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目的 用毕氏酵母 (Pichiapastoris)表达人可溶性Flt3配体 (rhFL) ,并观察rhFL对恶性造血细胞增殖的作用 ;探讨地塞米松 (DXM)对Flt3受体 (Flt3R)表达及rhFL介导的恶性造血细胞增殖的影响。方法 人工合成FL基因片段 ,经基因重组技术获得重组质粒pPICZα FL ,电转化毕氏酵母 ,筛选分泌rhFL的基因工程菌株。用流式细胞仪测定恶性造血细胞表面Flt3R的表达 ,用MTT法分析rhFL对恶性造血细胞的增殖作用及DXM的影响。结果 经筛选 ,获得稳定分泌rhFL的基因工程菌株 ,表达量达 30mg/L。rhFL在 10～ 10 0ng/ml时 ,刺激Raji和HL 6 0细胞的增殖 ,但对绝大多数的恶性造血细胞无刺激作用 ;18株恶性造血细胞株中 5株细胞表达Flt3R ;对部分病理类型的白血病新鲜标本的研究发现 ,6 0 %患者的外周血或骨髓细胞表达Flt3R ,急性单核细胞白血病及急性淋巴细胞白血病患者Flt3R的阳性率较高 ,但仅有部分患者的白血病细胞在体外对FL的刺激有增殖效应。DXM可抑制rhFL对Raji、HL 6 0细胞及部分新鲜白血病标本的白血病细胞的刺激增殖作用。结论 应用毕氏酵母表达具有生物学活性的rhFL ,rhFL可刺激Raji、HL 6 0和部分白血病患者白血病细胞在体外的增殖。DXM通过降低Flt3R的表达 ,抑制rhFL介导的恶性造血细胞的增殖。DXM有可能用于
Objective To express human soluble Flt3 ligand (rhFL) with Pichia pastoris and observe the effect of rhFL on the proliferation of malignant hematopoietic cells; To investigate the expression of Flt3 receptor (Flt3R) and rhFL-mediated malignant effect of dexamethasone (DXM) on malignant hematopoietic cells. Effect of hematopoietic cell proliferation. Methods The FL gene fragment was synthesized artificially. The recombinant plasmid pPICZα FL was obtained by gene recombination technology and transformed into Pichia pastoris by electroporation. The gene-engineered strain secreting rhFL was screened. The expression of Flt3R on the surface of malignant hematopoietic cells was measured by flow cytometry. The effect of rhFL on the proliferation of malignant hematopoietic cells and the effect of DXM were analyzed by MTT assay. Results After screening, a genetically engineered strain stably secreting rhFL was obtained with an expression level of 30 mg/L. When rhFL was between 10 and 10 ng/ml, it stimulated the proliferation of Raji and HL 60 cells, but it had no stimulatory effect on most malignant hematopoietic cells. Five of the 18 malignant hematopoietic cell lines expressed Flt3R; for some pathological types The study of fresh samples of leukemia found that 60% of patients had peripheral blood or bone marrow cells expressing Flt3R. The positive rate of Flt3R was higher in patients with acute monocytic leukemia and acute lymphoblastic leukemia, but only some patients had leukemia cells in vitro. FL stimulation has a proliferative effect. DXM can inhibit the proliferative effect of rhFL on leukemic cells of Raji, HL 60 cells and some fresh leukemia specimens. Conclusion The use of Pichia pastoris to express biologically active rhFL, rhFL can stimulate the proliferation of Raji, HL 60 and leukemia cells in some leukemia patients. DXM inhibits the proliferation of rhFL-mediated malignant hematopoietic cells by decreasing the expression of Flt3R. DXM may be used