摘要
目的:探讨整合素β1(β1-integrin)、蛋白激酶C(protein kinase C,PKC)与细胞骨架结构改变的关系及其对人脑胶质瘤细胞的生物学行为的影响和作用机制。方法:以U251胶质母细胞瘤细胞为研究模型,应用抗Inβ1抗体抑制内源性Inβ1作用,应用PKC激活剂PMA、PKC抑制剂Calphostin C干扰PKC的功能,通过细胞黏附实验、迁移实验和体外侵袭实验,检测及PKC对人恶性胶质瘤细胞黏附、迁移和侵袭能力的影响。通过免疫荧光化学染色结合激光共聚焦显微镜和扫描电镜方法,观察细胞微丝F-actin和胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)等骨架蛋白数量、分布和细胞表面伪足情况,比较整合素β1、PKC对微丝、GFAP等骨架蛋白的影响。结果:PKC激活剂PMA增加U251MG细胞在FN上的黏附能力(P<0.05),但PKC抑制剂Calphostin C及抗整合素β1抗体对细胞黏附能力无明显影响。PKC激活剂PMA增加U251MG细胞在FN的运动、迁移能力(P<0.01),PKC抑制剂Calphostin C及抗整合素β1抗体减弱U251MG细胞在FN上的运动、迁移能力(P<0.01)。PMA可促进U251MG细胞的体外侵袭能力(P<0.01),Calphostin C及抗整合素β1抗体可减弱此作用(P<0.01)。U251MG细胞内可见散在的微丝结构,在PKC激活剂PMA处理的细胞内,难以见到清晰的细胞微丝骨架,并常见大量絮团状的F-actin小体,但对GFAP的表达和结构并无影响。PKC抑制剂Calphostin C使微丝沿细胞膜成簇状排列,而抗整合素β1抗体可使细胞内F-actin微丝形成束状纤维(应力纤维),粗壮而密集,且GFAP表达增强。扫描电镜观察显示,加入PMA后,U251细胞表面的伪足数量明显增加,而PKC抑制剂Calphostin C及抗整合素β1抗体处理的细胞内,伪足数量明显减少,甚至消失。结论:整合素β1与FN协同作用,可改变U251MG细胞微丝骨架结构、数量和排列分布,以及细胞表面伪足结构和数量,从而影响肿瘤细胞的运动、迁移及体外侵袭能力,激活P
Objective： To investigate the correlations of β1- integrin and protein kinase C （PKC） with the inva-sive behaviour of gliomas. Methods ：The effects of β1-integrin, protein kinase C on cell adhesion, migration and in vitro invasion of U251MG malignant glioma cells were examined by adhesion assay, migration assay and invasion assay in vitro. The amount and distributions of cellular microfilaments, glial fibrillary acidic protein （ GFAP） and invadopidoas were demonstrated by immunoflorescent cytochemistry, confocal laser scanning microscope and scanning electron microscope, then the effects of β1- integrin and protein kinase C on cellular microfilament skeleton and GFAP were analyzed. Results：PMA stimulated cell adhesion of U251MG cells （P 〈 0 . 05 ） ,but Calphostin C and anti - β1- integrin antibodies had little effect on laminin - mediated cell adhesion. The cell migration of U251MG cells on dishes coated with FN was stimulated by PMA （ P 〈 0. 01） , Calphostin C and anti -β1 integrin antibodies inhibited cell mi-gration of U251MG on dishes coated with fibronectin （P 〈0. 01）. PMA disrupted the microfilament network and F - actin aggregated in these cells. Calphostin C and anti - β1 integrin made F - actins in U251 cells formed strong and dense stress fibers. PKC showed no effect on the structure and expression of GFAP. Moreover, the anti - β1 integrin antibodies improved the GFAP expression. PMA increased the invadopidoas number on cell surface,but Calphostin C and anti - β1 integrin antibodies reversed this function. The cellular invasion ability of U251 cells was enhanced by PMA （ P 〈 0. 01） , but inhibited by Calphostin C and anti - β1 integrin antibodies （P 〈 0. 0 1 ） . Conclusion ： The interactions between β1 - integrin and PKC may stimulate cell migration via changing the structure of microfilament skeleton and the number of invadopidoa in U251MG cells. PKC may be involved in FN - mediated cell adhesion of U251MG cells, β1 - integr
出处
《现代肿瘤医学》
CAS
2017年第10期1543-1548,共6页
Journal of Modern Oncology
