重组人Flt3配体在毕氏酵母中的表达及其对恶性造血细胞的体外作用

目的 用毕氏酵母（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）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ＭＴ

Expression of recombinant human soluble Flt3 ligand and its in vitro effects on malignant hematopoietic cells

OBJECTIVE: To express human soluble Flt3 ligand in a yeast expression system. Pichia pastoris, and investigate the effects of recombinant human soluble Flt3 ligand (rhFL) on the proliferation of malignant hematopoietic cells(MHC) and the effect of dexamethasone (DXM) on the expression of Flt3 receptor (Flt3R) and the rhFL induced proliferation of those cells. METHODS: An artificial gene for rhFL was synthesized by using favored genetic codons of the yeast Pichia pastoris. Flt3R was determined by flow cytometry. Proliferation of MHCs was measured by microculture tetrazolium (MTT) assay. RESULTS: 1. An expression vector, pPICZ alpha A-FL, was constructed and electroporated into Pichia pastoris. A yield of over 30 mg/L of yeast culture supernatant was obtained. The rhFL could stimulate the proliferation of Raji and HL-60 cells at concentrations of 10-100 ng/ml, but did not for most of other MHCs. 2. Flt3R was expressed in 5 of 18 MHC lines and the expression in leukemic blast cells was widespread and extremely heterogeneous. 3. The presence of the receptor on the surface of MHC did not necessarily imply a significant ligand-induced response, at least in terms of proliferation. By contrast, some Flt3R-negative MHC responded to rhFL. 4. DXM down-regulated the expression of Flt3R and inhibited the proliferation induced by rhFL in some MHC lines and fresh leukemia cell samples. CONCLUSION: A yield of 30 mg/L of rhFL was obtained. rhFL stimulated the proliferation of Raji, HL-60 cells and some fresh leukemia cells, which could be inhibited by DXM through down-regulation of Flt3R. A combination of FL and DXM might possibly serve to control hematopoietic defects in malignant hematopoietic diseases.