CAJ | 학술논문

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/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件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.
배경:유연구표명,재란황낭조혈、태간조혈화재배태간세포향조혈간세포분화과정중,감성성섬유세포생장인자가강렬표체.목적:재의배체배양계단시가감성성섬유세포생장인자,험증감성성섬유세포생장인자대집락형성세포산생적조공작용.방법:구매소서배태간세포D3세포계,취제3～5대소서원대배태성섬유세포,가입신배제함사렬매소C적DMEM생장배양기부육2.5 h,사사양층세포실거증식능력;가입이매소화괄도,리심후제성단세포현액,이10×104/cm2적밀도충지용명효포피적배양병중,방입부상내배양24 h지후사용.복소배태간세포D3세포병장기접충우사양층세포지상.재의배체배양계단안배양기성분불동분위2조:대조조(표준배양기+혈관내피생장인자+간세포인자조)、실험조(표준배양기+혈관내피생장인자+간세포인자+감성성섬유세포생장인자조).각조우배양3,6d시분별계수극륭형성수,통과면역형광법검측Flk-1+세포표체정황,병용IMAGE-PRO PLUS도상분석계통통계양건세포수급평균흡광도치.결과여결론:여대조조비교,재의배체배양계단가입감성성섬유세포생장인자가현저증가집락형성세포적수량(P＜0.01),Flk-1양성세포수급평균흡광도치야현저증가(P＜0.01).증명감성성섬유세포생장인자능구유효지촉진배태체적확증이급성혈관혈액간세포적산생여증식.
BACKGROUND:Some studies show that basic fibroblast growth factor(bFGF)strongly expresses dudng 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 pdmaw mouse embryonic fibroblasts were recovered,and then incubated with the DMEM medium containing mitomvcin 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.