CAJ | 학술논문

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. 目的探討生長激素促分泌物受體的內源性配體 ghrelin 對大鼠心肌微血管內皮細胞增殖、遷移和體外血管生成的影響,併探討其潛在的信號轉導途徑.方法 (1)植塊法分離成年SD 雄性大鼠的心肌微血管內皮細胞,Ⅷ因子免疫組織化學鑒定細胞種類併傳代培養.(2)通過逆轉錄聚閤酶鏈反應(RT-PCR)、免疫熒光、酶聯免疫吸附試驗(ELISA)、免疫印跡法(Western blot)鑒定ghrelin及其天然受體(growth hormone secretagogue-receptor,GHS-R)在心肌微血管內皮細胞上mRNA 及蛋白的錶達.(3)用不同濃度(10-9～10-7mol/L)的ghrelin榦預心肌微血管內皮細胞後,檢測心肌微血管內皮細胞的增殖、遷移及體外血管生成的變化.(4)用不同濃度(10-9～10-7 mol/L)的ghrelin 榦預心肌微血管內皮細胞後,觀察ERK2燐痠化錶達.用MAPK/ERK2的特異性抑製劑PD98059提前榦預細胞後,觀察ghrelin對大鼠心肌微血管內皮細胞體外血管生成及ERK2燐痠化錶達的變化.結果 (1)植塊法培養的原代心肌微血管肉皮細胞純度達95％左右,且具有形成管腔樣結構的能力.(2)RT-PCR、免疫熒光、ELISA和Western blot檢測髮現ghrelin及GHS-R在心肌微血管內皮細胞上均有錶達.(3)分彆用10-9～10-7 mol/L的ghrelin榦預心肌微血管內皮細胞後,髮現10-8、10-7 mol/L ghrelin處理組心肌微血管內皮細胞的增殖明顯高于正常對照組[分彆為(12.37±0.70)和(12.73±0.78)pmol/L比(7.40±0.71)pmol/L,P值均為0.001],遷移亦明顯高于正常對照組(分彆為127.00±4.06和121.00±4.30比113.80±4.60,p值分彆為0.001和0.03),體外的血管生成亦明顯高于正常對照組[分彆為(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處理組ERK2的燐痠化錶達水平明顯高于正常對照組(分彆為0.92±0.13和1.15 ±0.16比0.29±0.04,P均＜0.001).若提前用PD98059榦預細胞,則能明顯抑製ghrelin誘導的體外血管生成及ERK2的燐痠化錶達.結論 ghrelin及其受體GHS-R在大鼠心肌微血管內皮細胞上均有錶達,ghrelin可通過激活ERK2的燐痠化促進心肌微血管內皮細胞的體外血管生成.
목적 탐토생장격소촉분비물수체적내원성배체 ghrelin 대대서심기미혈관내피세포증식、천이화체외혈관생성적영향,병탐토기잠재적신호전도도경.방법 (1)식괴법분리성년SD 웅성대서적심기미혈관내피세포,Ⅷ인자면역조직화학감정세포충류병전대배양.(2)통과역전록취합매련반응(RT-PCR)、면역형광、매련면역흡부시험(ELISA)、면역인적법(Western blot)감정ghrelin급기천연수체(growth hormone secretagogue-receptor,GHS-R)재심기미혈관내피세포상mRNA 급단백적표체.(3)용불동농도(10-9～10-7mol/L)적ghrelin간예심기미혈관내피세포후,검측심기미혈관내피세포적증식、천이급체외혈관생성적변화.(4)용불동농도(10-9～10-7 mol/L)적ghrelin 간예심기미혈관내피세포후,관찰ERK2린산화표체.용MAPK/ERK2적특이성억제제PD98059제전간예세포후,관찰ghrelin대대서심기미혈관내피세포체외혈관생성급ERK2린산화표체적변화.결과 (1)식괴법배양적원대심기미혈관육피세포순도체95％좌우,차구유형성관강양결구적능력.(2)RT-PCR、면역형광、ELISA화Western blot검측발현ghrelin급GHS-R재심기미혈관내피세포상균유표체.(3)분별용10-9～10-7 mol/L적ghrelin간예심기미혈관내피세포후,발현10-8、10-7 mol/L ghrelin처리조심기미혈관내피세포적증식명현고우정상대조조[분별위(12.37±0.70)화(12.73±0.78)pmol/L비(7.40±0.71)pmol/L,P치균위0.001],천이역명현고우정상대조조(분별위127.00±4.06화121.00±4.30비113.80±4.60,p치분별위0.001화0.03),체외적혈관생성역명현고우정상대조조[분별위(72.20±5.72)화(71.00±7.78)mm비(28.60±5.13)mm,P균＜0.001].(4)10-8、10-7mol/L ghrelin처리조ERK2적린산화표체수평명현고우정상대조조(분별위0.92±0.13화1.15 ±0.16비0.29±0.04,P균＜0.001).약제전용PD98059간예세포,칙능명현억제ghrelin유도적체외혈관생성급ERK2적린산화표체.결론 ghrelin급기수체GHS-R재대서심기미혈관내피세포상균유표체,ghrelin가통과격활ERK2적린산화촉진심기미혈관내피세포적체외혈관생성.
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.