血红素结合蛋白通过上调HO-1表达促进EPCs分化以修复血管内皮损伤

Hemopexin protects endothelial integrity by promoting endothelial progenitor cells differentiation and new blood vessels formation through heme oxygenase-1 pathway

目的探讨血红素结合蛋白（HPX）是否通过上调血红素氧合酶．I（HO-1）的表达，促进内皮祖细胞（EPCs）分化和新生血管形成。以修复游离血红素引起的血管内皮损伤。方法从大鼠骨髓提取EPCs．培养鉴定后将处于对数生长期的细胞分为5组：对照组、HPX组、血红素组、血红素＋HPX组、锌原卟啉Ⅸ组。对照组以全培养基（M199培养基＋10％胎牛血清＋5μg／mL碱性成纤维细胞生长因子＋1％青链霉素混合液1培养，HPX组、血红素组、血红素＋HPX组、锌原卟啉Ⅸ组在此基础上分别加入20μmol／LHPX、20μmol／L血红素、20μmol／L血红素-HPX复合物、20μmol／L血红素-HPX复合物＋10μmol／L锌原卟啉Ⅸ。各组细胞培养24h后采用Westernblotting检测细胞内HO-1蛋白含量；采用CD31、血管性血友病因子（vWF）免疫荧光共染检测EPCs表型及向成熟内皮细胞分化程度：采用体外血管形成实验测定EPCs成血管能力。结果与对照组（0．60±0．15）相比，血红素组HO-1蛋白含量（1．07±0．03）明显增加，差异有统计学意义（P〈0．05）；与血红素组相比，血红素＋HPX组HO-1蛋白含量（1．57±0．06）明显增加，差异有统计学意义（P〈0．05）。与对照组相比，血红素组EPCs数量减少，表面CD31、vWF表达较少，免疫荧光较弱；血红素＋HPX组EPCs数量较多，表面CD31、vWF表达增加，免疫荧光较强。与对照组[（41．0±7．18）个／视野]相比，血红素组形成的管腔样结构（TLS）数目[（25．0±3．67）个／视野]较少，差异有统计学意义（P〈0．05）。与血红素组相比，血红素＋HPX组形成较多明显的TLS[（58．6±2．70）个／视野]，差异有统计学意义（P〈0．05）。结论HPX可通过上调HO-1表达，促进EPCs向成熟内皮细胞分化的程度，促进新生血管形成，修复游离血红素引起的血管内皮损伤。

Objective To investigate the effect of hemopexin （HPX） on promoting endothelial progenitor cells （EPCs） differentiation and neovascularization to protect endothelial integrity fi＇om toxic heme through heme oxygenase-1 （HO-1） pathway. Methods Rat EPCs were cultured routinely, and randomly divided into 5 groups （at their logarithmic phase）： control group, HPX group, heme group, heme＋HPX group, and Zn protoporphyrin IX （ZnpplX） group. Cells in the control group were cultured in M199 culture medium with 10% fetal calf serum, 5 /xg/mL basic fibroblast growth factor and 1% penicillin-streptomycin, and 20 μmol/L HPX, 20 μmol/L heme, 20 μmol/L heme-HPX compound, and 20 μmol/L heme-HPX compound and 10 μmol/L ZnpplX were added in the culture mediums of HPX group, heme group, heme＋HPX group and ZnpplX group, respectively. Cells were harvested after 24 h of culture; HO-1 expression was detected by Western blotting; differentiation of EPCs to mature endothelial cells was investigated using yon Willebrand factor （vWF） immunofluorescence; vasculogenic ability was tested using angiogenesis assay on Matrigel Basement Membrane Matirx. Results As compared with that in the control group （0.60±0.15）, HO-1 expression increased significantly in heme group （1.07±0.03,P〈0.05）; as compared with the heme group, the HO-1 expression in the heme＋HPX group （1.57±0.06） significantly increased （P〈0.05）. As compared with those in the control group, the EPCs number was smaller, CD31 and vWF expressions were decreased and immunofluorescence results were weaker in the heme group; as compared with those in the control group, the EPCs number was larger, CD31 and vWF expressions were increased and immunofluorescence results were stronger in the heme＋HPX group. As compared with that in the control group （[41.0±7.18]/field）, the number of tube like structure formation in the heme group （[25.0±3.67] /field） was significantly smaller （P〈0.05）; as compared with that in the heme group, the number of tube like structure formation in the heme＋HPX group （[58.6±2.70]/field） was significantly larger （P〈0.05）. Conclusion HPX shows significant effect on promoting EPCs differentiation and new blood vessels formation to protect the endothelial integrity from toxic heme by increasing the HO- 1 expression.