Lu L, Heinrich M C, Wang L S, Dai M S, Zigler A J, Chai L, Broxmeyer H E
Department of Microbiology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA.
Blood. 1999 Oct 1;94(7):2319-32.
The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34(+) cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34 and different levels of c-kit (++,+,Lo/-), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34KitLo+/- cells transduced with c-kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor-beta1 and tumor necrosis factor-alpha. c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.
c-kit受体及其配体——干细胞因子（SLF），对最佳造血功能至关重要。我们评估了用编码人c-kit cDNA的逆转录病毒转导脐血（CB）祖细胞的效果。将CD34(+)细胞分选成群体或按每孔1个细胞，用于分选表达高水平CD34和不同水平c-kit（++、+、低/ -）的细胞，进行转导，然后在无血清条件下于粒细胞-巨噬细胞集落刺激因子（GM-CSF）、白细胞介素-3（IL-3）、IL-6、促红细胞生成素（Epo）+/- SLF存在的情况下培养。在单细胞水平上，用c-kit而非对照病毒（仅neo）转导可显著增加集落形成，尤其是红系和多能祖细胞的集落形成。c-kit转导的增强作用与转导前c-kit蛋白的表达呈负相关。在用c-kit转导的CD34Kit低/ +细胞中观察到最大的增强作用。即使在没有外源添加SLF的情况下，但在有GM-CSF、IL-3、IL-6和Epo存在时，刺激作用也很明显。对SLF蛋白进行酶联免疫吸附测定（ELISA）、对CD34+细胞中SLF mRNA表达进行逆转录酶-聚合酶链反应（RT-PCR）分析以及使用针对SLF和/或c-kit的中和抗体表明，这些祖细胞存在内源性SLF表达，尽管可能水平非常低。c-kit转导显著降低了祖细胞对转化生长因子-β1和肿瘤坏死因子-α抑制作用的敏感性。c-kit转导的细胞c-kit蛋白表达增加，自发或细胞因子诱导的凋亡减少。我们的结果表明，将c-kit转导到选定的祖细胞中可增强增殖并减少凋亡，内源性SLF可能介导了这一效应。
