Institute of Cardiovascular Diseases, XinQiao Hospital, Third Military Medical University, Chong Qing 400037, PR China.
Int J Cardiol. 2010 Jan 7;138(1):70-80. doi: 10.1016/j.ijcard.2008.10.042. Epub 2008 Dec 17.
Endothelial repair is one of key events after vascular injury. The mechanisms by which hepatocyte growth factor (HGF) and endothelial progenitor cells (EPCs) may be responsible for re-endothelialization of injured blood vessel wall are poorly understood.
Primary culture SMCs were transfected with pcDNA3.0-HGF followed by G418 selection, one of G418-resistant colonies in well was picked, propagated and used as donor cells for further experiments. HGF and VEGF expression in SMCs were detected with western blot and enzyme linked immunosorbent assays (ELISA). Rat EPCs were cultured in untreated, pcDNA3.0 and pcDNA3.0-HGF transfected SMCs conditioned medium with or without anti-VEGF or exogenous recombinant HGF addition. eNOS, KDR and CD31 expression in EPCs was determined by real-time quantitative polymerase chain reaction (RT-qPCR) or flow cytometry; EPCs migration and proliferation were measured by using a modified Boyden chambers and MTT assay respectively.
Abundant and stable expression of HGF was found in G418-resistant colony-derived SMCs. VEGF expression significantly increased in HGF transfected SMCs. Exogenous recombinant HGF (rHGF) markedly up-regulated eNOS mRNA expression in EPCs and promoted EPCs migration and proliferation, but no significant changes were found in KDR and CD31 mRNA expression. HGF transfection in SMCs was more effective than exogenous HGF for EPCs differentiation, proliferation and migration.
Over-expression of HGF in SMCs can be helpful for promoting EPCs differentiation, increasing EPCs migration and proliferation. It may be responsible for angiogenesis of arteriosclerosis lesions and useful for blood vessel tissue engineering.
内皮修复是血管损伤后的关键事件之一。肝细胞生长因子（HGF）和内皮祖细胞（EPC）可能负责损伤血管壁再内皮化的机制尚不清楚。
原代培养的平滑肌细胞（SMC）转染 pcDNA3.0-HGF，然后进行 G418 筛选，从培养孔中挑选出一个 G418 抗性克隆，进行扩增，并用作进一步实验的供体细胞。用 Western blot 和酶联免疫吸附试验（ELISA）检测 SMC 中 HGF 和 VEGF 的表达。将大鼠 EPC 培养在未经处理、pcDNA3.0 和 pcDNA3.0-HGF 转染的 SMC 条件培养基中，或在添加抗 VEGF 或外源性重组 HGF 的情况下培养。通过实时定量聚合酶链反应（RT-qPCR）或流式细胞术检测 EPC 中 eNOS、KDR 和 CD31 的表达；通过改良 Boyden 室和 MTT 测定分别测量 EPC 的迁移和增殖。
在 G418 抗性克隆衍生的 SMC 中发现 HGF 大量且稳定表达。HGF 转染的 SMC 中 VEGF 表达显著增加。外源性重组 HGF（rHGF）显著上调 EPC 中 eNOS mRNA 的表达，并促进 EPC 的迁移和增殖，但 KDR 和 CD31 mRNA 的表达没有明显变化。SMC 中转染 HGF 比外源性 HGF 更有效地促进 EPC 的分化、增殖和迁移。
SMC 中 HGF 的过表达有助于促进 EPC 的分化，增加 EPC 的迁移和增殖。它可能与动脉粥样硬化病变的血管生成有关，对血管组织工程有用。
