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

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-1^ext)，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，G418筛选克隆，Western印迹检测hDll-1^ext。利用免疫磁性分离方法分离脐带血CD34^ 细胞。结果表明，RT-PCR方法检测到脐事业血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-1^ext，通过集落培养检测hDll-1^ext对造血祖细胞的扩增作用。含hDll-1^ext组中CFU-Mix和HPP-CFC数量是对照组的1.5倍，结论：重组的hDll-1^ext对原始的造血祖细胞具有扩增作用。

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

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.