Yamaguchi Y, Gunji Y, Nakamura M, Hayakawa K, Maeda M, Osawa H, Nagayoshi K, Kasahara T, Suda T
Department of Medical Biology and Parasitology, Jichi Medical School, Tochigi-ken, Japan.
Exp Hematol. 1993 Aug;21(9):1233-8.
To investigate how c-kit and c-kit ligand play a role in the function of hematopoietic stem cells, we determined the expression of c-kit in sorted human hematopoietic stem cells, CD34+CD33- cells and CD34+CD33+ cells. CD34+ cells constituted approximately 1% of the population of gated bone marrow cells and contained colony-forming cells. Two-color analysis by a fluorescence-activated cell sorter (FACS) revealed that about one-third to one-half of the total CD34+ cell population were positive for the CD33 antigen. To analyze the relative accumulation of c-kit mRNA in sorted cells, we used the reverse transcription-polymerase chain reaction (RT-PCR) method, followed by Southern blot analysis. There was a linear relationship between the amount of input RNA and products amplified in the range of 10(3) to 10(5) cells. Using this procedure, we carried out an analysis of c-kit mRNA expression in CD34+CD33-, CD34+CD33+, CD34-CD33+, and CD34-CD33- cells. Enhanced expression for c-kit mRNA was observed solely in CD34+CD33- cells. In contrast, flow cytometry shows that c-kit protein was expressed most abundantly in CD34+CD33+ cells. Colony-forming cells were generated on a human stromal cell layer for 5 weeks initiated with CD34+CD33- cells but not with CD34+CD33+ cells. During co-culture with stromal cells, CD34+CD33- cells differentiated into CD34+CD33+ cells. From these findings, it is concluded that CD34+CD33+ cells are direct progenies of CD34+CD33- cells. In this differentiation pathway, the expression of c-kit mRNA decreased and the c-kit protein increased.
为了研究c-kit和c-kit配体在造血干细胞功能中如何发挥作用，我们测定了分选的人造血干细胞、CD34+CD33-细胞和CD34+CD33+细胞中c-kit的表达。CD34+细胞约占门控骨髓细胞群体的1%，并含有集落形成细胞。通过荧光激活细胞分选仪（FACS）进行的双色分析显示，总CD34+细胞群体中约三分之一至二分之一的细胞CD33抗原呈阳性。为了分析分选细胞中c-kit mRNA的相对积累情况，我们采用逆转录-聚合酶链反应（RT-PCR）方法，随后进行Southern印迹分析。在10(3)至10(5)个细胞范围内，输入RNA的量与扩增产物之间存在线性关系。使用该程序，我们对CD34+CD33-、CD34+CD33+、CD34-CD33+和CD34-CD33-细胞中的c-kit mRNA表达进行了分析。仅在CD34+CD33-细胞中观察到c-kit mRNA表达增强。相比之下，流式细胞术显示c-kit蛋白在CD34+CD33+细胞中表达最为丰富。用CD34+CD33-细胞起始，在人基质细胞层上培养5周可产生集落形成细胞，而用CD34+CD33+细胞则不能。在与基质细胞共培养期间，CD34+CD33-细胞分化为CD34+CD33+细胞。从这些发现可以得出结论，CD34+CD33+细胞是CD34+CD33-细胞的直接后代。在这个分化途径中，c-kit mRNA的表达下降，而c-kit蛋白增加。
