【摘要】 目的研究整合素及纤粘连蛋白(fibronectin,FN)在卵巢癌细胞逃避失巢凋亡过程中的作用及其机制。方法2005年1月至2006年5月于山东省立医院采用间接免疫荧光染色检测人卵巢癌SKOV3细胞高转移亚克隆SKOV3-S1细胞中整合素蛋白αv、β1、α5β1的表达;建立细胞悬浮分离培养导致凋亡的模型,流式细胞仪检测细胞凋亡;抗体封闭法检测不同整合素蛋白在凋亡过程中的作用;逆转录PCR检测p53、bcl-2、bax基因表达的变化;比色法检测半胱氨酸天门冬氨酸蛋白酶-3(caspase-3)活性改变。结果(1)S1细胞表面普遍表达整合素αv、β1、α5β1。(2)FN使细胞凋亡率由63.7%降至29.0%,αv及β1抗体使凋亡率上升,α5β1抗体对凋亡没有影响。(3)FN使S1细胞中bcl-2基因表达上升,bax表达下降。αv封闭抗体能抑制FN的基因表达调节作用。(4)在悬浮生长状态下,caspase-3被激活;FN使caspase-3活性减弱;加入αv及β1封闭抗体后,酶活性增强。结论SKOV3-S1细胞表面整合素受体蛋白αv通过与FN结合,提高了细胞凋亡调节基因bcl-2/bax的比率,使caspase-3活性减弱,这是SKOV3-S1细胞避免失巢凋亡的重要原因之一。更多还原

【Abstract】 Objective To observe the effects of integrin αv,β1,α5β1 and fibronectin on survival ability of ovarian cancer cells under anchorage-independent growth and the associated mechanism.Methods Integrin subunits expression on the human ovarian cancer cell SKOV3-S1 surface was analyzed.The role of different integrin subunits with or without fibronectin during anoikis induction was detected by antibody blocking method.During this anoikis process,bcl-2、bax and p53 expression were measured by RT-PCR,and the activity of caspase-3 was measured by colorimetric assay.Results(1)Integrin αv,β1,α5β1 were commonly expressed in SKOV3-S1 cell.(2)Fibronectin protected S1 cells from anoikis by 54.4% and decreased apoptotic cells from 67.3% to 29%.Anti-integrin αv blocking antibody resumed the anoikis that was inhibited by fibronectin evidently,and anti-integrinβ1 blocking antibody also resumed the anoikis partly.(3)The treatment of fibronectin increased bcl-2 expression and decreased bax expression.Anti-integrin αv blocking antibody inhibited this modulation.(4)Caspase-3 was activated when cells grew in suspension.The activity of caspase-3 decreased with fibronectin in the culture media.An elevation of caspase-3 activity was observed when anti-integrin αv or β1 blocking antibody was added.Conclusion The binding of αv and soluble fibronectin protects SKOV3-S1 cells from anoikis apoptosis through increasing the ratio of bcl-2/bax and decreasing the activity of caspase-3.This is probably one of the important reasons for ovarian cancer cells’ resistance to anoikis.更多还原