人Delta-like-1胞外区在CHO细胞的表达纯化及对造血祖细胞的扩增作用

The Extracellular Domain of Human Delta-like-1Expressed and Purified from CHO Cells Promotes Expansion of Hematopoietic Progenitor Cells

Notch信号通路在调控造血干细胞的增殖分化中具有重要作用。本研究克隆表达人Notch配体Delta like 1(Dll 1)的胞外区 (hDll 1ext) ,并观察其对脐带血造血祖细胞的扩增作用。从人骨髓单个核细胞中提取总RNA ,用RT PCR的方法扩增Delta like 1基因胞外区 ,克隆到T载体。测序正确后 ,亚克隆到pcDNA3.1/Myc His(+)A表达载体。用脂质体转染CHO细胞 ,G4 18筛选单克隆 ,Western印迹检测hDll 1ext的分泌表达情况。选择表达量高的细胞克隆 ,扩增并收集上清。用金属亲和层析柱从上清中纯化融合蛋白hDll 1ext。利用免疫磁性分离方法分离脐带血CD34+ 细胞。结果表明 ,RT PCR方法检测到脐带血CD34+ 细胞表达Notch 1受体。在含rhIL 3,rhSCF及rhVEGF的脐带血CD34+ 细胞无血清培养体系中 ,加入纯化的hDll 1ext,通过集落培养检测hDll 1ext对造血祖细胞的扩增作用。含hDll 1ext组中CFU Mix和HPP CFC数量是对照组的 1.5倍 ,结论 :重组的hDll

Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta like 1 (hDll 1 ext ), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll 1 ext on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll 1 ext was amplified by RT PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll 1 ext in G418 resistant clones was assayed by Western blot. hDll 1 ext high expressed clone was cultured to collect supernatant. Fusion protein hDll 1 ext was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch 1 receptor was detected in cord blood derived CD34 + cells by RT PCR. Human umbilical blood CD34 + cells were cultured in serum free medium containing SCF, IL 3, VEGF, and with or without purified hDll 1 ext for 4 or 8 days. Effect of hDll 1 ext on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU Mix and HPP CFC generated from the culture system containing hDll 1 ext was 1.5 times of that from the control. In conclusion, the recombinant hDll 1 ext promotes the expansion of primitive hematopoietic progenitors.