摘要
为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .
In order to observe the molecular mechanism of aFGF in cell proliferation.The signal transduction pathway of aFGF after aFGF binds to the specific receptor was studied.Characterization of aFGF receptor on human umbilical vein endothelial cell(HUVEC)was determined by Scatchard plot analysis of binding of 125 I aFGF to HUVEC.After application of aFGF to HUVEC,the activity of tyrosine protein kinase(TPK)and protein kinase C(PKC) was determined by the incorporation of [γ 32 P] ATP into exogenous substrate,[Ca 2+ ] i was measured with Fura\|2/AM.Scatchard plot analysis showed a single class receptor with K d values of 3.6×10 -10 —9.6×10 -10 mol/L and receptor numbers/cell of 27090.The activity of TPK and PKC increased in a dose dependent manner.At 11.2 mg/L of aFGF,the activity of TPK and membraine PKC was about three times that of control group.Cytosolic PKC activity was 1.87 times that of control group.[Ca 2+ ] i was elevated significantly compared with that of control group.The results suggested that aFGF receptor possessed TPK activity.These tyrosine\|specific protein phosphorylation may initicate a cascade of biochemical events,which may ultimately promote gene expression and cell proliferation.PKC and Ca 2+ locate downstream of TPK in HUVEC.
出处
《中国生物化学与分子生物学报》
CSCD
2000年第2期267-270,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金课题!( 3 90 70 45 4)
关键词
AFGF
HUVFC
TPK
PKC
细胞增殖
细胞内信号转导
Tyrosine protein kinase
Protein kinase C
Intracellular free [Ca
2+ ]([Ca
2+ ] i)
Cell proliferation
