CAJ | 학술논문

背景：如何提高胚胎干细胞诱导效率、促进胚胎干细胞源造血干细胞体外增殖成为目前急需解决的课题。 　　目的：以外源性Wnt3a作为诱导剂，激活培养中的小鼠胚胎干细胞Wnt/β-catenin信号通路，观察该通路的激活是否促进胚胎干细胞向造血祖细胞的定向分化。 　　方法：用外源性wnt3a(100μg/L)持续作用ES-E14TG2a小鼠胚胎干细胞21 d，通过细胞免疫荧光及蛋白免疫印迹检测细胞内β-catenin蛋白含量，QRT-PCR检测Wnt下游靶标基因的表达量来确定经典Wnt/β-catenin信号通路是否被激活，然后采用单层贴壁培养法诱导其向造血干细胞分化，流式细胞仪检测造血发育相关表面标志CD34+/Sca-1+，同时以QRT-PCR法检测造血相关基因的表达情况。 　　结果与结论：ES-E14TG2a小鼠胚胎干细胞经wnt3a(100μg/L)连续培养21 d后发现β-catenin蛋白在细胞内积累；Wnt信号通路的下游靶标基因Pitx2、Frizzled、Sox17、Oct4的表达量均出现不同程度的增加，可见经典 Wnt/β-catenin 信号通路有被激活；单层贴壁培养法诱导其向造血干细胞分化的过程中检测到CD34+/Sca-1+细胞含量在14 d时占总细胞量高达20.2%，而对照组的仅占11.9%。造血相关基因骨形态发生蛋白4、FLK2及CD34的表达量均增加，而Smad5的表达则明显受到抑制。说明Wnt3a持续作用可激活Wnt/β-catenin信号通路，并促进ES-E14TG2a小鼠胚胎干细胞向造血干细胞的定向分化。
배경：여하제고배태간세포유도효솔、촉진배태간세포원조혈간세포체외증식성위목전급수해결적과제。 　　목적：이외원성Wnt3a작위유도제，격활배양중적소서배태간세포Wnt/β-catenin신호통로，관찰해통로적격활시부촉진배태간세포향조혈조세포적정향분화。 　　방법：용외원성wnt3a(100μg/L)지속작용ES-E14TG2a소서배태간세포21 d，통과세포면역형광급단백면역인적검측세포내β-catenin단백함량，QRT-PCR검측Wnt하유파표기인적표체량래학정경전Wnt/β-catenin신호통로시부피격활，연후채용단층첩벽배양법유도기향조혈간세포분화，류식세포의검측조혈발육상관표면표지CD34+/Sca-1+，동시이QRT-PCR법검측조혈상관기인적표체정황。 　　결과여결론：ES-E14TG2a소서배태간세포경wnt3a(100μg/L)련속배양21 d후발현β-catenin단백재세포내적루；Wnt신호통로적하유파표기인Pitx2、Frizzled、Sox17、Oct4적표체량균출현불동정도적증가，가견경전 Wnt/β-catenin 신호통로유피격활；단층첩벽배양법유도기향조혈간세포분화적과정중검측도CD34+/Sca-1+세포함량재14 d시점총세포량고체20.2%，이대조조적부점11.9%。조혈상관기인골형태발생단백4、FLK2급CD34적표체량균증가，이Smad5적표체칙명현수도억제。설명Wnt3a지속작용가격활Wnt/β-catenin신호통로，병촉진ES-E14TG2a소서배태간세포향조혈간세포적정향분화。
BACKGROUND:A variety of embryonic stem cells induction and differentiation systems have been established so far, while the research that promotes embryonic stem cells to differentiate into hematopoietic stem cells is stil at an initial stage, and the induction efficiency needs to be improved. OBJECTIVE:To active the Wnt/β-catenin signal pathway in mouse embryonic stem cells with exogenous win3a as an inducer, and then to observe whether the activation of this pathway wil promote the directional differentiation of embryonic stem cells into hematopoietic progenitor cells. METHODS:The ES-E14TG2a mouse stem cells were cultured with the exogenous wnt3a (100 μg/L) for 21 days, the content ofβ-catenin was tested by cellimmunofluorescence and western blot, and expression of Wnt downstream target gene was detected by quantitative reverse transcription PCR to determine the activation of Wnt/β-catenin signal pathway. Single-layer adherent culture method was used to induce the directional differentiate of above-mentioned cells into hematopoietic stem cells, and detection of hematopoietic development associated surface marker CD34+/Sca-1+was achieved by flow cytometry;meanwhile, the expression of hematopoietic associated gene was measured by quantitative reverse transcription PCR. RESULTS AND CONCLUSION:We found thatβ-catenin accumulated in ES-E14TG2a mouse stem cells after cultured with wnt3a (100 μg/L) for 21 days;the expressions of Wnt downstream target genes such as Pitx2, Frizzled, Sox17 and Oct4 showed the different degrees in increase, meaning the activation of Wnt/β-catenin signal pathway. Furthermore, during the time that we used single-layer adherent culture method to induce hematopoietic stem cells, the CD34+/Sca-1+cells accounted for 20.2%of total cells at day 14, and control cells only accounted for 11.9%. Again, expression quantity of hematopoietic associated gene BMP4, FLK2 and CD34 increased while Smad5 was suppressed significantly. Our data suggest that sustaining action by wnt3a wil active Wnt/β-catenin signal pathway, and also promote the directional differentiation of ES-E14TG2a mouse stem cells into hematopoietic progenitor cells.