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目的构建稳定表达人膜型FL分子的转基因细胞株,观察膜型FL和可溶性FL对单核细胞性(M5型)白血病细胞株的体外促增殖效应。方法利用PCR方法克隆人FL全长基因,继而重组入逆转录病毒表达载体pEGZ-Term,转染包装细胞293T,用含有完整重组逆转录病毒颗粒的293T细胞培养上清感染L929细胞,筛选获得Zeocin抗性的转基因细胞。采用RT-PCR和流式细胞仪表型分析等方法进行鉴定。MTT法分析了L929/FL细胞和可溶性FL体外刺激U937细胞的增殖效应。结果成功构建了稳定表达人膜型FL的转基因细胞L929/FL,膜型FL在体外能够有效促进U937细胞增殖,而可溶性FL并不能有效促进U937细胞的体外增殖。结论成功构建了稳定表达人膜型FL的转基因细胞,并且提示膜型FL在白血病发病机理中可能具有重要作用。
OBJECTIVE: To construct a transgenic cell line stably expressing human membrane-type FL molecules and observe the proliferative effects of membrane-type FL and soluble FL on monocyte-type (M5) leukemia cell lines in vitro. Methods The full-length human FL gene was cloned by PCR. The retroviral vector pEGZ-Term was then recombined into 293T cells. The 293T cells were transfected with 293T cells and the supernatant of 293T cells were infected with L929 cells. Zeocin Resistant transgenic cells. RT-PCR and flow cytometry analysis methods such as identification. MTT assay L929 / FL cells and soluble FL stimulated proliferation of U937 cells in vitro. Results The transgenic L929 / FL stably expressing human membrane type FL was successfully constructed. The membrane type FL could effectively promote the proliferation of U937 cells in vitro. Soluble FL did not effectively promote the proliferation of U937 cells in vitro. Conclusion Transgenic cells stably expressing human membrane-type FL were successfully constructed and suggested that membrane-type FL may play an important role in the pathogenesis of leukemia.