifth generations of the primary mouse embryonic fibroblasts were recovered, and then incubated with the DMEM medium containing mitomycin C for 2.5 hours in order to lose the proliferative capacity. Then cells were suspended into single cell by trypsinization and inoculated in the gelatin-coated bottle at the density of 10×104/cm2. After culturing for 24 hours, mouse embryonic stem cells (mESC) of D3 were recovered and placed on the feeder layer cells. According to the composition of medium in embryoid body generation, mESCs were divided into two groups: group A: standard medium + VEGF + SCF; group B: standard medium + VEGF + SCF + bFGF. Each group was cultured for 3 days and 6 days respectively, and the cloning number of BL-CFC was quantified, as well as Flk-1+ expression was observed by immunofluorescence staining. Positive number and average absorbance were analyzed using IMAGE-PRO PLUS imaging analysis system. RESULTS AND CONCLUSION: Adding bFGF in the course of embryoid body growth could significantly increase the number of BL-CFC (P < 0.01), and the positive results of Flk-1 and the average absorbance were also increased significantly (P < 0.01). bFGF effectively promoted embryoid body amplification and proliferation of BL-CFC.