Ohta M, Anklesaria P, Wheaton M, Ohara A, Pierce J H, Holland C A, Greenberger J S
Department of Radiation Oncology, University of Massachusetts Medical School, Worcester 01655.
Leukemia. 1989 Mar;3(3):206-26.
To define the action of the retroviral src gene on hematopoietic stem cells, C57BL/6 x DBA/2 (B6D2F1) mouse long-term marrow cultures were infected at initiation with Moloney murine leukemia virus (MuLV) pseudotypes of src-recombinant retroviruses with the src gene inserted in the env region of an amphotropic MuLV (src-Ampho), or in the gag region of Moloney MuLV (src-Mo). Other cultures were infected with Friend spleen focus-forming virus polycythemia-inducing strain (SFFVp), Moloney MuLV, or amphotropic MuLV, or were uninfected controls. Harvested nonadherent cells were tested weekly for multilineage, granulocyte-erythroid-megakaryocyte macrophage (CFU-GEMM) colony formation in vitro in recombinant murine IL-3 and erythropoietin, and individual colonies were removed, split 1:2, with half of each replated for in vitro self-renewal and the other half examined morphologically for number of hematopoietic cellular lineages, or tested for release of MuLV and src virus. Cultures infected with src-Ampho, src-Mo, or SFFVp demonstrated a significant increase in cumulative nonadherent cell and CFU-GEMM production. There was prolonged self-renewal over seven serial transfers of individual CFU-GEMM from src virus-infected cultures over seven serial transfers, and five of 61 individual colonies from the second or third generations contained detectable v-src gene sequences, but none released detectable src virus. Self-renewal of CFU-GEMM was similar to that with permanent IL-3-dependent cell line B6SUtA. In contrast, MuLV-infected or control uninfected cultures produced fewer cells, and self-renewal of CFU-GEMM did not exceed three generations. IL-3-dependent clonal hematopoietic progenitor cell lines, derived from each culture group, formed no detectable tumors in vivo; however, each released the original helper and/or transforming virus. Adherent cell lines, derived from src-Ampho-infected cultures released src virus and formed fibro-sarcomas in vivo. The data support the conclusion that src-recombinant virus expression in long-term marrow cultures increases the self-renewal capacity of multilineage hematopoietic stem cells.
为了确定逆转录病毒src基因对造血干细胞的作用，在起始时用src重组逆转录病毒的莫洛尼鼠白血病病毒（MuLV）假型感染C57BL/6×DBA/2（B6D2F1）小鼠长期骨髓培养物，其中src基因插入到嗜异性MuLV的env区域（src-Ampho）或莫洛尼MuLV的gag区域（src-Mo）。其他培养物用弗氏脾集落形成病毒红细胞增多症诱导株（SFFVp）、莫洛尼MuLV或嗜异性MuLV感染，或作为未感染的对照。每周对收获的非贴壁细胞进行检测，以观察其在重组鼠白细胞介素-3和促红细胞生成素存在下体外多谱系、粒细胞-红细胞-巨核细胞-巨噬细胞（CFU-GEMM）集落形成情况，将单个集落取出，按1:2分开，一半重新接种用于体外自我更新，另一半进行形态学检查以确定造血细胞谱系数量，或检测MuLV和src病毒的释放情况。用src-Ampho、src-Mo或SFFVp感染的培养物显示，累积非贴壁细胞和CFU-GEMM产量显著增加。来自src病毒感染培养物的单个CFU-GEMM在七次连续传代中自我更新延长，来自第二代或第三代的61个单个集落中有5个含有可检测到的v-src基因序列，但没有一个释放出可检测到的src病毒。CFU-GEMM的自我更新与永久性依赖白细胞介素-3的细胞系B6SUtA相似。相比之下，MuLV感染的或未感染的对照培养物产生的细胞较少，CFU-GEMM的自我更新不超过三代。从每个培养组衍生的依赖白细胞介素-3的克隆造血祖细胞系在体内未形成可检测到的肿瘤；然而，每个细胞系都释放出原始的辅助病毒和/或转化病毒。从src-Ampho感染培养物衍生的贴壁细胞系释放src病毒，并在体内形成纤维肉瘤。这些数据支持以下结论：长期骨髓培养物中src重组病毒的表达增加了多谱系造血干细胞的自我更新能力。
