生长激素促分泌物受体的内源性配体ghrelin促进大鼠心肌微血管内皮细胞体外血管生成

Ghrelin stimulates in vitro angiogenic capacity of rat cardiac microvascular endothelial cells

目的 探讨生长激素促分泌物受体的内源性配体 ghrelin 对大鼠心肌微血管内皮细胞增殖、迁移和体外血管生成的影响,并探讨其潜在的信号转导途径.方法 （1）植块法分离成年SD 雄性大鼠的心肌微血管内皮细胞,Ⅷ因子免疫组织化学鉴定细胞种类并传代培养.（2）通过逆转录聚合酶链反应（RT-PCR）、免疫荧光、酶联免疫吸附试验（ELISA）、免疫印迹法（Western blot）鉴定ghrelin及其天然受体（growth hormone secretagogue-receptor,GHS-R）在心肌微血管内皮细胞上mRNA 及蛋白的表达.（3）用不同浓度（10-9～10-7mol/L）的ghrelin干预心肌微血管内皮细胞后,检测心肌微血管内皮细胞的增殖、迁移及体外血管生成的变化.（4）用不同浓度（10-9～10-7 mol/L）的ghrelin 干预心肌微血管内皮细胞后,观察ERK2磷酸化表达.用MAPK/ERK2的特异性抑制剂PD98059提前干预细胞后,观察ghrelin对大鼠心肌微血管内皮细胞体外血管生成及ERK2磷酸化表达的变化.结果 （1）植块法培养的原代心肌微血管肉皮细胞纯度达95％左右,且具有形成管腔样结构的能力.（2）RT-PCR、免疫荧光、ELISA和Western blot检测发现ghrelin及GHS-R在心肌微血管内皮细胞上均有表达.（3）分别用10-9～10-7 mol/L的ghrelin干预心肌微血管内皮细胞后,发现10-8、10-7 mol/L ghrelin处理组心肌微血管内皮细胞的增殖明显高于正常对照组[分别为（12.37±0.70）和（12.73±0.78）pmol/L比（7.40±0.71）pmol/L,P值均为0.001],迁移亦明显高于正常对照组（分别为127.00±4.06和121.00±4.30比113.80±4.60,p值分别为0.001和0.03）,体外的血管生成亦明显高于正常对照组[分别为（72.20±5.72）和（71.00±7.78）mm比（28.60±5.13）mm,P均＜0.001].（4）10-8、10-7mol/L ghrelin处理组ERK2的磷酸化表达水平明显高于正常对照组（分别为0.92±0.13和1.15 ±0.16比0.29±0.04,P均＜0.001）.若提前用PD98059干预细胞,则能明显抑制ghrelin诱导的体外血管生成及ERK2的磷酸化表达.结论 ghrelin及其受体GHS-R在大鼠心肌微血管内皮细胞上均有表达,ghrelin可通过激活ERK2的磷酸化促进心肌微血管内皮细胞的体外血管生成.

Objective To clarify whether ghrelin could promote in vitro rat cardiac microvascular endothelial cells（CMECs）angiogenesis and related mechanisms.Methods CMECs were isolated from myocardial tissue of adult male SD rats and characterized by the immunocytochemistry staining with Factor Ⅷ and the capacity of in vitro capillary tube-like formation.The mRNAs and protein expressions of ghrelin and its receptor（growth hormone secretagogue receptor,GHS-R）of CMECs were determined by RT-PCR,Immunofluorescence,ELISA and Western blot.Proliferation,migration and in vitro angiogenesis as well as ERK2 phosphorylation of CMECs were tested in the presence of ghrelin（10-9-10-7 mol/L）with or without pretreatment with specific MAPK/ERK2 inhibitor PD98059.Results Purity of CMECs characterized by immunocytochemistry staining with Factor Ⅷ was about 95%,and the cells showed a high ability to form the capillary tube-like structures on Matrigel.Ghrelin and GHS-R were constitutively expressed in CMECs.Proliferation,migration and in vitro angiogenesis capacities of CMECs（72.20 ±5.72 vs.28.60 ±5.13,P 〈0.001;71.00±7.78 vs.28.60 ±5.13,P 〈0.001）as well as ERK2 phosphorylation（0.92 ±0.13 vs.0.29 ± 0.04,P 〈 0.001 ; 1.15 ± 0.16 vs.0.29 ± 0.04,P 〈 0.001）were significantly enhanced by exogenous ghrelin（10-8-10-7 mol/L）.PD98059 abolished ghrelin-induced ERK2 phosphorylation and in vitro angiogenesis.Conclusions Ghrelin and its receptor are expressed in CMECs and ghrelin could stimulate CMECs in vitro angiogenesis through activation of MAPK/ERK2 signaling pathway.