Ghrelin对骨髓源性内皮祖细胞迁移能力的影响

目的 探讨ghrelin对体外培养的内皮祖细胞(EPCs)迁移能力的影响及相关的信号转导途径.方法 密度梯度离心法获取大鼠骨髓单个核细胞层,接种至纤维连接蛋白包被的培养板上,培养7～ 10d后观察细胞形态学改变,以免疫组织化学染色法检测Dil标记的乙酰化低密度脂蛋白(Dil-acLDL),FITC标记的荆豆凝集素1(FITC-UEA-1),以及CD34、CD133、人血管性血友病因子(vWF)和血管内皮细胞生长因子(VEGF)特异的酪氨酸激酶受体(Flk-1),鉴定细胞种类后传代培养.采用不同浓度(10-9～10-6mol/L)的ghrelin干预EPCs后,通过Transwell小室迁移实验检测EPCs迁移能力的变化.用不同浓度(10-9～10-6mol/L)的ghrelin干预EPCs l5min或10-7mol/L的ghrelin干预0～ 60min,Western blotting 检测蛋白激酶B(Akt)及内皮型一氧化氮合酶(eNOS)及其磷酸化状态的表达；分别用磷脂酰肌醇3激酶(PI3K)的特异性抑制剂LY294002和eNOS的特异性抑制剂L-NAME预处理EPCs,检测ghrelin对EPCs迁移及Akt、eNOS及其磷酸化蛋白表达的变化.结果 新分离24h内的EPCs近似圆形,培养第4～7天转化为梭形贴壁细胞,培养至第9天后梭形细胞呈条索样排列生长.Dil-acLDL和FITC-UEA-1双染色阳性,CD34、CD133、vWF和flk-1免疫组织化学染色均呈阳性反应.分别用10-9～10-6mol/L的ghrelin干预内皮祖细胞,发现10-8、10-7mol/L的ghrelin可明显促进内皮祖细胞的迁移(P＜0.001),而更高浓度( 10-6mol/L)的ghrelin则可显著抑制EPCs的迁移(P＜0.05).Ghrelin呈时间和剂量依赖性地促进EPCs表达磷酸化Akt和eNOS；PI3K特异性抑制剂LY294002能明显抑制ghrelin诱导的Akt、eNOS的磷酸化表达(P＜0.05);eNOS的特异性抑制剂L-NAME能明显抑制ghrelin诱导的eNOS磷酸化表达(P＜0.05),但对Akt的磷酸化表达无影响.LY294002和L-NAME均能显著抑制ghrelin诱导的内皮祖细胞迁移(P＜0.05).结论 Ghrelin可通过激活PI3K/Akt/eNOS信号途径促进骨髓源性EPCs迁移,但其效应呈现一定的浓度依赖性.

Effect of ghrelin on directional migration of bone-marrow-derived endothelial progenitor cells in vitro

Objective To explore the influence of ghrelin on migration function of rat endothelial progenitor cells(EPCs) and potential signal transduction pathway in vitro.Methods Mononuclear cells were collected from rat bone marrow by density gradient centrifugation.The cells were identified by Dil-acLDL and FITC-UEA-1 double staining,and immunohistochemical staining was performed for CD34,CD133,vWF and flk-1.The effects of 10-9–10-6mol/L ghrelin on directional migration of EPCs were determined by Transwell assay.EPCs were incubated with 10-9–10-6mol/L ghrelin for 15min,or incubated with 10-7mol/L ghrelin for 0–60min for assay of Akt and eNOS phosphorylation.EPCs were pretreated with PI3K or eNOS inhibitors LY294002 or L-NAME for the assay of Akt and eNOS phosphorylation,as well as directional migration of EPCs by Western blotting.Results Isolated cells were approximately round in shape within 24h of culture,and they assumed a spindle-shaped morphology after culture for 4–7 days,then they transformed into cordlike shape after 9 days.The cells were positive for double staining with Dil-acLDL and FITC-UEA-1 and identified as EPCs.They were additionally confirmed by demonstration of the expression of well-established cell surface markers such as CD31,CD34,CD133,vWF and flk-1.10-8–10-7mol/L ghrelin significantly induced directional migration of EPCs(P<0.001).However,a higher concentration of ghrelin(10-6mol/L) significantly inhibited directional migration of EPCs(P<0.05) instead.Treatment of EPCs with ghrelin increased phosphorylation of Akt and eNOS in a time-and dose-dependent manner.PI3K inhibitor LY294002 attenuated ghrelin-induced phosphorylation of Akt and eNOS(P<0.05),eNOS inhibitor L-NAME blocked ghrelin-induced phosphorylation of eNOS(P<0.05),but it did not exert effect on Akt phosphorylation.Ghrelin-induced directional migration of EPCs was suppressed by LY294002 or L-NAME(P<0.05).Conclusions Ghrelin can stimulate directional migration of EPCs via activation of the PI3K/Akt/eNOS signaling pathway.However,the above effect of ghrelin is concentration-dependent.