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  

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

作　　者：许志祥[1] 徐颖[1] 朱剑昆[1] 李颖[1] 施勤[1] 蒋令瑜 黄伟达[2] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所免疫研究室,215007 [2]复旦大学生物化学系

出　　处：《中华血液学杂志》2000年第11期595-599,共5页Chinese Journal of Hematology

基　　金：国际原子能机构 (IAEA)资助项目! (CRP/9/0 2 5 )

摘　　要：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。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 ①An expression vector, pPICZ αA 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.②Flt3R was expressed in 5 of 18 MHC lines and the expression in leukemic blast cells was widespread and extremely heterogeneous.③The 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;④DXM 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.

关 键 词：重组人Flt3配体 造血细胞 地塞米松 白血病 

分 类 号：R733.7[医药卫生—肿瘤]