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可移植性大鼠急性粒系白血病(Brown Norway rat acute myelocytic leukemia,BNML)是研究人急性粒系白血病的颇为完备的实验模型。由于重组人IL-6(rhIL-6)可作用于大鼠,然而大鼠BNML细胞系LT12缺乏rhIL-6受体(rhIL-6R)表达。为了在BNML模型探讨rhIL-6在体外以及体内对白血病细胞增殖和分化的影响,本文应用改良的磷酸钙共沉淀法将携带rhIL-6R cDNA的逆转录病毒表达质粒XM6/IL-6RNeo导入LT12细胞,经过G418筛选获得G418抗性克隆,建立了表达rhIL-6R的LT12亚细胞系(称之为R cell lines)。我们分别应用三种不同的方法(rhIL-6R的单克隆抗体和FACScan检测、~(125)I-rhIL-6的配基结合分析测定、地高辛配基标记的rhIL-6R cDNA探针的细胞原位杂交技术)分折了R细胞系在体外不同条件下传代过程中及给BN大鼠尾静脉回输后其rhIL-6R的表达。研究结果表明,用改良的磷酸钙共沉淀法将编码rhIL-6R cDNA和Neo~R基因的逆转录病毒表达质粒XM6/IL-6RNeo导入LT12细胞系后,受体细胞在体外含适当浓度G418的基质液中传代培养,可长期稳定表达rhIL-6R,给BN大鼠尾静脉回输后20天左右,其脾脏和骨髓细胞均可检测到rhIL-6R的明显表达,而且~3H-TdR掺入分析证明细胞膜表面所表达的rhIL-6R可以介导外源性rhIL-6信号传到细胞核内。
Transplantability Brown Norway rat acute myelocytic leukemia (BNML) is a quite complete experimental model for the study of human acute myeloid leukemia. Since recombinant human IL-6 (rhIL-6) acts in rats, rat BNML cell line LT12 lacks rhIL-6 receptor (rhIL-6R) expression. In order to investigate the effect of rhIL-6 on the proliferation and differentiation of leukemic cells in vitro and in vivo, a retroviral expression plasmid XM6 / IL-6RNeo carrying rhIL-6R cDNA was introduced into LT12 cells by modified calcium phosphate co-precipitation method G418-resistant clones were screened by G418 to establish the LT12 sub-line (called R cell lines) that expresses rhIL-6R. We used three different methods (rhIL-6R monoclonal antibody and FACScan assay, ~ (125) I-rhIL-6 ligand binding assay, digoxigenin-labeled rhIL-6R cDNA probe Cell in situ hybridization) to analyze the expression of rhIL-6R in the R cell line during the passage of different conditions in vitro and after the tail vein of BN rats were transfused. The results showed that the recombinant plasmids XM6 / IL-6RNeo encoding rhIL-6R cDNA and Neo ~ R gene were transfected into LT12 cell line with modified calcium phosphate co-precipitation method. The recipient cells were cultured in vitro with proper concentration of G418 After rhIL-6R was stably expressed for a long time, the rhIL-6R was stably expressed in the spleen and myeloid cells of rats after 20 days of tail vein reinfusion, and ~ 3H-TdR incorporation It is demonstrated that the rhIL-6R expressed on the cell membrane surface can mediate the exogenous rhIL-6 signaling to the nucleus.