Notch配体的原核表达与纯化及其对四氯化碳损伤后造血的影响

Prokaryotic Expression and Purification of the Notch Ligand and Its Effect on Hematopoiesis after Carbon Tetrachloride Damage

目的:通过原核表达并纯化靶向内皮细胞的小鼠Notch重组配体m D1R蛋白,观察其对四氯化碳损伤后造血的影响。方法:PCR克隆并构建p ET22b(+)-m D1R表达载体,转化大肠杆菌,IPTG诱导,镍珠亲合层析纯化,SDS-PAGE确认目的蛋白表达。应用流式细胞术、细胞粘附实验、免疫荧光染色及实时定量RT-PCR检测蛋白的内皮靶向性和Notch激活程度。建立四氯化碳损伤小鼠模型,应用流式细胞术观察m D1R蛋白对造血干细胞(HSC)、髓系和淋系细胞的影响,应用磁珠分选Lin^-Scal-1^+c-Kit^+细胞并分析细胞周期的变化,RT-PCR检测IL-10的表达。结果:成功克隆并构建原核表达载体,获得高纯度且有生物学活性的m D1R重组蛋白。m D1R能激活四氯化碳损伤小鼠造血干/祖细胞的Notch信号途径,内源性扩增HSC和长期HSC达2.96倍和6.18倍,促进了髓系祖细胞及髓源性抑制细胞的自体扩增,尤其有利于粒/单核细胞进入血液循环,其具体机制可能与Notch信号促进应激损伤后造血干细胞增殖和上调IL-10表达有关。结论:成功构建了连接造血干细胞和造血微环境的新型Notch配体活性蛋白,证实Notch信号途径可能通过促抗炎因子表达,实现应激损伤后自体造血动员。

Objective： To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein roD1 R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. Methods： PCR was performed to clone and construct the expression vector pET22b（ ＋ ）-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni2＋ - beads affinity chromatography. The target protein was detected by SDS- PAGE. The fluorescence-activated cell sorting analysis （FACS）, cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of naDIR on the hematopoietic stem cell （HSC）, myeloid cells and lymphoid cells by flow cytometry. The Lin- Scal-1＋ c-Kit＋ cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin （IL）-10. Results： The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2. 96 - fold and 6. 18 - fold. In addition, mD1R improved the amplification of the myeloid progenitor ceils and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury. Conclusions： A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem ceils and hematopoietic microenvironment. The Notch - mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.