中华创伤骨科杂志
中華創傷骨科雜誌
중화창상골과잡지
CHINESE JOURNAL OF ORTHOPAEDIC TRAUMA
2011年
11期
1072-1076
,共5页
于留钱%张宁%胡志毅%殷国勇
于留錢%張寧%鬍誌毅%慇國勇
우류전%장저%호지의%은국용
受体,G-蛋白偶联%成骨细胞%载体蛋白质类%磷酸转移酶类
受體,G-蛋白偶聯%成骨細胞%載體蛋白質類%燐痠轉移酶類
수체,G-단백우련%성골세포%재체단백질류%린산전이매류
Receptors,G-protein-coupled%Osteoblasts%Carrier proteins%Phosphotransferases
目的 探讨G蛋白偶联受体激酶结合蛋白1(GITl)的SHD结构域对成骨细胞内局部黏附激酶(FAK)活性的影响,并分析其机制.方法 取1～2dSD大鼠颅盖骨进行成骨细胞原代培养,传至第3代.体外构建野生型GIT1和删除了SHD结构域的GIT1的质粒并组成慢病毒.血小板衍生生长因子(PDGF)刺激成骨细胞后,采用免疫共沉淀法检测GIT1、删除了SHD结构域的GIT1与FAK的相互关系,采用免疫荧光法检测GIT1和FAK在成骨细胞内的位置、删除了SHD结构域的GIT1对成骨细胞内FAK位置和活性的影响.结果 PDGF刺激成骨细胞后,GIT1与FAK的相互结合明显增加,且随着时间的增加,结合量逐渐增加(F=293.105,P=0.000).免疫荧光染色结果表明:PDGF刺激后,成骨细胞局部黏附内活性形式的FAK(FAK酪氨酸397磷酸化)明显增加.PDGF刺激成骨细胞后,删除了SHD结构域的GIT1与FAK的结合明显被抑制,且随着时间的增加,二者的相互关系无明显变化(F=0.322,P=0.737);删除了SHD结构域的GIT1抑制成骨细胞局部黏附内FAK酪氨酸397的磷酸化,同时降低FAK在局部黏附内的表达.结论 删除了SHD结构域的GIT1通过降低与FAK的相互结合,从而抑制成骨细胞局部黏附内FAK酪氨酸397的磷酸化,影响成骨细胞的功能.
目的 探討G蛋白偶聯受體激酶結閤蛋白1(GITl)的SHD結構域對成骨細胞內跼部黏附激酶(FAK)活性的影響,併分析其機製.方法 取1～2dSD大鼠顱蓋骨進行成骨細胞原代培養,傳至第3代.體外構建野生型GIT1和刪除瞭SHD結構域的GIT1的質粒併組成慢病毒.血小闆衍生生長因子(PDGF)刺激成骨細胞後,採用免疫共沉澱法檢測GIT1、刪除瞭SHD結構域的GIT1與FAK的相互關繫,採用免疫熒光法檢測GIT1和FAK在成骨細胞內的位置、刪除瞭SHD結構域的GIT1對成骨細胞內FAK位置和活性的影響.結果 PDGF刺激成骨細胞後,GIT1與FAK的相互結閤明顯增加,且隨著時間的增加,結閤量逐漸增加(F=293.105,P=0.000).免疫熒光染色結果錶明:PDGF刺激後,成骨細胞跼部黏附內活性形式的FAK(FAK酪氨痠397燐痠化)明顯增加.PDGF刺激成骨細胞後,刪除瞭SHD結構域的GIT1與FAK的結閤明顯被抑製,且隨著時間的增加,二者的相互關繫無明顯變化(F=0.322,P=0.737);刪除瞭SHD結構域的GIT1抑製成骨細胞跼部黏附內FAK酪氨痠397的燐痠化,同時降低FAK在跼部黏附內的錶達.結論 刪除瞭SHD結構域的GIT1通過降低與FAK的相互結閤,從而抑製成骨細胞跼部黏附內FAK酪氨痠397的燐痠化,影響成骨細胞的功能.
목적 탐토G단백우련수체격매결합단백1(GITl)적SHD결구역대성골세포내국부점부격매(FAK)활성적영향,병분석기궤제.방법 취1～2dSD대서로개골진행성골세포원대배양,전지제3대.체외구건야생형GIT1화산제료SHD결구역적GIT1적질립병조성만병독.혈소판연생생장인자(PDGF)자격성골세포후,채용면역공침정법검측GIT1、산제료SHD결구역적GIT1여FAK적상호관계,채용면역형광법검측GIT1화FAK재성골세포내적위치、산제료SHD결구역적GIT1대성골세포내FAK위치화활성적영향.결과 PDGF자격성골세포후,GIT1여FAK적상호결합명현증가,차수착시간적증가,결합량축점증가(F=293.105,P=0.000).면역형광염색결과표명:PDGF자격후,성골세포국부점부내활성형식적FAK(FAK락안산397린산화)명현증가.PDGF자격성골세포후,산제료SHD결구역적GIT1여FAK적결합명현피억제,차수착시간적증가,이자적상호관계무명현변화(F=0.322,P=0.737);산제료SHD결구역적GIT1억제성골세포국부점부내FAK락안산397적린산화,동시강저FAK재국부점부내적표체.결론 산제료SHD결구역적GIT1통과강저여FAK적상호결합,종이억제성골세포국부점부내FAK락안산397적린산화,영향성골세포적공능.
Objective To study function and mechanism of the Spa2 homplogy domian(SHD)of G protein coupled receptor kinase interacting protein 1(GIT1)on focal adhesion kinase(FAK)activation in rat osteoblasts.Methods Osteoblasts were prepared from the calvaria of SD rats 1 to 2 days old.The third generation of osteoblasts was treated with platelet-derived growth factor(PDGF)to detect the relationship between GIT1 and FAK.Localizations of GIT1 and FAK were determined by immunofluorescent staining with or without PDGF stimulation.We made GIT1 and GIT1(del SHD)lentiviruses to detect the relationship between GIT1(del SHD)and FAK by imnunoprecipitation.The FAK activation was checked in osteoblastic cells infected with GIT1 and GIT1(del SHD)lentiviruses by immunofluorescent staining.Results The association between GIT1 and FAK was increased after PDGF stimulation.GIT1 and FAK were localized in focal adhesion in osteoblastic cells after PDGF stimulation.The interaction between GIT1(del SHD)and FAK was not changed after PDGF stimulation(F =0.322,P =0.737).The GIT1(del SHD)inhibited the FAK activation in focal adhesion in osteoblastic cells.Conclusion GIT1(del SHD)can inhibit the association with FAK and decrease the FAK activation in focal adhesion in osteoblast cells after PDGF stimulation,affecting the capacity of osteoblasts.
